Virology Exam 1

decidua

"maternal component" of the placenta; the differentiated, outer layer of the uterus to which the developing blastocyst attaches and invades

why is VSV used in the lab as a model RNA virus

- Ability to infect a wide variety of cell types - Rapid replication cycle - Susceptibility to interferons

how a phylogenetic tree is made

1) Align sequence 2) Calculate the number of differences between each sequence (Genetic distance) -nucleotide changes frequencies (HKY85) -Insert of deletion events can weigh heavier on tree structure 3) Group sequences together that are similar -Many methods UPGMA, Neighbor-Joining, Bayesian •Many trees possible! None are perfect. 4) Bootstrapping-> robustness (make trees with a subset of sequence data: how often get the same branch points?)--confidence check

what happens when PRR is activated

1) Interferon response factors (e.g. IRF3 and IRF7) control expression of anti-viral cytokines such as IFN-a and IFN-b (so, activate transcription of IFNs) 2) NFkB controls expression of pro-inflammatory cytokines such as IL-6, IL-12 and TNF-a

describe the 3 strategies viruses use to assemble their capsid

1) assembly from individual protein molecules: subunits that were translated separately self-assemble into structural unit, which assemble into capsid due to high affinity 2) assembly from a polyprotein precursor: The capsid proteins are translated sequentially into a polyprotein and then fold and form the individual structural unit for the capsid. the structural units then associate 3) chaperone-assisted assembly: some capsid proteins need the assistance of chaperones to assemble properly • Chaperones can be cellular or viral and can help in forming the structural units

What are the 4 ways that budding can occur

1) glycoproteins insert into cell membrane, then nucleocapsid associates with internal side of membrane, budding occurs 2) Matrix proteins migrate to internal side of cell membrane, then genome comes and matrix proteins drive budding. Glycoproteins added post-maturation 3) envelope proteins drive budding (doesn't need genome to bud)--useful for vaccines 4) glycoproteins and matrix protein required for budding

Virus Life Cycle

1. Attachment (of virion to host cell): glycoprotein spikes on envelope or conformational determinants on capsid bind to host cell receptors that have normal cell function. Specificity of attachment is a key determinant of host range 2. Entry (of nucleocapsid to host cell) Two major pathways: • Receptor-mediated endocytosis • Direct membrane fusion 3. Uncoating: May be triggered by pH changes within endosomes 4. Replication and protein production: All viruses must generate positive strand mRNAs that can be read by host ribosomes in order to produce proteins and replicate themselves: No exception. 4. Assembly (of viral components into progeny virions) and Release (of progeny virions from host cell): budding, lysis

what are the major steps of virus entry into host cells

1. Binding to Surface Receptors 2. Internalization of Virus 3. Trafficking 4. Uncoating Viral Transcription and Replication

theories of SARS-CoV-2 origin

1. Natural selection in an animal host before zoonotic transfer 2. Natural selection in humans following zoonotic transfer Original reservoir--bats, could have been passed to intermediate host, like the pangolin, and then us

what are some viral transcription factors

1. VP16 - Herpes Simplex Virus 1: VP16 Forms complex with cellular transcription factors OCT1 and HCF1 to cause gene expression 2. Zebra - EBV - Regulatory protein, enhances viral transcription; doesn't need host transcription factors to promote viral transcription 3. NF-κB - Cellular transcription factor used by many viruses to enhance viral gene transcription (ex: KSHV)

why does immune selection have little influence on HIV phylogenies at the population level

1. there is an extreme bottleneck in viral diversity at the time of transmission. 2. Transmission occurs early in infection before immune selection has had a chance to operate. 3. Finally, the replicative fitness of a viral strain (measured in transmissions per host) is largely extrinsic to virological factors, depending more heavily on behaviors in the host population.

two ways host can use innate immunity defense against viruses

1: Generation of an anti-viral state at the initial site of viral infection and surrounding cells. This anti-viral state depends mainly on type I/III interferons. 2: Infiltration and activation of "innate immune cells", such as macrophages (Mø), monocytes (Mo), dendritic cells (DC), innate lymphoid cells (NK and ILC) and neutrophils. Key functions include secretion of inflammatory cytokines; phagocytosis of viral particles and virus/antibody immune complexes; and phagocytosis and/or lysis of virus-infected cells. Innate cells (especially dendritic cells) are also critically important for activation of the adaptive immune response.

what does the influenza nucleocapsid look like

A double-helical conformation in which two NP strands of opposite polarity are associated with each other along the helix. Both strands are connected by a short loop at one end of the particle and interact with the polymerase complex at the other end.

leaky scanning

A translation strategy used by almost all viruses. During translation initiation the 40s ribosome can scan past a weak Kozak sequence and initiate translation at a further downstream AUG. Allows the production of different proteins in specific quantities from a single mRNA by shifting the reading frame to have different start codons

defensins

AMPs. found on epithelial. create pores in microbial membranes, disrupting their integrity. Only effective at killing enveloped viruses.

what happens when macrophages are overwhelmed by TLR stimulation

Activated macrophages can generate huge amounts of pro-IL-1beta very rapidly, which are then stored in the cytoplasm in an inactive state. Overwhelming TLR stimulation leads to inflammasome activation, IL-1b release, and death.

systems biology and systems virology

An approach to studying biology that aims to model the dynamic behavior of whole biological systems based on a study of the interactions among the system's parts. Whole system approach to investigate the body's response to viruses. •Properties or biological outcomes that cannot be predicted by an understanding of the individual parts of a system alone, but rather become apparent only with knowledge of the specific organization of, and interactions between, components •Assumptions about interactions can be included in statistical models and tested by model selection methods. The immune system is highly interregulated. Hence, systems level investigation is critical for understanding immune responses to infections and diseases

why are B cells not enough?

Antibodies are generally ineffective against non-cytopathic viruses with cell-cell transmission. It's also a good idea to kill virus-infected cells before they can make huge numbers of viral particles, which is why T cells are important

How do antibodies help the immune response/help fight viruses

Antibodies block virus invasion of target cells (block virus binding to cell receptor and clump them into aggregates), and enhance virus particle phagocytosis bc phagocyte binds to Fc region of antibody and engulfs antigen it is bound to. Antibodies can reduce viral spread after new virions are released by infected cells

how is the adaptive immune response activated

Antigen is carried in lymph from the site of infection/vaccination to the nearest SLO by dendritic cells. Then, need to activate rare-antigen specific lymphocytes. These lymphocytes then multiply and differentiate, but bear receptors of identical specificity.

how can pathogens reach the placenta

Ascending infection from the maternal uterine tract through the decidua (uterine wall): Sexually transmitted pathogens such as HIV, syphilis, HSV and CMV; ZIKV Systemic infection by way of maternal blood passing through the intervillous space and decidua: targets syncytiotrophoblast and extravillous trophoblast (EVT) Antibody-mediated transfer: ZIKV? --antibodies that have virus attached can bind to syncytiotrophoblasts and then can then be transferred into placenta • Infection can also occur during the process of birth as the baby transits through the uterus: • HIV • HSV

what are the two ways that viruses can bind to the cell surface

Attachment factors: • Bind viral particles to the cell surface • Interactions usually non-specific Cell receptors: • Specific interactions with viral proteins, specific • Actively promote viral entry; allows virus to target cells its capable of infecting

Receptors on B and T cells

B cells express membrane-bound antibody on their cell surface. This antibody molecule is also called the B cell receptor, or BCR. T cells express a membrane-bound receptor on their cell surface (TCR) that recognizes antigen fragments bound to antigen presenting molecules (MHC). Both are constructed from immunoglobulin domains.

how do CD4+ T cells start the process of somatic hypermutation

B cells with really good BCR that are binding viral antigen are endocytosed, and this antigen is presented on MHC II of B cell. T cell can bind to MHC II + antigen and make this cell proliferate. T cell won't bind to B cell that doesn't have good BCR.

how are unique BCRs and TCRs formed

BCR: The Ig heavy and light chain loci have multiple immunoglobulin gene segments. Generation of antibody diversity is due to random recombination of Ig gene segments. Also, nucleotides can be added or taken away from junctions between gene segments. TCR: random recombination of multiple immunoglobulin gene segments. Also, nucleotides can be added or taken away from junctions between gene segments.

why are bats major reservoirs of viruses

Bats have many anti-ageing genetic tricks: Most bats live less than 20 years in the wild, but six species live more than 30 years, allowing viruses to evolve within them for a long time. Bats don't get sick from viruses: Bats are stocked with many genes responsible for anti-viral activity and they always have their anti-viral mechanisms switched on. Also, Unique immune system of bats forces viruses to rapidly evolve within them, creating a unique breeding ground for viruses that could potentially hop between species.

what does the TCR need in order to bind to MHC with high affinity

CD4 and CD8 are expressed by T cells, and are TCR co-receptors for MHC molecules. Expression of these co-receptors is essential for T cell activation: the TCR alone has very, very weak affinity for MHC. The co-receptors bind to the non-polymorphic site of the MHC, whereas the TCR binds to the variable region of the MHC (where the antigen is)

Helical Nucleocapsid Assembly

Capsid is assembled around the nucleic acid genome and relies on self and nucleic acid interactions. When one capsid protein binds, it increases the affinity for more proteins to bind

what are characteristics of naked capsid viruses that make them more stable?

