Hthsci 3K03
PFU/mL =
# of plaques x amount plated x dilution factor
SV40: circular genome and replication fork
- 1 origin of replication - semi-discontinuous DNA replication from bidirectional origin - circular process (creates bubble) - circle is the solution to the 5' end problem
bigger icosahedral capsid
- 180 identical protein subunits - pentamers and hexamers - 3 modes of subunit packaging - bonding interactions are quasiequivalent ( all engage tail to tail and head to head)
Medusavirus
- 260nm - discovered in hot springs - has external spikes - turns amoeba into 'stone-like cysts'
reverse transcription
- coated with NC protiens - 50-100 molecules of RT per virus particle
Splicing takes place in the spliceosome
- is a very large complex of proteins and RNAs - enzymatic reactions are catalyzed by RNAs
Viruses are a tremendous source of biodiversity
- outnumber cellular life 10:1 - viruses drive global cycles
central dogma
DNA -> RNA -> Protein
cellular enzymes responsible for transcription
DNA dependent RNA polymerases (DpRp) - RNA pol 1 = no viral RNA - RNA pol 2 = pre-mRNA, pri-miRNA, HDV genome, RNA, and mRMA - RNA pol 3 = Ad-2 VA RNAs, EBV, MHV68npri-miRNA RNA pol 2 is the most important for viral RNA
Mechanisms of initiation of RNA synthesis
De novo or primer dependent (protein or capped primer)
regulation of transcription by viral proteins
- auto regulatory loop - cascade
ds RNA genomes
- have (+) RNA strand but cannot access it - must be synthesized fresh by bringing in RdRp
infectious cycle
- replication cycle - things happen at the same time
influenza virus genome
- subgenomic mRNAs (like VSV) - each segment codes for one or more proteins - splicing gives rise to alternative mRNAs
key rules of viral RNAs
- the RNA genome must be copied in its entirety with NO LOSS OF SEQUENCE - MUST produce viral mRNAs that can be translated by the host machinery
Adenovirus cascade regulation
- three viral proteins and DNA synthesis govern phase transitions - E1A necessary for transcription of all E transcription units - E2 required for DNA synthesis and entry into L phase (increase initiation from late promotor)
structure of a typical mRNA: 5'-untranslated region
- typically 50-70nt (can be 3-1000) - often contains RNA secondary structures must be unwound to allow passage of ribosome - length influences translation efficiency
Synthesis of subgenomic mRNAs
1. RNA polymerase binds at 3' end go N gene 2. initiation of mRNA synthesis at 3' end of N gene 3. Synthesize N mRNA and terminate at intergenic region (ig) 4. reinitiate at 3' end of P gene
Influenza virus replication considerations
1. segmented genome 2. replicates in the nucleus (complicated)
DNA size
2nm
poliovirus size
30nm
What cellular enzyme exists to make RNA from RNA templates?
NONE
Human Endogenous Retroviruses (HERVs)
Virus genes that are incorporated into human chromosomes and comprise around 8% of the human genome.
(-) strand
- complement of the (+) strand - the (-) strand cannot be translated it must be copied to make a (+) strand
Adenovirus: protein priming and strand displacement
- double stranded linear genome - ends are inverted terminal repeats (ITR) - replication by strand displacement
how do dsDNA genomes replicate?
- dsDNA is the only template for TRANSCRIPTION - DNA is transcribed into mRNA, mRNA encodes for proteins
suppression of termination
- eRF1 and eRF3 recognizes all 3 stop codons (UAA, UAG, UGA) - stop codons may be recognized by charged tRNA - misreading, or charged suppressor tRNA (ex. selenocysteine for UGA) - retroviruses = misreading - alpha viruses = read through translation
ss RNA (-) genomes
- ebolavirus, influenza - segmented (influenza A) - non-segmented (Newcastle disease virus)
techniques to study virus structure
- electron microscopy - x-ray crystallography - cry-electron and cry-electron tomography - nuclear magnetic resonance spectroscopy
RNA genomes
- every RNA virus on the planet encode RNA dependent RNA polymerases (RdRp)
Fusion is regulated
- must happen at the right location (want destabilization to happen in the right place, in the right cell - there are different requirements for fusion at the plasma membrane and fusion in an endosomal compartment
Subgenomic mRNAs are short, what is the solution?
- nucleoproteins - NP binds newly synthesized RNAs and makes them extend all the way to the 3' end
dsRNA
Reoviridae
retroviruses
use reverse transcriptase to copy their RNA genome into DNA - contain RT, protease, and integrase
Virus movement within cells
virus particles are moving within the cell as particles or vesicles on motor proteins
location
- RNA pol 1, 2, and 3 live in the nucleus - DNA viruses that replicate in the cytoplasm MUST encode their own DpRp
Stimulation of transcription by Adenovirus E1A proteins: Rb
- Rb (co-binding molecule) binds to the DNA binding molecule (Dp-1 and the host protein E2f) and represses transcription - Rb represses by stimulating Hdacs - if viral protein E1A binds to Rb allows the DNA binding molecule to properly bind allowing the stimulation of transcription
characteristics of RT
- SLOW (4hr/9kb genome) - error prone (1 mistake/10kb genome) - 1 misincorportation pre 10^4 to 10^6 nucleotides
resistant
- a cell that lacks the requisite viral receptor - may or may not be able to support replication (could force virus into cell)
additional functions of viral structural proteins
- specific recognition and packaging of the nucleic acid genome - bind host cell receptor - interaction with host cell membranes to form the envelope - fusion with cell membranes
viruses to human cells
10x more bacteria and 100x more virus particles
Hepatitis B virus
1970: RT discovered 1982 (another surprise) - a RNA as an intermediate in the replication of a virus with a DNA genome
alpha helix in protein size
1nm
ss RNA (+) genomes with a DNA intermediate
2 human pathogens: HIV and HTLV
SV40: Autoregulatory loop regulation
2 phases (promoters) early and late which are defined by mRNAs - Large T antigen
ribosome size
20nm
other things in virus particles
- activators of mRNA synthesis, enzymes that degrade mRNA that are required for efficient infection - cellular components: histones, tRNAs, lipids
the surface of a cell is decorated with proteins
- all of the proteins have a role for the cell, they DO NOT exist for the viruses - viruses have evolved to bind to host cell receptors for entry (evolutionary adaptation)
alternative splicing
- allintrons are spliced out but only selected axons are spliced in - the result is mRNAs having different coding information derived from a single gene - expands the coding capacity
enveloped viruses
- capsids can be covered by host membranes (HOST DERIVED) - envelope acquired by budding of the nucleocapsid through a cellular membrane (not limited to the plasma membrane) - nucleocapsid inside the envelope may have helical or icosahedral symmetry
how can non-enveloped viruses attach to surface proteins?
- cell surface receptor fits into a groove (canyons), ex. poliovirus - binding at the five-fold axis, ex. rhinovirus (conserved lysine binds to low-density lipoprotein (LDL) receptors) - contact with a fiver (not capsid), ex. adenovirus
fusion of enveloped viruses
- fusion at the plasma membrane (HIV) - fusion in an endosomal compartment (influenza)
packaging signals - segmented RNA genomes
- how do you get the right number of segments? - random mechanism would yield 1 infectious particle per 400 assembled within known particle:pfu ratio - evidence for SPECIFIC packaging sequence on each RNA segment
tailed bacteriophages
- icosahedral head - tail is attached at one of 12 vertices of the capsid - tail is a complex rod using helical symmetry in many places
Cyro-EM
- if you freeze virus quickly in very cold water it gives particle contrast - takes hundreds of photos of viral particles and if you take enough pictures you can computationally assemble a 3D image
how to build a virus
- symmetry - Watson and crick used EM to describe 2 types of symmetry
Sequence specific transcriptional activators
1. DNA binding molecule 2. Dimerization molecule - typically exist as dimers 3. activation molecule - interacts with polymerase or interacts with a protein that then interacts with polymerase
The Baltimore system
1. dsDNA 2. ssDNA 3. dsRNA 4. ss (+) RNA 5. ss (-) RNA 6. ss (+) RNA with DNA intermediate (retroviruses) 7. gapped dsDNA
how does a virus find the right cell?
1. early interactions are mainly electrostatic (non-specific bumping) 2. attach to a specific receptor molecule (or molecules) 3. transfer genome inside of cell
Sandwich ELISA
An ELISA designed to capture the antigen of interest between an immobilized anti-target capture antibody and the same or another anti-target antibody in solution which is then viewed using a change in colour or fluorescence
Physical measurements of virus
Hemagglutination, electron microscopy, viral enzymes, serology (antibodies), nucleic acids
When it comes to serological assays: how can we differentiated between vaccinated individuals and infected?
Infected: has antibodies for all parts of covid Vaccinated: only antibodies for spike protein
pandoravirus
Infects amoebas Giant virus Large virus @ 1nm, larger than some bacteria Largest genome of any known virus
internal initiation
Initiating at IRES; requires eIF2, eIF4, other proteins; Does not require Cap binding protein - polioviruses (really long UTR)
What switches the making of mRNA to full length genomes?