Capsid is resistant to: • Drying • Heat • Detergents • Acids • Proteases Consequences: • Can survive in the gastrointestinal tract • Retain infectivity on drying • Survive well on environmental surfaces • Spread easily via fomites • Must kill host cells for release of mature virus particles • Humoral antibody response may be sufficient to neutralize infection

How do capsids with helical symmetry form

Capsid proteins engage in identical, equivalent interactions with one another and with the viral genome to allow construction of a large, stable structure from a single protein subunit. TMV Virus Purified TMV RNA and coat (capsid) protein can assemble by themselves to form functional viruses! Self-assembly

how are innate immune cells activated

Constitutively expressed molecules (alarmins) are activated by cleavage and released by dying host cells. Other cell contents, including virions, viral particles and host molecules (e.g. DNA) are released. Many of these molecules (PAMPs, DAMPs) can bind to receptors expressed by innate immune cells.

How are orthomyxoviruses (enveloped virions with helical capsids) assembled?

Contains segmented genome with multiple nucleocapsids. Helical structure is slightly different than usual because RNA is exposed, which allows for interaction between the different segments/nucleocapsids

Flu viral infection is normally restricted to the respiratory tract, so why are pregnant women more susceptible and at a greater risk of death?

Current thinking is that pregnant women's respiratory capacity is decreased due to the placenta impinging on the diaphragm, and this in conjunction with excessive production of inflammatory cytokines leads to increased incidence of death

How do ssDNA viruses replicate their genome

DNA dependent DNA polymerase uses (+) strand as template to make complimentary (-) strand. (-) strand in double stranded DNA is used as template by DNA dependent DNA polymerase to make (+) strand that is packaged into viral genome Transcription comes from dsDNA intermediate

What are the 3 ways that viral proteins can interact with viral envelope proteins

Direct: internal domain of glycoprotein interacts with/anchors to capsid Via a matrix protein: internal domain interacts with/anchors to internal matrix protein Via a multiprotein layer: complex icosahedrals--interacts with/anchors to layers of internal proteins that are covering the capsid

how do dying neutrophils help the host

Dying neutrophils form neutrophil extracellular traps (NETs) from their DNA that are sticky and can capture bacteria and viruses

Icosahedral Capsid Assembly

Empty protein shell (procapsid or prohead for tailed bacteriophages) is assembled first. • The procapsid is then filled with the virus genome becoming a capsid • Ex: Many large dsDNA viruses have ATP-dependent molecular machines that pump the DNA into the capsid, creating a pressurized particle

how do the majority of animal viruses enter the cell

Endocytosis because it makes viral genome easier to travel through cell to particular spot without being degraded (bc cell can detect viral genome floating around in cytoplasm and destroy it)

Macropinocytosis

Endocytosis by actin rearrangement. viral attachment activates intracellular signaling which initiates actin filaments to rearrange, making the plasma membrane protrude and form an enclosure around the virus. The virus is then taken into the cell in a macropinosome, which fuses with a lysosome. Low pH causes fusion at endosomal membrane (envelope) or lysis (naked) so that the viral genome can be released into the cytoplasm

what are characteristics of enveloped viruses that make them less stable

Envelope is sensitive to: • Drying • Heat • Detergents • Acid Consequences: • Must stay wet during transmission • Transmission in large droplets and secretions • Cannot survive in the gastrointestinal tract • Do not need to kill cells in order to spread • May require both a humoral and a cellular immune response

How do viruses use direct cell-to-cell fusion to infect cells

Example: HIV infected cell accumulates Gag-GFP at cell surface, which fuses infected cell with neighboring, uninfected CD4+ cells Syncytia formation: RSV infected cell can fuse with 4 neighboring uninfected cells

how can researchers use a cellular approach/assay to measure immune response/status and collect data

Flow cytometry: cell counting, cell sorting by suspending cells in a stream of fluid and passing them by an electronic detection apparatus

why are there increased rates of schizophrenia and autism in the progeny of pregnant females infected with flu

Flu virus not found in the fetus, so symptoms did not arise from infection. interleukin-6 (IL-6) produced by the maternal immune system in response to flu infection is associated with this phenotype • Thus, cytokines meant to protect the host appear to result in pathology of the progeny

what process ensures the integrity of the outer layer of the placenta and what impedes this integrity?

Fusion of Cytotrophoblast into Syncytiotrophoblast • This process is continuous during pregnancy and is necessary to maintain the integrity of the syncytiotrophoblast layer and its functions • Fusion requires proteins called "syncytins", which are envelope-glycoprotein-derived genes of endogenous retroviruses • Thus, the integrity of the outer layer of the placenta requires genes originally encoded by retroviruses IFNβ Inhibits Fusion of Primary Human Villous Cytotrophoblast Cells

What information is encoded in a viral genome?

Gene products and regulatory signals for: • Replication of the viral genome • Assembly and packaging of the genome • Regulation and timing of the replication cycle • Modulation of host defenses • Spread to other cells and hosts

how did researchers test where the viral infection is localized within the 1st trimester placental villi

Get 1st trimester placental explants, infect with VSV-GFP, fix and stain with different antibodies of different colors that bind to either SYN or CYT cells, observe with florescence microscopy. VSV-GFP was observed in villous cytotrophoblast but not syncytiotrophoblast. Then it moved to the stroma. So, SYN not being infected, CYT is. Results!!: syncytiotrophoblast from 1st trimester placentas is resistant to infection, BUT it does not serve as an effective barrier to viral infection of the placenta, at least in the 1st trimester (it could be barrier in later trimesters). Indeed, the placenta is significantly more susceptible in the 1st trimester.

How does neuraminidase help influenza be released externally after budding

HA on influenza can accidentally bind to sialic acid on cell membrane. Neuraminidase cleaves the receptor to release the virion

how can the virus inhibit PRR signaling and IFN production

HIV and rotavirus can degrade IRF7 and IRF3, which are host transcription factors that are responsible for inducing transcription of IFNs

which is a virus that experiences its first replication and transcription in the capsid

HIV reverse transcription

how does HIV target the immune response

HIV targets CD4+ T cells. Without these cells, Impaired CD8+ T cell immunity to all antigens because CD4+ T cell interactions with dendritic cells are essential for the development of fully functional CD8+ T cell immunity. Also, decreased somatic hypermutation, so decreased antibodies. Also, decreased activity of macrophages, so impaired clearance of infected cells.