Nucleoprotein
SARS-CoV-2 Antigen Tests
Use colloidal gold as detection system and monoclonal antibodies with capillary flow to show if substrate is bound to monoclonal antibody the gold will react and form a second line
What can viruses infect?
all living things (even other viruses)
quasiequivalent
all structural units interact the same but the subunits within the structural units may interact differently
Distribution of virus particles
can be describes by Poisson distribution P(k)=(e^-m)(m^k)/k!
auto regulatory loop regulation
can be positive (increases transcription) or negative (inhibits transcription) - the generation of the protein product causes this
how do we grow viruses?
chicken eggs are used in vaccine manufacturing but we often use cell culture
RNA has extensive secondary and tertiary structure
roles in genome expression, RNA protein synthesis and expression
subunit
single folded polypeptide chain
Alphaviruses
subgenomic mRNAs
How is virus stability achieved?
symmetry
initiation complex transcription
template strand, TF, binding protein, RNA pol 2 - enhancer binding proteins bend the DNA to contact both the initiation complex and the enhancing region - enhancer is bound by activators that increase transcription through the contact with RNA pol 2 or the complex - the initiation complex ensures that everything happens in the correct order and orientation
assembly
the structure of the virus particle determines how it is formed
Eclipse period
the time it takes to go through the infectious cycle
Parvovirus DNA replication
1. hairpin formation 2. elongate 3. nick (Rep 78/68) 4. elongation from nick 5. hairpin formation 6. reinitiating from 3' OH 7. displacement of genomic DNA
Assembly is dependent on host cell machinery
• Cellular chaperones • Transport systems • Secretory pathway • Nuclear import and export machinery
Particle to PFU ratio
# of physical particles/# of infectious particles
viral origins of replication
- A-T rich segments (less bonds to unwind) - recognized by viral origin binding proteins - viruses can have many origins or as little as 1
What do all viruses have in common?
- All viral genomes are obligate molecular parasites that can only function after they replicate in a cells - all viruses must make mRNA that can be translated by the host ribosome (NO EXCEPTIONS) - all viruses are reliant on the host protein synthesis machinery
viral encoded proteins that participate in DNA synthesis
- DNA pol and accessory proteins - origin binding protein (recruitment) - helices (separation of 2 strands) - exonucleases (error correction - only DNA viruses) - enzymes of nucleic acid metabolism
5' end problem for DNA replication
- DNA polymerized in short fragments - 3' hydroxyl can fill in gap - leaves no free 5' hydroxyl - don't want to lose any genome
large complex capsids
- Ex. Adenovirus T=25 with fibres at 12 vertices - distinct components with distinct symmetries, in addition to the proteins that make up the capsid that are other proteins interspersed throughout - specialized roles of proteins: some bind DNA, some are thought to "glue" the particle together, but are not used to build the shell
Endpoint dilution assay (TCID50)
- For viruses that don't plaque - caveat: similar to the plaque assay where a virus must form plaques here a virus MUST cause CPE
icosahedral symmetry
- Icosahedron: solid with 20 faces, each an equilateral triangle - 5X (12 vertices), 3X (20 sides), 2X (30 edges) of symmetry - Allows formation of a closed shell composed of identical subunits
viral envelope glycoproteins
- Integral membrane glycoproteins - Ectodomain: attachment, antigenic sites, fusion - Internal domain: assembly - SARS-CoV-2 has spikes that are perpendicular to the virus surface while Flavivirus E dimer has "spikes" that are lying flat (spikes are glycoproteins)
Triangulation Number (T)
- Number of facets per triangular face - increasing capsid size increases the T number
Multiplicity of Infection (MOI)
- Number of infectious particles added per cell (# infectious particles/# of cells) - this is not the same as the # of infectious particles each cell receives
susceptible
- a cell that possesses the requisite viral receptor (i.e. the virus can get in) - this does NOT mean that the cell will be able to support viral replication (might not be able to create progeny)
splicing is value added
- alternative splicing creates different mRNAs, proteins - coding information of small genome is expanded - regulation of gene expression
influenza virus RNA packaging
- always 8 RNA segments - segments orientated perpendicular to budding tip - RNA-RNA or RNA-protein interactions - mutants with packaging defects
nucleoprotein act as the switch
- at the start of infection the amount of nucleoprotein is low - later in infection levels rise as nucleoprotein is synthesized (anti-terminators)
How do we grow viruses?
- before refrigeration = animals - before advances in cell culture = animals - would inject the animal with virus and could send it to another lab and then could retrieve the virus from animal (could be a dead or alive animal it really doesn't matter)
electron microscopy
- biological materials need to be stained for contrast - negative staining with electron dense material and scatter electrons - resolution is 50-70 angstroms - can see overall size and shape of virus particles, some details, resolution not great
ssDNA
- can be circular or linear genomes - either strand of DNA can be packaged into a virus particle and initiate infection but a single strand of DNA CANNOT be transcribed
structure of a typical mRNA: 3'-untranslated region
- can regulate translation, initiation, translation efficiency and mRNA stability - poly(A) tail necessary for efficient translation
Fluorescent focus assay
- cells are permeabilized and incubated with an antibody recognizing a viral antigen - a second antibody conjugated to fluorescent molecule is used to recognize the first antibody - cells are examined using a fluorescent microscope
Classes of fusion proteins
- class 1 (vertical alpha helices) - class 2 (beta sheet dimers lie flat) - class 3 (mix of 1 and 2) - for all classes a fusion protein is inserted into the target membrane and the fusion protein refolds to bring the 2 membranes into close contact - within each class fusion may be triggered by receptor binding, low pH, or a combination of both
Fusion at the plasma membrane HIV
- co-receptor interaction triggers fusion of the viral and cellular membranes - sequential binding of gp120 to primary receptor CD4 induces conformational changes that result in high affinity binding to a second co-receptor CCR5 (or CXCR4)
What information is NOT contained in a viral genome?
- completed protein synthesis machinery (some giant viruses encode parts but no virus encodes it all) - proteins involved in energy production - proteins involved in membrane biosynthesis - no telomeres or centromeres
sub-assemblies
- ensure orderly formation of viral particles and virion subunits - formation of discrete intermediate structures - cannot proceed unless previous structure is formed: quality control
Hepadnaviral DNA and RT
- even though gapped dsDNA and retroviruses both possess RT, exactly how and when RT is used in the viral life cycle is very different - gapped DNA molecule enters the nucleus and is repaired (seen as damaged, acquires cellular histones) - RNA pol 2 transcribes viral RNA - pregenome RNA serves as template for reverse transcription - P protein provides all the activities required for reverse transcription
Rendondoviridae
- family of 19 viruses that live in the human lung - discovered in the bronchiolar lavage fluid of human lungs - second most common virus in human respiratory virome samples
Tobacco Mosaic Virus
- first virus to be discovered in the 1800s - 1892 dimitrii ivanosky found that the causative agent of tobacco mosaic disease not retained by filter - he ground up infected plant and passed it through a filter - he then put the poisonous soup on a healthy plant and it became infected
Trimer hairpin fusion mechanism
- form trimers and initiate fusion through hairpin structures 1. prebundle trigger 2. bundle 3. trimer of hairpins 4. close apposition (bends out pulling the membranes) 5. hemifusion (membranes begin to fuse and spontaneous rearrangement begins) 6. fusion pore created
why are 2 RNAs packaged in the virion?
- gives the viruses some amount of resistance to mutations (ex. UV and ionizing radiation, two copies of every gene) - COPY-CHOICE rebuilds one functional genome (promotes recombination)
Herpesvirus transcriptional programs (cascade)
- initiated by VP16 (packaged in virus particle) - activates IE transcription - IE proteins control transcription from all virus genes - expression of E genes and DNA synthesis - expression of DL and L genes, DNA dependency - ensures coordinated production of DNA genomes and structural proteins
5'-end dependent initiation
- initiation factor binding - binding of ternary complex - 43S preinitiation complex - binding of eIF4G - binding of eIF4E (cap binding protein) - 48S initiation complex - scanning - hydrolysis of GTP in ternary complex - joining viruses can shut this off by cleaving initiation facts on eIF4G (site recognized by viral proteases)
herpes simplex virus: a unique example
- linear double stranded DNA - 2 oriS and a unique oiL sequence - DNA enters as a linear molecule and converts to a circle (mediated by host cell ligase)
retroviral LTR co-option
- long terminal region - strong promoter - corticotropin releasing hormone - responsible for timing of birth
simple icosahedral capsid
- made of 60 identical proteins - the protein subunit is the structural unit - interactions of all the molecules with their neighbours are identical (head to head, tail to tail)
symmetry and self-assembly
- many capsid proteins can self-assemble and from virus-like particles (VLPs) - we rely on this property for many important vaccines such as hepatitis B and HPV vaccines - Hepatitis B virus surface antigen (HBsAg) self-assembles into VLPs
viral proteins have addresses