How do dsDNA reverse transcription viruses replicate their genome

Hepatitis B • Host cell DNA-dependent RNA-polymerase transcribes RNA from DNA genome. This RNA is used as template by the viral RNA-dependent DNA-polymerase to replicate the DNA genome in a circular fashion. Complete replication of the genome is not completed prior to packaging. A "relaxed-circular" form of the dsDNA genome which has a gap in the DNA is packaged. After infection the rcDNA is transported to the nucleus and the host DNA repair machinery fills in the gap to make the final closed-circular DNA. Cycle starts again.

why does Medium from Human SYN cells Confer Viral Resistance to Recipient Cells

Human placental syncytiotrophoblast in contact with maternal blood (and underlying villous cytotrophoblast) secrete exosomes containing a family of microRNAs (miRNAs) encoded by a large cluster on chromosome 19 (C19MC) • These C19MC function to induce autophagy in recipient cells, which subsequently inhibits viral infection inhibits translation of RNA it binds to

Baltimore system of classification

I. Double-stranded DNA viruses: Some replicate in the nucleus using cellular proteins. Poxviruses replicate in the cytoplasm and make their own enzymes for nucleic acid replication. II. Single-stranded (+) sense DNA viruses: Replication occurs in the nucleus, involving the formation of a (-) sense strand, which serves as a template for (+) strand RNA and DNA synthesis. III. Double-stranded RNA viruses: These viruses have segmented genomes. Each genome segment is transcribed separately to produce monocistronic mRNAs. RNA dependent RNA polymerase can attach to negative strand to make positive sense RNA strand IV. Single-stranded (+) sense RNA viruses: Polycistronic mRNA: Genome RNA = mRNA. Since the RNA is the same sense as mRNA, the RNA alone is infectious, no virion particle associated polymerase. Translation results in the formation of a polyprotein product, which is subsequently cleaved to form the mature proteins V. Single-stranded (-) sense RNA viruses :The virion RNA is a negative sense (complementary to mRNA) and must, therefore, be copied into the complementary plus-sense mRNA to make proteins. This group of viruses must code for RNA-dependent RNA-polymerase and also carry it in the virion so that they can make mRNAs upon infecting the cell. VI. Single-stranded (+) sense RNA viruses with DNA intermediate in life-cycle: RNA genome is (+) sense but does not serve as mRNA. It is used as a template for reverse transcription. The viruses (Retroviruses), therefore, encodes an RNA-dependent DNA polymerase (reverse transcriptase) to make the DNA provirus which then is transcribed to genomic RNA by a host enzyme, RNA polymerase II. VII. Double-stranded DNA viruses with RNA intermediate: This group of viruses also relies on reverse transcription, but unlike the Retroviruses, this occurs inside the virus particle on maturation. On infection of a new cell, the first event to occur is a repair of the gapped DNA genome, followed by a transcription.

How does the body maintain tissue homeostasis and prevent over-damaging? Essentially, how do we regulate/control the IFN response?

IFN responses through the JAK-STAT pathway are eventually attenuated to prevent induction of programmed cell death and/or adaptive immune responses against healthy cells (autoimmune disease) because prolonged cytokine production is causes tissue damage. Failure of cells to attenuate IFN responses can lead to programmed cell death that damages organs (ISGs can induce apoptosis). Moreover, long duration IFN signaling can activate inappropriate innate and adaptive immune responses against host tissues. JAK-STAT signaling in response to IFNs is controlled at multiple levels and by distinct mechanisms: • IFN Cell surface receptors can be internalized (i.e., IFNa/bR) • Protein tyrosine phosphatases, i.e., PTPs (SHP1, SHP2, PTP1B, CD45) inhibit JAK activity and STAT-P by causing JAK degradation (PTP is an ISG, so feedback loop) • Suppressor of cytokine signaling-1 (SOCS1) and SOCS3 inactivate JAKs (SOCS are ISGs, so feedback loop) • Protein inhibitors of activated STAT (PIAS) inactivate STATs SOCS1 knockout mice died because can't turn off inflammatory response

which IFN protect mother and fetus from viral infection, not just placenta

IFN-1 important for protecting mother and preventing death

how do IFNs signal to cells to go into an antiviral state

IFNs mediate their responses through activation of JAK-STAT pathways. JAK is kinase bound to IFN receptor. When IFN binds, JAK phosphorylates STAT, which forms a complex and activates transcription factors (IRF9) that are responsible for transcription of ISG. This whole complex can enter the nucleus to increase transcription of ISGs. Each family of IFNs utilize distinct receptors but overlapping JAK and STAT molecules Overlapping but distinct sets of "interferon stimulated genes (ISGs) are activated by each of the IFNs; some genes also repressed

inhibitory cytokines

IL-10 is involved in the suppressive function of regulatory T cells and play critical roles in maintaining immune homeostasis to prevent over-damaging. Suppresses inflammatory cytokines. • Inhibits effector T cell responses due to down-regulation of MHC and co-stimulatory molecules on antigen resenting cells. • Impairs DC maturation viruses can upregulate IL-10 to decrease dendritic cell antigen presentation and decrease activation of T cells and antibody response

3 different strategies viruses use to initiate translation

IRES, Cap snatching, Leaky scanning

in what ways could a virus have a functional outcome/could branch off?

If gained function in: •Virulence phenotypes •Phenotypes associated with viral transmissibility •Cell or tissue tropism phenotypes Antigenic phenotypes that can facilitate escape from host immunity

when are innate immune cells activated

If infection isn't contained/limited by IFN antiviral state, cells are damaged, virus spreads, innate immune cells in tissue are activated. Then, Plasmacytoid DC (pDC), NK cells, monocytes and neutrophils are recruited from blood into the infected tissue

discovery of interferons

Infect one chick egg membrane with heat inactivated virus and one with buffer Collect fluid post-infection and lay on fresh chick egg membrane Infect with real flu virus Chick egg membrane that was infected w heat inactivated virus had reduced viral production; "interference" identified as a soluble factor(s). Chick egg membrane that was infected w buffer had optimal viral production

Cap-snatching

Influenza steals the 5' cap from host RNAs to prime viral translation and disguise viral mRNAs also promotes degradation of host mRNA and allows viral mRNA to be translated by ribosome and host mRNA to not

one way viruses prevent cells from going into antiviral state

Inhibition of JAK-STAT Signaling is an Important Mechanism of Immunoevasion • A multitude of pathogens, including many viruses, Toxoplasma, bacteria, mycobacteria, etc., block cellular responses to IFNs by inhibiting the JAK- STAT pathways dephosphorylate activated, phosphorylated STAT1 degradation of STAT2 HIV (and multiple other viruses) activate expression of SOCS1 to block JAK activity Multiple viruses activate expression of SOCS3 SARS-COV-2 ORF-6 blocks STAT1 nuclear translocation

how did MERS emerge

It is believed that the virus originated in bats and was transmitted to camels sometime in the distant past. Became stable in camels. Camels are likely to be a major reservoir host for MERS-CoV and an animal source of MERS infection in humans.

how do viruses inhibit MHC I antigen presentation to CD8+ T cells

Key stages of the pathway are targeted by immunomodulatory proteins. 1. Proteasomal processing inhibitors: evasion of proteosomal processing so that peptides are not generated 2. TAP function inhibitors: viral protein binds to cytoplasmic or ER side of TAP, generating conformational change 3. Tapasin function inhibitors 4. ER-associated degradation inducers 5. ER retainers or retrievers of MHC class I molecules

how do viral infected cells get killed?

MHC I molecules can bind viral peptides that can be recognized by CD8+ cytotoxic cells, and the infected cell is killed.

MHC

MHC molecules are membrane-associated peptide-sampling devices. They show which peptides are inside the cell, whether they are self or non-self. Have polymorphic peptide binding cleft.

macrophages, cDC, pDC--what do they release when activated? What PRR do they use?