- membrane targeting: signal sequences, fatty acid modifications - membrane retention signals - nuclear localization sequences (NLS) - nuclear export signals contained in amino acid sequences that dictate where it ends up in the cell
Giant viruses
- mimivirus (400nm) - megavirus (440nm) - pandoravirus (1000nm)
regulation of DNA synthesis
- most of our cells in our body reside in G0 BUT viruses don't fare well in non-diving cells - viruses want our cells to be in G1 - viruses must force cell division in order to utilize host replication proteins - achieved by early viral gene products
Fundamental properties of viruses
- most viruses are very small - viruses replicate in an assembly line fashion
reverse transcriptase (RT)
- needs a primer (DNA or RNA) - template can be RNA or DNA - only dNTPs are incorporated (DNA product ONLY) - both bacteria and archaea have RT activity - therefore RT evolved before the operation of Archaea, bacteria, and eukaryotes (in common ancestor) - RT might be the bridge between early RNA world and modern DNA world - RT also in HBV (and our genomes)
integration
- no LTRS in mRNA - integrase is brought in with the virus particle - a bit of LTR is removed to expose the 3' OH
X-ray crystallography for viruses
- not used for viruses until 1970s - resolution 2-3 angstroms - x-ray source, crystal, diffraction pattern, electron density map, atomic model
RT summary
- one DNA produced from 2 RNAs by RT - strong promotor (LTR) built during RT - proviral DNA directs the host transcriptional machinery to synthesize many copes of viral mRNA - viral mRNA is translated into viral proteins or encapsidated into virus particles THERE IS NO VIRAL DNA REPLICATION AND NO VIRAL RNA REPLICATION
overview of reverse trasncription
- one DNA produces from RNAs by RT - strong promotor (LTR) is built during RT - proviral DNA directs the host transcription machinery to synthesize many copies of viral mRNA - viral mRNA is translated into viral proteins or encapsidated into virus particles THERE IS NO VIRAL DNA REPLICATION AND NO VIRAL RNA REPLICATION (cell does all the work)
reverse transcriptase
- opposite of the central dogma - RNA to DNA - retroviruses are so named because of their ability to reverse the flow of genetic information - revolutionized molecular biology
Plaque assay
- originally developed studying bacteriophages - agar/gel overlay restricts the spread of virus to neighbouring uninfected cells - 1 plaque = 1 infectious particle = 1 PLAQUE FORMING UNIT (PFU)
enzymes within virus particles
- polymerases, integrases, associated protiens - proteases - poly(A) polymerase - capping enzymes - topoisomerase
integration happens everywhere
- preference for DNA wrapped around nucleosomes and not "free" DNA in between histones - cellular protein are required
Primary vs continuous cell lines
- primary cells are from animal tissues and have a finite number of replications (hayflick limit) because the telomeres get too short with time - continuous cell lines are immortal cell lines that are abnormal with odd numbers of chromosomes (ex. HeLa cells)
Influenza viral RNA synthesis
- primer dependent mRNA synthesis - enzyme primer comes from cellular RNAs - it is short by like 20nt but still needs to be copied completely
Adenovrius DNA replication
- primer is a protein, starts the complex of the polymerase - pTP = p Terminal protein - once the polymerase starts to copy the template it dissociates from the pTP which remains linked to the 5' end - DNA binding protein covered strand can be recognized by the viral polymerase
DNA synthesis in cytoplasm
- recall fusion at the plasma membrane - RT takes place in the cytoplasm - first of 2 template exchanges - ppt = polypurine tract
fusion in endosomal compartments influenza
- receptor binding at the cell surface leads to endocytosis - fusion occurs in the ENDOSOME and is catalyzed by a LOW pH - for fusion to occur HA must be cleaved by cellular protease - low pH induces a conformational change in HA that releases the fusion peptide
Retroviral protein co-option
- retroviral envelope protein - tissue layer - used to exchange nutrients in the placenta
viruses of the dame family can bind different receptors
- rhinoviruses and retroviruses have multiple receptors - one virus may bind multiple receptors or a co-receptor may be needed - receptors for attachment and entry can vary in different tissues
translation: the machinery
- ribosome, tRNAs - initiation proteins (eIFs) - elongation proteins (eEFs) - termination proteins (eRFs)
helical symmetry
- rod shaped viruses - engagement of coat proteins with one another and the genome allows for the construction of a large, stable structure from a single subunit - protein protein and protein nucleic acid interaction
Cleavage and polyadenylation
- same as in our cells - polyadenylation: produce pre-mRNA then bring in host macherinery to create poly(A) tail
Parvovirus: DNA priming and strand displacement
- single stranded with terminal repeat sturctures - primer is built in but need to ensure that the end sequence is propagated to daughter strands - continuous replication and happens via strand displacement
co-trancriptional capping
- start to incorporate nucleotides then we add 5' cap - capping enzyme is recruited when the polymerase is phosphorylated 1. initiation and incorporation of 20-30 nucleotides 2. CTD phosphorylation 3. synthesis of 5' cap