Macrophage: cytokines, TLR cDC: cytokines, TLR pDC: IFN-alpha, TLR

which innate immune cells are tissue resident cells and which circulate the blood

Macrophages, dendritic cells (cDC) and innate lymphoid cells (ILC) are tissue-resident cells Plasmacytoid DC (pDC; the principal source of IFN-a), NK cells, monocytes and neutrophils circulate in the blood and come to site of infection when need be.

how does second-time infection get squashed so quickly by immune system

Much higher frequency of antigen-specific B and T cells .Antibodies have appropriate effector functions (e.g. class switch to IgG, IgA) and are in blood. Antibodies have high affinity for antigen (somatic hypermutation)

how do ISG Protein Products Promote the Antiviral State

Multiple mechanisms are employed to block viral replication: • Degradation of viral nucleic acids • Inhibition of viral protein translation • Inhibition of viral budding • Apoptosis of virally-infected cells • Some of the ISGs are JAK-STAT pathway components, which is thought to confer heightened sensitivity to viral onslaught • Certain IFNs themselves are also ISGs

What do negative sense RNA viruses need that positive sense RNA viruses don't

Must contain an RNA dependent RNA polymerase in the virion (influenza and other orthomyxoviruses)

how does the host respond to viruses that interfere with MHC I presentation/downregulate MHC I production to prevent CD8+ cells from killing

NK cells --> cytotoxic cells NK cells express two kinds of receptor: activating (ITAMs) and inhibitory (ITIMs) receptors. If cell has MHC I, inhibitory receptor binds to it, which tells NK cell not to kill. If cell doesn't have MHC I, inhibitory receptor does not bind and activated NK cell releases granules, inducing apoptosis in target cell

how are NK cells activated and what do they do

NK cells proliferate in response to type 1 interferons that are produced by viral infected cells, and differentiate into cytotoxic cells.

Information NOT contained in viral genomes

No genes encoding the complete protein synthesis machinery (AARS, eIFs, tRNAs) • No genes encoding proteins involved in energy production or membrane biosynthesis • No classical centromeres or telomeres found in standard host chromosomes • Probably we haven't found them yet - 90% of giant virus genes are novel

is genetic variation the same across viral proteins and across a single viral protein

No. Same mutation rate, but some viral proteins are more likely to evolve than others because some proteins are essential and lose function completely with mutation. Also, a single protein can have essential and non-essential domains--essential domains won't evolve as much as non-essential domains will

what happens when B cells are activated

Once activated by antigen, B cells secrete a soluble form of the BCR (i.e. antibodies) into the blood

what is a way that researchers can do primary data analysis

PCA (principle component analysis) transforms data to convert a set of observations of possibly correlated variables into a set of values of linearly uncorrelated variables called principal components. The number of principal components is less than or equal to the number of original variables. Shows correlations •Unsupervised clustering allows evaluation of similarity between samples and genes especially for high-throughput datasets

clustering algorithm

PCoA and PCA. Just like phylogenetic tree, but different visual. Clusters viral genome sequences that are closely related and separates clusters that have viruses that are more genetically distant

cGAS/STING

PRR in cytoplasm. Senses viral DNA, phosphorylates IRF3, which promotes transcription of INF-1

TLR for viral products

PRR that binds to microbial properties. TLRs specific for viral products are expressed inside endosomal vesicles because this is where the viral PAMPs can be detected. This is important, because viral DNA/RNA often isn't accessible until virions are degraded or uncoated in phagolysosomes. When activated, activates IRF3, IRF7, and NFkB

where can PRRs be found

PRRs are expressed inside endosomes, on the mitochondria, the cytoplasm (RIG-I), and on the cell surface, in accordance with the fact that different pathogens (viruses) have different intracellular life cycles and thus can be localized to different sites within the cell

what are fusion proteins and what are the three classes of fusion proteins

Participate in receptor-mediated fusion. Fusion proteins on virions start out in a "prefusion conformation" - inactive • Fusion proteins becoming fusion competent is typically irreversible • Usually fusion is triggered by low pH (proteolytic cleavage). Class I: • Proteins are trimers • Perpendicular to the membrane (spikes) • α-helix is the major secondary structure • Requires proteolytic cleavage to become fusion competent • Examples: Influenza virus (HA protein) Class II: • Proteins are dimers • Parallel to the viral membrane • β-sheets are the major secondary structure • Also require proteolytic cleavage to become fusion competent • Examples: Dengue virus (E protein) Class III: • Complex, not well defined • Made up of α-helices and βsheets • No obvious "priming event" like proteolytic cleavage • Examples: Vesicular Stomatitis Virus (G protein)

how do cells detect viral infection

Pathogens, including viruses, express a variety of molecules collectively termed "Pathogen-Associated Molecular Patterns" (PAMPs). PAMPs can also be viral nucleic acid, like dsRNA, uncapped ssRNA PAMPs are recognized by "Pattern Recognition Receptors " (PRRs) expressed by a variety of different cell types. Each PRR recognizes and binds to distinct features of nucleic acids (dsRNA, etc.) derived from pathogens. upon binding to PAMPs, PRRs activate intracellular signal pathways to activate IRFs, which are transcription factors for the production of IFNs and other cytokines. Viral genomes and proteins that are released after cell lysis can bind to surrounding PRRs, activating them to start making and releasing IFNs

How do pandemic influenza viruses occur

Pigs can be naturally infected with both avian and human viruses. • Epithelial cells in pig tracheas contain both human- (a2,6-) and avian (a2,3-)-type receptors. • Pigs as "mixing vessels" hypothesis: pigs are simultaneously infected with avian and human viruses, which allow for the generation of reassortants capable of causing pandemics. These reassorted influenza virions have novel surface proteins (HA or NA), so the human population is entirely non-immune

how did researchers test viral infection of the placenta between the 1st, 2nd, and 3rd trimesters?

Placenta explant cultures taken from 1st, 2nd, and 3rd trimester pregnancies were cultured with VSV modified to have GFP. Florescence microscopy used to see where virus was replicating in the placenta. Found that 1st trimester placenta villi were more susceptible to infection bc of high florescence and term placenta had no florescence at all, so were completely resistant.

What are the functions of viral proteins

Protect viral genome - assembly of a stable, protective protein shell - specific recognition and packaging of viral genome Deliver viral genome - bind to the specific host receptors - transmit specific signals that induce uncoating of the genome - fuse with host cell membrane (enveloped viruses) - interact with cellular components to transport the genome to the appropriate site for replication • Other interactions - with cellular components to ensure efficient infection cycle - with cellular components to transport structural components during assembly - with the host immune system to regulate immune response

What is a virus capsid?

Protective protein coat surrounding the nucleic acid • Usually symmetrical & comprised of repeating subunits; either (1) helical (rod-like) or (2) icosahedral (sphere-like)

RLR sensor

RIG-1. PRR that senses viral RNA, then activates IRF3 and NFkB to turn on transcription of INF-1 and cytokines

what are the major classes of PRRs that bind viral products

RIG-I-like receptors (RLR), NOD-like receptors (NLR), Toll-like receptors (TLR)

how do (+)ssRNA RT replicate their genome

RNA dependent DNA polymerase comes packaged in virion because cannot use (+)ssRNA as mRNA RT uses +ssRNA as template to make -ssDNA. DNA dependent DNA polymerase uses -ssDNA as template to make +ssDNA, so dsDNA is formed. Then, DNA dependent RNA polymerase copies (-) DNA strand to make +ssRNA genome. Transcription occurs from dsDNA intermediate

What kind of genome do retroviruses have

RNA single stranded, positive sense (like mRNA)

how do (-)ssRNA replicate their genome

RNA-dependent RNA polymerase comes packaged in virion and is used to make mRNA and (+)ssRNA template that is used to make new (-)ssRNA genome Rabies and Flu Transcription occurs from (-)ssRNA template

what are the two viral entry pathways

Receptor-mediated fusion: only enveloped viruses Endocytic pathway Viruses can use both, depending what receptors are present on the cell surface: EBV does fusion in epithelial cells but endocytosis in B cells

Steps of reovirus disassembly

Reovirus virions enter cells by receptor-mediated endocytosis. Within an endocytic compartment, the viral outer-capsid undergoes acid-dependent proteolysis. The first disassembly intermediate is the ISVP, which is characterized by loss of σ3. Further conformational change expose hydrophobic residue on µ1, which mediates penetration through the endosomal membrane releasing the transcriptionally active core into the cytoplasm 1) endocytosis 2) Shedding and ISVP exposed 3) Uncoating and penetration through endosomal membrane into cytoplasm

why were we able to stop SARS-CoV 1 but not SARS-CoV 2

SARS-1 starts symptoms early and high fever SARS-2 starts asymptomatic and can remain asymptomatic in some

Why do we need secondary lymphoid organs? (lymph nodes)

SLO provide a place that maximizes the chances of interaction between antigen and rare, antigen-specific B and T cells. SLO also provide an environment where lymphocytes activated by antigen can proliferate and differentiate into effector cells.

how are term SYN resistant to infection by ZIKA

SYN secretes IFN-lambda, which can protect surrounding SYN cells by promoting the antiviral state in the pregnant uterus. Zika infection increased when IFN-lambda receptor is not expressed and injection of IFN-lambda increased protection in placenta and uterus

How does HIV use packaging signals to package its genome into the virion

Secondary structure of RNA genome has region called psi that is folded in specific way. Psi region from two HIV RNA genomes associate to form a complex. HIV nucleocapsid recognizes the complex for packaging

How do some non-enveloped icosahedral viruses get their genome into the cytoplasm

Some non-enveloped viruses inject their genome into the host cytoplasm through creation of a pore in the host membrane. This is mediated by a viral pore-forming peptide associated with the viral capsid. Binding to receptor (yellow) leads to a conformational change in capsid structure during which the VP4 is externalized and creates a pore

subunit vs. structural unit vs. capsid vs. nucleocapsid

Subunit: Single folded polypeptide chain Structural unit (protomer, asymmetric unit): Unit from which capsids are built; one or more subunits Capsid: Protein shell surrounding genome--proteins are non-covalently bound so can break apart easily inside cell Nucleocapsid: Nucleic acid - protein assembly within virion

Placental Mechanisms Proposed to Prevent Viral Infection

Syncytiotrophoblast and cytotrophoblast lack expression of the relevant receptors necessary for viral attachment/internalization ex., HSV • Trophoblast cells express microRNAs (miRNAs) encoded on chromosome 19, which promote an antiviral state • Term syncytiotrophoblast constitutively express IFN-3s, which also promotes an antiviral state

how are T cells activated and what happens when T cells are activated

T cells are activated when TCRs see viral antigen fragments bound to MHC molecules on the cell surface. T cells activated by [MHC:antigen] complexes secrete effector cytokines, or kill infected target cells.

cytotoxic CD8+ T cells

TCR specific for MHC I/peptide

helper CD4+ T cells

TCR specific for MHC II/peptide

Overall, how do trophoblast cells prevent viral infection

TERM Syncytiotrophoblast and cytotrophoblast cells appear to utilize overlapping mechanisms for preventing viral infection, including production of C19MC and IFN-lambda. 1st trimester cells don't have this antiviral activity, so zika can be deadly early on in pregnancy.

How do pDC cells generate IFN-alpha

TLR in endosome is activated by viral nucleic acid; activation leads to the activation of IRF7, which then increases transcription of IFN-alpha

MHC II

Targeted by CD4+ T cells. MHC II molecules are loaded in endosomes, and sample proteins from the extracellular environment

MHC I

Targeted by CD8+ T cells. MHC I molecules are loaded in the endoplasmic reticulum, and sample proteins made by the cell itself.

why is there an increased rate of birth defects that occur when mothers are infected during the 1st trimester.

The human placenta appears to be more susceptible to viral infections during the first trimester. It does not produce IFN-1 or IFN-3 until it gets to the 2nd and 3rd trimesters

alarmins

These proteins expressed constitutively (often as pro-proteins). They are activated by cell stress or damage and released into the local environment. Examples: IL-1a, IL-1b, IL-18, IL-33.

how do macrophages make inflammatory cytokines/induce inflammation

Tissue-resident macrophages at sight of infection stimulated by TLR ligation secrete inflammatory cytokines. TLR in endosome is activated by viral nucleic acid; activation leads to the activation of NFkB, which then promotes transcription of inflammatory cytokines

how did researchers figure out if IFN treatment could Play a Role in Promoting an Antiviral State in the 1st trimester Placenta?

Took placental explants from 1st trimester and cultured them with IFN-alpha or IFN-lambda. Then waited a few hours and added VSV-GFP. Then did immunofluorescence. Found that pre-treatment with IFN-alpha or IFN-lambda resulted in anti-viral state in placenta. alpha was better than lambda. Suggested that, as pregnancy progresses, placental cells start making IFNs to protect the fetus

describe the complex capsids with two icosahedral protein layers? What are the function of the two layers

Two concentric shells: The outer capsid shell plays important roles in maintaining the stability of the thin innermost capsid shell and sequestering the dsRNA genome, as well as in conferring host specificity and mediating entry into host cells Inner shell: protect genome outer shell: attachment Reoviruses: Reoviridae is the largest and most diverse family of double-stranded RNA (dsRNA) viruses

Do icosahedral virions typically have bigger or smaller genomes?

Typically bigger--need to use a lot of their genome (1/3) to code for the capsid subunit

What are the consequences of viral infection of placental villi in the 1st trimester? In what way do viruses cause placental damage?

VSV/GFP infection of 1st trimester villi results is massive apoptosis within the placenta

How are rhabdoviruses (enveloped virions with helical capsids) assembled?

VSV: Viral RNA associated with N protein subunits to form helical nucleocapsid. Matrix proteins covers nucleocapsid, which interacts with lipid membrane/envelope

How can viral proteins initiate replication of genome

Viral proteins (ex: LT for SV40): • Bind to viral origin and direct assembly of cellular proteins into replication complex • SV40 LT protein - Contains binding sites for cellular proteins. Also has helicase and ATPase activities to unwind the origin of replication

phagocytosis

Virus engulfed into a phagosome. Fuses with lysosome. Low pH allows breakdown and inner membrane fusion with phagolysosome and viral seed is released

how do CD4+ T cells get activated and what do they do after activation

Virus-specific CD4+ T cells are activated in SLO (e.g. draining lymph nodes) by dendritic cells loaded with viral antigens (MHC I* + antigen and MHC II + antigen). Virus-specific CD4+ T follicular helper cells interact with antigen-specific B cells in SLO, driving: a) immunoglobulin class switch (e.g. IgM -> IgG, IgA) b) somatic hypermutation and selection for increased antibody affinity (help B cells mutate their immunoglobulin genes to make antibodies with higher affinity to antigen) Virus-specific CD4+ Th1 cells migrate to infected tissues, where they secrete IFN-gamma and cytokines, enhancing macrophage phagocytosis of dead and dying virus-infected cells (supercharge macrophages)

virus definition

Viruses are small, infectious, obligate intracellular parasite, capable of replicating itself in host cells • Minimally they must have two components: • A genome • A proteinaceous structure to protect the genome • An individual particle is called a virion

Internal Ribosome Entry Sites (IRES) and why do viruses utilize it

Viruses don't need 5' cap for translation initiation!! • IRES are structures in the 5' UTR (untranslated region) of viral mRNA • Allow viral mRNA to bypass cap-dependent translation initiation (eIF4E not required, which is always required for host cell translation) • Direct recruitment of other factors or ribosomes to mRNA • Why? - Selective translation of viral mRNA • Many viruses which use IRESs inhibit cap-dependent translation to stop cellular translation (destroy elF4E)

how do researchers model interactions among components of a system

We assume that genes which are up-regulated at similar times are regulated by common TFs. generate transcriptional network of TFs and at what time they are expressed Can do a GRN Clustering coefficient

The relative risks for both miscarriage and congenital infection/birth defects, particularly by ZIKV, are highest when the mother is infected during the 1st trimester of pregnancy. Why?

ZIKV Alters Morphology and Halts the Growth of Human Neurospheres, which are comprised of neural stem cells that differentiate and form organized spheres. So, ZIKV infects and damages neural stem cells, which are present at the start of pregnancy. Conclusion: ZIKV productively infects neuroprogenitor cells, leading to compromised growth and differentiation of these cells and associated structures. Dogma: Elevated risk for adverse outcomes in the 1st trimester from viral infections such as ZIKV and Rubella is due to sensitivity of neuroprogenitor cells to viral infections.

how do viruses interfere with NK cells

a. MHC I homologs: Some viruses encode surrogate ligands for inhibitory NK cell receptors. b. Regulators of MHC I: At the cell surface, HLA-E can interact with inhibitory receptor on NK cells, thereby delivering an inhibitory signal. • hCMV UL40 protein upregulates cell surface expression of HLA-E. c. Release of virus-encoded proteins to bind to activating ligand to block activating receptor from binding. Several viral proteins are able to keep activating ligands inside cells and prevent their surface expressions. d. Antagonist of activating receptor: release proteins that counteract or modulate the interactions between cytokines and their receptor on the infected cell (release proteins that bind to cytokines and inhibit them) e. Infection of NK cell can lead to disruption of function or cell death

how are innate lymphoid cells (ILCs) activated

activated by cytokines released by other innate immune cells, NOT by PAMPs. When activated, secrete IFN-gamma which enhances macrophage phagocytosis and clearance of viruses and virus-infected cells.

how/where do MHC II molecules bind peptides

antigen taken up from extracellular space into vesicle. Vesicle at neutral pH has proteases that are inactive, but acidification activates proteases to degrade antigen into fragments. This vesicle fuses with vesicle that contains MHC II + CLIP. Antigen peptide fragment replaces CLIP, and MHC II + antigen is brought to cell membrane surface.

what animal is the main reservoir of influenza A

aquatic birds, but can infect tons of different animals

why does the adaptive immune response take so much time

because first, need to activate antigen-specific lymphocytes. Then, these lymphocytes need a few days to divide to significant levels.