(+) strand synthesis
- tRNA is removed PBS is hybridized - this serves as primer to make the rest of the (+) strand - double stranded DNA BUT ends are different
metastability
- virus particles have not yet achieved a minimum free energy conformation - unfavourable energy barrier must be overcome, by putting energy in (done during virus assembly) - potential energy used for disassembly if cell provides the right trigger
manipulating viral genomes
- we can manipulate the viral genome to insert genes that can later be used to track or assess the infectious cycle - reporter viruses are simply recombinant viruses expressing a foreign gene - fluorescent proteins, luciferase
a second function of RT: RNAseH
-can cleave RNA when in duplex - makes endonucleolytic cleavage - produces short oligos with 5' phosphates
universal rules of DNA replication
1. ALWAYS primer dependent 2. DNA dependent DNA polymerase (DdDp) adds bases to the 3' end of the primer and synthesizes in the 5'-3' direction dictated by the template 3. Semi-conservative 4. replication initiates at specific sites on template called origins of replication (one or many) 5. catalyzed by DdDp and accessory proteins 6. a host is required 7. viral DNA replication ALWAYS requires synthesis of at least one viral protein (explains why DNA synthesis is delayed after infection) 8. simple viruses rely on more host proteins (no room to encode) 9. complex viruses encode many but not all proteins required for replication
three strategies for making sub-assemblies
1. Assembly from individual protein molecules 2. Assembly from a polyprotein precursor 3. Chaperone-assisted assembly
proteins that regulate transcription
1. DNA binding proteins - host or viral 2. co-activating molecules - no direct DNA binding - many co-activators modulate the structure of the nucleosomal template (alters the accessibility of this template to the transcriptional machinery)
rules of symmetry
1. Each subunit has identical bonding contacts with its neighbours. - the nature of this arrangement (i.e. repeated interaction of complementary surfaces at the interfaces of subunits) leads to a symmetric virion 2. These bonding contacts are usually NON-COVALENT - Reversible, quality control
(+) strand RNA viruses RNA synthesis
1. Genome = mRNA - translated and cleaved 2. production of subgenomic mRNAs - pieces synthesized by promotor (smaller mRNAs)
Types of virus symmetry
1. HELICAL symmetry for rod-shaped viruses 2. ICOSAHEDRAL (platonic polyhedra) symmetry for round shaped viruses
Common COVID-19 tests
1. Molecular - detecting viral RNA using RT-PCR (most sensitive) 2. Antigen (rapid test) - detects viral proteins using sandwich ELISA 3. Antibody tests (serological) - Detects antibodies your body has mounted against the virus/vaccine using ELISA
circulation of genome - poliovirus
1. Poly-(A) binding protein (Pabp) interacts with 3' poly A 2. polymerase relocates to 3' end 3. VPg used as a primer for synthesis 4. elongation and synthesis of negative strand - IF CIRCULARIZATION IS PREVENTED THERE IS NO GENOME REPLICATION
Universal rules for RNA-directed RNA synthesis
1. RNA synthesis relies on start and stop signals 2. RdRp may initiate synthesis de novo or rely on a primer 3. some viral or cellular proteins may be needed 4. RNA is synthesized by template-directed stepwise incorporation of NTPs in the 5'-3' direction
RdRp
1. copies the genome 2. makes mRNA from RNA templates - DNA viruses need two separate enzyme to carry out of these functions
all virions complete a common set of assembly reactions
1. formation of individual structural units of the protein shell from one or several viral proteins 2. assembly of the protein shell by appropriate and sometimes variable, interactions among structural units 3. selective packaging of the nucleic acid genome and other essential virion components 4. acquisition of an envelope (common to many but not all viruses) 5. release from host cell 6. maturation of virus particles (common to many but not all viruses)
poliovirus genome replication key points
1. if circularization is prevented there is no genome replication 2. primer is a protein
how to quantify viruses
1. infectivity: we can measure infectious viruses 2. Physical: virus particles and their components # virus particle ≠ # of infectious particles
viral fusion at the plasma membrane
1. receptor binding 2. virus with host membrane fusion 3. release of vision contents into host cytoplasm - occurs at a NEUTRAL pH
2 mechanisms of dsDNA synthesis
1. replication fork - synthesis on both strands (one continuous and one discontinuous) - RNA acts as a primer 2. strand displacement (primer) - one strand is displaced as the other is being copid - primer is protein or DNA (NEVER RNA)
Large T-antigen functions
1. required for viral DNA synthesis (binds origin of replication) 2. inhibits its own synthesis when present in high enough quantities (NEGATIVE autoregulatory)
How many viruses in the biosphere?
10^31 viruses
VSV non-segmented (-) RNA viruses
5 regions including 5' cap and poly(A) tail
twists on translation
5' dependent intiaition - all eIFs type 1 or 2 IRES - all eIFs except eIF4E Hepatitis C virus - eIF2, eIF3
the human virome: how infected are we?
67.7% viruses, 3.6% eukaryota, bacteria 9.5%, other sequences 4.4%, and unknown 14.7%
Reassortment
A mixing of the genetic material of two viruses that are infecting the same cell - this is how we get influenza PANDEMICS
problem with subgenomic mRNAs
CANNOT MAKE A FULL LENGTH GENOME
DNA vs RNA viruses: polyadenylation
DNA - mechanism: cleavage of pre-mRNA followed by by polyadenylation - Post transcriptional - cellular enzyme - Viral: adenovirus, HBV, HDV, herpesvirus, polymavirus, retrovirus RNA - 2 ways: stuttering or copying a long U stretch in RNA template - occurs during mRNA synthesis - both done by viral enzymes (RdRp?) - stuttering occurs in influenza and VSV - U's occur in poliovirus and alpha virus
Constitutive splicing
Every intron is spliced out and every exon is spliced into the mature mRNA
True or False: all RNA viruses ENCODE an RdRp in their genome and ALL RNA viruses package translated RdRp in the virus particle
FALSE: all RNA viruses ENCODE an RdRp in their genome; however NOT all RNA viruses package that translated RdRp in the virus particle
True or false: ALL DNA viruses need to make at least 2 proteins to replicate their genome
False: ALL DNA viruses need to make at least 1 protein to replicate their genome - coordination and temporal regulation
What information is contained in a viral genome?
Gene products and regulatory signals for: - replication of viral genome - assembly and packaging of the genome - regulation and timing of the replication cycle - modulation of host defences - spread to other cells and hosts
poliovirus genome
Genome = mRNA - protein linked to 5' end (VPg) which serves as the PRIMER for RNA synthesis - genome is translated into a long protein and then virally encoded protease chop it up to give individual viral proteins - has a cloverleaf, 5' VPg, cre, and pseudo knot
regulation of late phase promotors SV40
IBP = initiator binding protein - in early phase there is enough IBP to repress the genome - late phase dilutes out the repression (not enough IBP to repress all the genomes) - DNA synthesis can act as an anti-repressor for the late phase promotor
What do we need to make new virons?
Protein and Genomes
rolling circle replication
Replication of circular DNA that is initiated by a break in one of the nucleotide strands, producing a double-stranded circular DNA molecule and a single-stranded linear DNA molecule, the latter of which may circularize and serve as a template for the synthesis of a complementary strand 1. only one strand is nicked 2. circular fragment has continuous DNA synthesis while the linear piece has discontinuous DNA synthesis 3. this creates a rolling conatemer
what kind of cell can a virus infect and replicate in?
SUSCEPTIBLE AND PERMISSIVE CELL
SARS-CoV-2 Antibody Tests
Similar to antigen test but using antibodies
where does the polymerase come from?
Small DNA viruses - genome copied by HOST DNA polymerase - need a way to attract the DNA synthetic apparatus to their genomes - must be in nucleus Large DNA viruses - genomes encoded DNA polymerase - still rely on at least 1 viral protein made by host cell machinery - could be in nucleus or cytoplasm
Are viruses the good or bad guys?
Viruses impact on human health is pretty much neutral
permissive
a cell which supports viral replication (may or may not get in)
How is a plaque assay performed?
a suspension of phage is serially diluted, an aliquot of a young broth culture of host cells is added to each dilution, suspension incubated for the phage to attach to and infect the host cells then an aliquot of tempered semisolid agar (soft agar), resulting mixture quickly poured over a base layer of solid nutritional medium where it hardens
virus particles are metastable
a virus particle is stable when travelling from host to host however a trigger from the host cell (receptor binding, low pH, protease in cells etc) will result in the virus particle becoming unstable and fall apart
cytopathic effect (CPE)
a visible effect on a host cell, caused by a virus, that results in changes in morphology
dsDNA genomes
adenoviridae, herpesviridae, papillomaviridae, polyomaviridae, poxviridae
packaging signals - DNA genomes
adenovirus - packaging signal near left inverted repeat and origin - signal is complex: a set of repeated sequences; overlapping with enhancers that stimulate late transcription - recognized by viral protein IVa2
Pseudotyped Vector Virus
are safe surrogates of highly infectious and/or deadly viruses. this involves the manipulation of the viral genome such that surface proteins of one virus are displayed on the backbone of a different virus. these can be used to study binding and neutralization in vitro
ss RNA (+) genomes
covid, west nile etc
syncytia
fusion of multiple host cells into single large cells containing multiple nuclei caused by viral infections (due to fusogenic viral protein)
other genome class that uses RT
gapped dsDNA viruses - hepatitis B virus
transcriptional cascade
genes in each step code for transcription factors that control synthesis of later-acting transcription factors - can be activating or inhibiting
simple retroviral genome
genomic expression - gag, pol, env proviral DNA (integrated ds copy) - LTR, gag (core porteins), pol (enzymes), env (envelope)
enveloped viruses bind via
glycoproteins
CAR protein
group B cocksackie viruses and subgroup C adenoviruses receptor
mRNA
has a 5' cap (m^7 G) that protects from degradation and has a poly(A) tail and UTRs (untranslated regions)
Hdacs
histone deacetylases - removes acetyl groups from histones tails - allows the DNA to be wrapped more tightly and reduces transcription
herpes simplex virus genome circularization
host DNA ligase IV/XRCC4 is responsible for ligating 5' and 3' ends
envelope
host cell-derived lipid bilayer, viral glycoproteins are often embedded in the envelope
Burst/Yield
host cells release many viral particles after cycle is complete - multiple bursts can happen when a few cells are infected
HeLa cells
human epithelial cells of a strain maintained in tissue culture since 1951 and used in research, especially in virology - cells were nonconsentually taken from Henrietta Lacks
discovery of mRNA splicing in adenovirus infected cells
hybridization of heron mRNA to complementary adenoviral DNA revealed unhybridized loops
cell receptors for viruses
infection of cells by many, but not all viruses, require binding to cellular receptors (exceptions plant viruses of yeast & fungi) - receptor biology is a relatively new field
virion
infectious virus particle
cap snatching
influenza steals the host 5' cap that lets them produce mRNA
IRES
internal ribosomal entry site - secondary structure that increased translation - directly recruits 40s subunit decreases host dependency on initiation factors
Packaging signals - RNA genomes
kissing loop complex?