Receptor-mediated endocytosis

binding of ligands to receptors triggers vesicle formation. Once endosome forms from intake of virus, it fuses with a lysosome, whose acid breaks down the envelope and releases the nucleocapsid into the cytoplasm

IFN-1 (alpha/beta)

both IFN-I and their cell surface receptors tend to be ubiquitously expressed, although dendritic cells (DCs) are the major producers of IFN-a during viral infection

what can phylogenetic trees tell us

can tell us if virus is mutating fast or is stable (can tell us if exponentially growing or stable in population)

where are the different sites that viruses can assemble

cell membrane, golgi, ER, nucleus

epitopes

certain regions of an antigen molecule that stimulate immune responses. A single viral antigen may have multiple epitopes and, therefore, can be bound by several various antibodies. So, one virus can have multiple different antibodies generated against its multiple different epitopes

clathrin vs. caveolin

clathrin: virus binds to receptor protein, adaptor protein migrates to the inside of the cell surface and marks the virus for entry, clathrin associates with adaptor protein and grows around vesicle as it is being taken in, dynamin allows vesicle to pinch off of cell membrane and enter the cell, clathrin dissassociates before being taken in by endosome caveolin: virus binds to receptor on lipid raft of cell membrane (cholesterol enriched domain), cavin-1 and caveolin grow around vesicle as it is being taken in, dynamin allows vesicle to pinch off of cell membrane and enter the cell, cavin-1 and caveolin target it to endosome Differences: clathrin uncoats early, caveolin targets virus to endosome caveolin targeted to lipid raft

How do CD8+ T cells kill their target cells

cytotoxic effector molecules (granules) punch holes in infected cells to lyse them. A tight junction is produced by the CD8+ cells so that these effector molecules are only killing the targeted cells, not all the cells around them. T cells interact with their target cells through the immunological synapse. This directed secretion is important to prevent collateral damage to neighboring cells.

how do CD8+ T cells get activated

dendritic cells can process and present peptides from extracellular antigens on MHCI which is an exception. Then, the MHC I + antigen can activate CD8+ T cells to proliferate. So DC cells can have MHCI and MHC II on them, even when they aren't infected.

how do dsRNA replicate their genome

dsRNA uses viral RNA dependent RNA polymerase to copy the + and - RNA strands to make a new dsRNA genome (also uses this polymerase for transcription) (+)sense RNA can either serve as mRNA or be used as a replication intermediate for new dsRNA Transcription occurs from dsRNA - strand template

What is conserved across all icosahedral virions

eight-stranded antiparallel beta-sandwich is conserved across all icosahedral virion capsid proteins the subunit building block found in many RNA viruses is known as the eight-stranded antiparallel b-sandwich

DAMPs

endogenous danger molecules that are released from damaged or dying cells and activate the innate immune system by interacting with pattern recognition receptors (PRRs) like TLR --> activate NFkB to produce inflammatory cytokines

Direct membrane fusion

enveloped viral membrane fuses with cell membrane, which results in uncoating of the viral genome at the cell membrane. The nucleocapsid is then released into the cytoplasm

clustering coefficient

if genes form a group, how closely connected/related are neighbors of group? •an example of a graph theoretical measure that allows identification of group of correlated genes

what are the 3 mechanisms of virus release

lysis: cell death releases viruses; enveloped and non-enveloped exocytosis: Can occur for enveloped or nonenveloped virus • Release virion from vesicle that came from golgi. If enveloped viruses exit by endocytosis, typically need to acquire envelope from other cellular compartments, like the ER or ER-Golgi intermediate compartment budding: enveloped viruses

IFN antagonist

made by virus to prevent the production of IFN or binding of IFN to cell receptors Viral defense against IFN

what kind of structures are virions

metastable--To be infectious, the particle must protect the genome (stable) and come apart quickly on infection (unstable)

how do antibodies, BCRs, and TCRs bind to target molecules

non-covalently. Since these structures are proteins who have variable regions made up of amino acids, they can bind to almost any complementary surface. This is because of the amino acid's acidic, basic, aliphatic properties. The variable regions on antibodies contain multiple antigen binding sites.

What are the helical capsids of enveloped (-) ssRNA virions called

nucelocapsids Flu, Rabies, VSV • Rhabdoviridae (rabies virus, VSV) • Paramyxoviridae (measles, parainfluenza viruses) • Orthomyxoviridae (influenza A, B, C viruses) • Filoviridae (Ebola virus)

What are different ways that viruses can be endocytosed

phagocytosis, macropinocytosis, clathrin vs caveolin

Immune selection

phylogenetic tree of virus appears ladder-like (when a single predominant trunk lineage persists through time and has side branches that only last for 1-5 years before splitting/going extinct--like the flu). Virus in constant battle with the immune system.

IFN-3 (lambda)

production restricted primarily to epithelial cells, keratinocytes and subsets of DCs; functions are also restricted to these cell types, due to cell-type specific expression of the receptors

IFN-2 (gamma)

production restricted primarily to immune cells (T cells and NK cells) but reported to be expressed by many other cell types under certain conditions; most cells can respond to IFN-g

how are NK cells activated

proliferate in response to type 1 interferons (IFN-alpha) that are released by other infected cells, and differentiate into cytotoxic cells that kill virus infection cells by apoptosis. Also releases IFN-gamma

proof that host immunity shapes evolution

put virus with antibody in a dish and allow to culture. sequence the virus, and find that virus mutated. put virus with NO antibody in a dish was allowed to culture and didn't end up mutating

How do inflammatory cytokines help?

recruits neutrophils, monocytes, and NK cells from blood to infected tissue. Also generates heat at site of infection

why is IL-1beta useful?

resident macrophages can bind IL-1beta, which activates them to generate inflammatory cytokines also initiates cDC (who are bearing antigen) migration from tissues to lymph nodes to active adaptive immune response via T cells

what are the general interactions of hosts and viruses

stable evolving dead-end resistant host

placental villous tree structure

syncytiotrophoblast cells outline the placenta (are on outer surface touching mother's blood bath). Right underneath these cells are the cytotrophoblast cells. EVTs attach placenta (villous tree) to uterine wall

how can researchers use a proteomics approach/assay to measure immune response/status and collect data

test serum proteins, like cytokines. ELISA and multiplex arrays

phylodynamics virology

the study of how epidemiological (e.g. incidence, distribution, and control of diseases.), immunological (e.g. antibodies, B cells, T cells), and evolutionary processes (e.g. founder effect, population mixing) act and interact to shape viral phylogenies (evolution

what is true about people with serious COVID, IFN speaking

those with life threatening COVID either had mutations in IFN-1 pathway genes (like IRF7, IRF9) or were producing antibodies against IFN-1

what is the function of the capsid

to protect the nucleic acid and provide specificity to virus unstable so easy to release genome into cell

differences between type I and type III IFNs

type I induces massive but short antiviral response type III induces induce steady, prolonged antiviral response

What are the three viral uncoating strategies to get the genome into the cell

uncoating at the plasma membrane uncoating within endosomes uncoating at the nuclear membrane: virus taken into endosome, then lysis occurs, then docking onto nuclear membrane (allows virus to go undetected in cell, so can reach the nucleus with more success)

how do MHC I go from binding self-antigen to viral antigen

under inflammatory conditions (IFN gamma), the constitutive proteasome is replaced by the immunoproteasome. This alters the proteasome's enzymatic specificity. There is increased peptide cleavage after hydrophobic residues, and decreased cleavage after acidic residues. This generates peptides that have higher affinity for binding most MHC I molecules, and also preferred structures for transport by TAP. Proteasome-generated peptides are translocated into the ER lumen by TAP. The MHC I is produced in the ER lumen and TAP delivers the peptide to be bound to MHC 1 to make it mature. Then MHC 1 is exported from a vesicle to the cell membrane.

problem with clustering algorithm

unique viral clusters may be part of bigger whole cluster but just didn't sequence enough viruses

how do segmented viruses ensure only one of each segment is packaged?

untranslated regions of segmented genomes interact with each other; one specific segment only interacts with another specific segments which interacts with another specific segment (makes the 8 segments interact together and get packaged together)

How do enveloped viruses assemble at the cell membrane

viral transmembrane glycoproteins are targeted to the lipid raft (cholesterol enriched domains) of the cell membrane so that budding can occur.

how are antibodies formed

when a naive b cell is activated after infection, it starts proliferating, and then some of those cells differentiate and start generating and releasing antibodies instead of having BCRs

Do capsid proteins self-assemble

yes Viral proteins have structural properties that permit regular and repetitive interactions among them • The repetition of these interactions among a limited number of proteins results in a regular structure • The symmetry rules are elegant in their simplicity • They provide rules for "self-assembly" • Many capsid proteins can self assemble into 'virus‐like particles (VLPs)'

why is mRNA capping important

• 5' Cap structure is critical for initiating mRNA translation • 5' cap protects mRNA from degradation and prevents innate immune responses • Not all viruses utilize 5' caps however (many RNA virus genomes don't have 5' cap, so either host will attack or virus finds way to evade host attack)

what are common features across viruses for their origin of replication in their genome

• AT-Rich Sequences • Binding Sites for Activators (Viral & Cellular)

How do viruses decrease IFN production/activity

• Avoid recognition by PRRs (cGAS and RIG-I) by hiding the viral genome--replicating in nucleus instead of cytoplasm, viral binding proteins to sequester genome away from PRRs, formation of membranous web around viral genome • Inhibit signaling pathway that leads to IFN synthesis--bind viral proteins to PRRs to block receptors, inhibit phosphorylation in signaling pathway to IFN and ISG synthesis (JAK/STAT pathway), cleave/degrade proteins involved in pathway, like PRRs (STING) • Express IFN receptor decoys to bind to IFN and block them from binding to their cell surface receptors • Block function of ISGs

stable host-virus relationship

• Both virus and host survive and multiply, can be maintained in host population without killing the host • Some are effectively permanent: Humans are sole natural host for measles virus, herpes simplex virus, HCMV, smallpox • May include infection of more than one species: Influenza A virus, flaviviruses, togaviruses

why would a virus want to attach to multiple receptors on a cell

• Cell type specificity • Induce step-wise conformational changes in viral glycoproteins to allow entry (ex: can bind receptor, conformational change occurs, which allows binding to co-receptor) • Overcome energy barrier for entry: • For enveloped viruses, typically direct fusion of viral envelope with cellular membrane has a very high energy cost. • This energy cost is offset by having specialized fusion proteins and usage of multiple receptors

how do viruses evade anitbodies

• Continuous mutation of the target epitopes to escape neutralizing antiviral antibodies is an immune evasion mechanism used by HIV.

What is a virus envelope and are viruses more or less stable if they have one

• Derived from the host cell membrane during budding. Can be regular or irregular bc lipid envelopes allow for some irregularities in construction Envelopes include a lipid bilayer plus virally-encoded proteins (often glycoproteins/spike proteins); they interact with the capsid via matrix proteins, which helps interaction between capsid and envelope. • Viruses with envelopes are usually less stable than those without envelopes. This influences transmission (fecal-oral vs. direct transfer). • Envelope proteins often play a role in virus attachment and entry/uptake. They are also often the major antigenic target for virus-neutralizing antibodies.

what are general viral evasion strategies of the immune system

• Disarm innate immunity: Block PRR signaling and IFN production • Regulate MHC molecules: Responsible for antigen presentation Interfere with CTL and NK cells

Fidelity of Viral RNA Polymerases

• Do NOT have intrinsic proof reading ability • Error frequency, one mutation per 103 to 104 nucleotides • 1000 fold higher error frequency than DNA polymerases • High mutation rate during RNA replication is important for RNA virus evolution

How can segmented genome of influenza lead to pandemics and new strains

• Each individual segment needs to be replicated • Segmented genome can lead to reassortment • Co-infection of two viruses and progeny virions contain genomic segments from both parental viruses

what are different ways virus egresses/leaves the cell

• Egress is often polarized and has important consequences for pathogenicity • Apical membranes - release virus into outside world (ex: sneezing to shed flu) • Basal membranes - release virus into contact with blood, lymph, nerves, etc. (allow for systemic spread) • Laterally - Sites of cell-cell contact to maximize infection and cell-cell spread, so no virus ever released into the outer membrane space

How do viruses target the nucleus

• For targeting to the nucleus, viruses use Nuclear Localization Signals (NLS) • NLS are recognized by host importin proteins and bring cargo through the nuclear pore complex and into the nucleus Four different ways: 1) small viruses, like parvoviruses, can enter nucleus via the nuclear pore 2) Big viruses, like HSV, releases its genome across the nuclear pore (injection) 3) Like 2, except virus needs to be completely disassembled before DNA translocation 4) segments individually imported into the nucleus (Flu)

dead end host-virus interaction

• Frequent outcome of cross-species infection, but not of intra-species infections • No sustained transmission from new infected host to others of the same species • Often host is killed so quickly that there is little or no transmission to others: • Ebolavirus: humans, chimps, gorillas • Highly pathogenic avian H5N1 influenza A virus • Contribute little to the spread of a natural infection West nile virus is stable in birds and mosqitoes but dead end in humans

how does HIV evade the immune system

• HIV immune evasion is due to (1) escape of virus from the specificity of initially generated humoral memory (escape from antibodies by rapid mutation) (2) the inability of the immune system to create protective humoral memory for the mutated virus by impairment of T cell help. (killing of CD4+ T cells)

Evolving host-virus relationship

• Hallmarks are instability and unpredictability • Outcome of infection may range from benign to death. Usually when new virus enters host population. • Introduction of smallpox and measles to native Americans by Old World colonists • Introduction of West Nile virus into Western Hemisphere.

which forms can a capsid/nucleocapsid take

• Helix • Icosahedron • Cone (HIV, enveloped)

What makes icosahedrons special/unique

• Icosahedral symmetry is the most economical way to build a symmetric shell and have maximal internal volume with nonsymmetric proteins. Can make a virion with 60 subunits but bigger ones can have more subunits

factors that drive viral emergence

• Increasing number of people living and moving on earth. • Ecological, environmental, or demographic factors; increased contact with a previously unfamiliar microbe or its natural host. • Ongoing evolution of viral variants and selection for drug resistance. • Expanding populations: Mega cities • Poverty • Uncontrolled urbanization Globalization: Rapid air travel allows viruses to move around quickly Altered ecosystem: • Climate change • Dams, water impoundments, irrigation, all leading to expansion of mosquito habitat Deforestation, which forces forest animals closer to man in search of food Human activities: Wildlife parks • Long-distance transport of livestock and birds • Blood transfusion • Societal changes with regard to drug abuse and sex • Microbial evolution: Rapid mutation of viruses and adaptation to new host • Leads to the biodiversity of pathogens existing in nature • Adaptation to new hosts and environments (through variation and selection)