how is virus instability achieved?
lacks covalent bonds
low MOI
like 0.001
high MOI
like 10
How old are viruses?
likely billions of years old
Viral transcriptional control regions
local regulatory sequences of promotors are different from virus to virus and even within a virus - could have few or many regulatory sequences
LTR
long terminal repeat - strong promotor - built into the genome by reverse transcriptase
(+) RNA
mRNA that's able to be directly translated by the ribosome
structural unit
made up of 1 or more subunits
DNA-dependent DNA polymerase (DdDp)
makes DNA from an DNA template
DNA dependent RNA polymerase (DdRp)
makes RNA from DNA templates
Splicing marks mRNAs for
nuclear export - proteins from spliceosomes are left on mRNA and these are recognized by nuclear export pathway
nucleocapsid
nucleic-acid protein assembly within the particle - used when it is a discrete substructure - is in enveloped viruses
juxtaposition of mRNA ends
pea enation mosaic virus, barely yellow draws virus - CITE = cap independent translation enhancer
Poly A stuttering
polymerase begins to make A but cannot progress and keeps churning out A's - polymerase is stationary - reaches a point where the mRNA can no longer get pulled through, stutters, adds A's, and then product falls off
Acquiring polyA tails
prevents degradation of mRNA and helps in translation
genome packaging
problem: viral genomes must be distinguished from cellular DNA or RNA molecules where assembly takes place - solution: packaging signals in the viral genome
Main functions of viral structural proteins
protect the genome and delivery of viral genomes
capsid
protein shell surrounding the genome
the cell cycle is tightly controlled
retinoblastoma (Rb) controls entry into S phase - either binding to Rb (viral products) or phosphorylation of Rb allows for entry to S phase - Rb bound results in the inhibition of synthesis of cellular and some viral replication proteins - Rb not bound leads to increased synthesis of cellular and some viral replication proteins
retroviruses vs gapped dsDNA viruses
retroviruses - RT in cytoplasm first; imported into nucleus - INTEGRATION into host DNA - utilizes host machinery to produce new genomes and mRNA hepadnaviruses - repair in nucleus first, exported to cytoplasm for RT - NO INTEGRATION - utilizes host machinery to produce viral RNAs but, new genomes produced through RT
moving in heavy traffic
short distance = energy dependent protein channels long distance = energy dependent motor proteins on cytoskeletal tracks
ribosome shunting
skipping over part of the UTR - stable stem loop can't be unwound by helices - so the ribosome jumps past it
transcription is tightly regulated
there are a host of sequences surrounding the initiator sequence that help to specify initiation and its efficiency - distant regulatory sequences: silencers and enhancers - local regulatory sequences - TATA sequence (binds TFIID) - initiator sequence
Gapped dsDNA genomes
this genome cannot be transcribed as is. It needs to first be repaired and it relies on cellular repair enzymes to achieve this
what is the first biosynthetic event to occur in our cells infected with dsDNA viruses?
transcription - all DNA viruses need to make at least 1 protein to replicate their genome
true or false: dsRNAs never leave the particle
true - mRNA synthesis happens right in the particle - all mRNA synthesis happens in the particle - exits from the 5-fold axis of symmetry
Tissue cultures today
use either monolayers or suspension methods
non-enveloped viruses cell entry
use receptor-mediated endocytosis
stability conundrum
virus particles must be able to survive outside of a cell BUT they also need to essentially fall apart and release their genome once inside a cell
Do viruses have a membrane?
viruses are a bit of nucleic acid (DNA or RNA) surrounded by protein, some viruses also have lipid membrane (enveloped) that surrounds the protein coat
What is a virus?
viruses are infectious, obligate intracellular parasites composed of genetic material surrounded by a protein coat and.or an envelope derived from a host cell membrane