Viral envelope glycoproteins

• Integral membrane glycoproteins • Ectodomain: attachment, antigenic sites (for antibodies), fusion • Internal domain: assembly • Oligomeric: spikes

what is virion maturation

• Irreversible processing of viral proteins to activate the virion and make it infectious: Rearrangement or cleavage • For some viruses the glycoproteins or fusion proteins undergo maturation (conformational change due to pH change, for example) • Ex: Dengue • Other viruses finish the assembly of genome structures: proteolytic cleavage of capsid proteins to create mature capsid after budding • Ex: HIV

functions of the placenta

• Mediates gas, nutrient and waste exchange between the maternal and fetal bloodstreams. Acts as barrier that prevents mother and fetus's blood from mixing. Prevents transfer of toxins/xenobiotics to fetus • Secretes hormones necessary for maintaining pregnancy • Protects the fetus from infection and maternal immune system • Anchors itself to the maternal uterine wall • Thus, at some level the placenta simultaneously acts as the lungs, liver, kidneys and innate immune system for the developing embryo/fetus

overall, what are some viral immune evasion mechanisms

• NK cell evasion • MHC class I and II inhibition • Interference of IFN signaling • Block apoptosis • Expresses decoys

assembly and release of viruses for naked vs enveloped

• Naked viruses (non-enveloped): Release typically occurs by a passive process (host cell lysis). Enveloped viruses: Virus-encoded glycoproteins are inserted into cell membranes (most often the plasma membrane), where they form "patches". Matrix proteins are recruited to the cytoplasmic side of these patches, and form a site to which viral nucleocapsids attach. The virus nucleocapsid is then enveloped by a process of "budding", resulting in release of a virus particle.

what are capsids inside of enveloped virions called

• Nucleocapsids-- may have helical or icosahedral symmetry

packaging signals

• Packaging signals are specific sequences in the viral genome that are recognized by viral packaging proteins so that the virus knows which nuclear material to package inside of the virion • In single stranded genomes, the signal is typically within secondary structure • Packaging signal must be present only in the strand to be packaged • It's important that not all of the genome are packaged, some need to serve as template for replication or that host genome is not packaged

Polyadenylation of Viral mRNAs

• Poly (A) tail increases mRNA stability and aids in translation • For (+)ssRNA viruses, poly (A) tail is already in the genome • For (-)ssRNA viruses, viral genome has a poly (U) stretch to make the poly (A) tail • Most DNA viruses acquire poly (A) tail similar to cellular mRNA processing

How can we determine that a cell surface molecule is a viral receptor

• Receptor-neutralizing antibodies: Block the receptor on the target cell • Natural ligands of the receptor: Block the receptor on the target cell • Soluble form of the receptor: Neutralize the virus before it can infect target cells • Exogenous expression of a receptor renders cell susceptible to infection • siRNA, or CRISPR/Cas9: Knockdown or knockout expression of the receptor on the target cell Transfect cell that doesn't typically express receptor (and therefore doesn't get infected) with DNA of receptor and see if gets infected

What are the symmetry rules for capsid assembly

• Rule 1: Each subunit has identical bonding contacts with its neighbors • The repeated interaction of chemically complementary surfaces at the subunit interfaces naturally leads to a symmetric arrangement • Aggregates, clumps, disordered complexes form when nonidentical bonds form between identical subunits Rule 2: These bonding contacts are usually non-covalent • The reversible formation of non-covalent bonds between properly folded subunits leads naturally to error-free assembly and minimizes free energy

type I and type III interferons, generally

• Serve as a critical first line of defense against viruses • Slow down the progression of viral spread prior to activation of innate and adaptive immune responses • Critical component of innate immunity in vertebrate species • Produced following host cell exposure to viral components • Defects in IFN production and/or responses significantly increase morbidity and/or mortality Each of the IFNs is produced by distinct sets of cells • Each of the IFN types bind to distinct cell surface receptors (IFN-a/bR, IFN-gammaR, IFN-lambdaR) • Mediate their functions by inducing transcription of interferon stimulated genes (ISGs), aka host restriction factors

what is the fidelity of viral DNA polymerases

• Similar to cellular DNA replication = High fidelity, typically only one mistake in 109 bases • Intrinsic proof reading ability: 3' - 5' Exonuclease activity removes errors

what has increased incidence of emerging viruses

• Since the rise of agriculture - 11,000 years ago - new infectious agents have invaded human populations because of increase in contact with animals, so viruses from animals could expand their host range

Difference btween DNA virus size and if they code their own polymerase? What does this mean about where they replicate?

• Small DNA viruses require more host proteins • Papillomaviruses, Polyomaviruses, Parvoviruses • These viruses require the cell's DNA dependent DNA polymerase • Large DNA viruses encode many of their own replication proteins • Adenoviruses, Herpesviruses, Poxviruses • These viruses encode their own DNA dependent DNA polymerase. Large DNA viruses usually, but not always, replicate in cytoplasm because don't need host polymerase found in nucleus

how do (+)ssRNA replicate their genome

• Viral genome can serve as mRNA (mRNA can code for RNA dependent RNA polymerase) • Replication requires this RNA dependent RNA polymerase to generate a (-)sense intermediate, which can be used as a template to make a new (+)ssRNA

How do viral particles encounter the 'right cell'

• Viral particles don't have a way to specifically hone in on a cell • Rely on: Random collisions • Brownian motions • Hundreds of thousands of viral particles on constantly bouncing off your cells because they are not landing on the 'right' one This is why a certain concentration of viral particles is required to get you sick

How Do Viral Proteins Reach Assembly Sites?

• Viral proteins have "addresses" built into them • Membrane proteins go to the appropriate membranes: Signal sequences, fatty acid modifications • Membrane proteins stay in the appropriate membrane: Retention signals • Nuclear proteins go to the nucleus: Nuclear localization sequences • Viral mRNA or nucleoprotein complexes move to the cytoplasm: Nuclear export signals • Capsid and protein complexes exhibit directed motion, not diffusion: Microtubules and intermediate filaments are the tracks, dyneins, kinesins, myosins are the motors

what are RNA virus strategies to get around their small genome size

• mRNA Splicing • One mRNA encodes for multiple proteins (cleavage of polyprotein post-translation)• Programmed ribosomal frameshifting: create alternate versions of proteins--• Certain codons (like UUU) or sequence elements make it very likely that ribosomes can "slip" and lead to an alternate reading frame being translated if certain codons are repeated or if the reading frame is shifted Leaky Scanning in ribosome

steps for systems level investigations

•Designing experiments to define a system to address a question: ex-an effort to build a comprehensive, standardized knowledge repository of SARS-CoV-2 virus-host interaction mechanisms to be able to predict symptomatic vs. asymptomatic infection •Large-scale data generation: collect tons of blood samples, molecular and cellular assays facilitate profiling of immune status--can test genomic, cellular or proteomic components Can do a microarray to investigate the expression state of a large number of genes •Primary data analysis: notice which genes are being transcribed for symptomatic vs. asymptomatic patients and decipher molecular pathways involved in symptomatic versus asymptomatic infections •Modeling interactions among the components of the system •Experimental validation

how can researchers use a genomic approach/assay to measure immune response/status and collect data

•Estimating variation in human genome: single nucleotide polymorphism (SNP) chips permit the identification of common polymorphisms or rare mutations associated with diseases. -E.g. autoimmune diseases, resistance to infection Microarrays, Nanosequencing, RNA seq--most commonly used to see which genes are being expressed because sequences all mRNA (high throughput gene expression)

Gene regulatory networks

•Gene-gene association networks can be used to represent a system •Graph theoretical measures allow investigation of signal integration •Networks can be used to represent interactions between multiple pathways •Dynamic modeling techniques can be used to investigate flow of signals Build gene co-expression networks to see which gene expressions are correlated with each other

fitness landscape

•Random genetic changes can result in higher fitness (more replication or transmission). A peak occurs on the landscape when a virus gains a functional mutation and peak goes down when host gains immunity against the viral mutation. So on and so forth.

why do we need systems approach to virology

•Study responses to viral infections in humans •Why do we need systems approach? -To find novel regulators/ targets/ genes -Highly variable immune responses -Baselines are different and are dependent on Exposure, Environment, previous infections, gender, etc..

negative outcomes associated with infection during pregnancy

•blindness • Spontaneous miscarriage • In utero growth restriction (IUGR) • Preterm and still birth • Increased rates of schizophrenia and autism in the newborns (in response to Influenza) • Maternal death metabolic syndrome, immunodeficiency