Micro biology fina
- How does poliovirus generate multiple viral proteins from a single mRNA?
1)viral genomic RNA is translated into a single large polyprotein - precursor to all viral proteins, generated by cleavage 2) polyprotein gets autocatalytically cleaved by viral proteases - 2A and 3C 3) 2A and 3C are active within the polyprotein - generate P1, P2 and P3 (properly fold and act as protease while part of polyprotein!)
Enveloped or not?
Non enveloped
Type of genome?
enclosing ss, (+) sense genomic RNA(around 7.7 kb)
What catalyzes integration of proviral DNA?
integrasemediates the integration of the proviral DNA into the host cell DNA -cleavage-ligation reaction
What are the 2 outcomes after HIV-1 provirus integration?
(1) Active infection-production of progeny virus, occurs only in activated T cells (2) Latent infection-viral RNA not expressed, but is maintained for long periods in genome -barrier to full elimination of virus by immune system/drug therapy -immune system can't recognize that cell is infected -drugs target actively replicating virus
What are the major categories of viral vaccines? (including new approaches). Be able to describe them and understand their advantages and disadvantages.
(1) Live wild-type virus (mostly historical now!!): • a virus that infects one species - can sometimes be used to infect another species to protect against a closely related pathogen • viruses adapted to replicate in natural host - replicate poorly, not pathogenic in other species • but shares immunogenic determinants with the related virus • Ex. Vaccinia (Cowpox) virus - vaccine for smallpox - not really in use anymore as smallpox vaccine, but used as a viral vector
Describe the proviral DNA. How is it different from the retrovirus genome?
-linear, dsDNA with long terminal repeats (LTRs) at both ends = proviral DNA Retrovirus genome is positive sense RNA.
What is a viral vector and when is it used? What are the advantages of using a viral vector?
A viral vector is a virus that is genetically modified to deliver and express a foreign gene. A viral vector is used: - (Expression/production): To study the effect or function of a protein by expressing it in cells or tissue. - Gene Therapy:To replace a mutated gene - Vaccination of humans/animals against specific diseases - Anticancer agents - that specifically kill human cells.
Be able to explain antigenic shift and drift of Influenza viruses.
Antigenic Drift: •results from accumulation of point mutationsin HA and NA that don't affect protein function but allow virus to escape Ab recognition •error prone RNA polymerase is responsible for a lot of the accumulating point mutations in influenza HA and NA genes Antigenic Shift: •results from reassortment of influenza virus gene segmentsduring mixed infections with 2/more virus subtypes •reassortant virions-some genome segments from 1 strain, some from other strain •if resassortant virus can replicate in host, it will be propagated •reassortment between animal and human strains(ie. birds, humans, pigs) could lead to human replicating strain with HA or NA derived from birds -no Abs in human population that recognize avian HA -pandemic
What drugs have been generated to target these stages?
Azidothymidine(AZT)-nucleoside analog (thymidine analog) -AZT is a nucleoside inhibitor of RT altered sugar group-normal 3'OH converted to azido (N3) group •AZTtaken up by cell and phosphorylated by cellular enzymes •when AZT triphosphate incorporates into the growing DNA strand it causes chain terminationdue to the missing OH group •other nucleoside inhibitors of RT: dideoxycytidine (ddC) and dideoxy- inosine (ddI) (8 currently available for treatment of HIV-1 infection)
Fusion inhibitor of HIV
Enfuvirtide(T-20, Fuzeon) -prevents gp41 from promoting fusion of viral envelope and cellular membrane -used in combination with RT/protease inhibitors when other treatments not effective, start developing resistance -injected 2X a day, very expensive
What does the Influenza A NS2 protein do?
Export of influenza nucleocapsids from the nucleus.Matrix Protein (M1)imported into nucleus -binds to nucleocapsids •NS2 imported into nucleus -binds to nucleocapsids also •M1 and NS2 have nuclear localization signal (NLS) •NS2 has a nuclear export signal (NES)-transports the whole complex through the nuclear pore to the cytoplasm
Be able to explain how SV40 can induce cellular transformation.
If the cell is non-permissive for viral replication- no viral genome replication occurs, the virions don't assemble and there is no lysis. T ags still get expressed leading to transient transformation of the cell. Or if the viral DNA integrates into the cellular chromosome there can be stable transformation and tumors through continuous Ag expression.
How is the initiation of translation of poliovirus mRNAs different from that of cellular mRNAs?
Initiation of translation of host cell mRNAs: Step 1: 5' end of host cell mRNA is capped -initiation of translation is cap dependent. -capping involves addition of a 7-methyl guanine nucleotide to the 1st base of the mRNA transcript. Step 2: eIF-4F complex ( eukaryotic initiation factors eIF, eIF-4A, eIF-4E and eIF-4G) recruits 40 S ribosomal subunit to the capped 5' end of an mRNA Step 3: 40 S subunit scans mRNA searching for an AUG codon in appropriate sequence context. Step 4: 60 S ribosomal subunit joins in to form an 80S ribosome complex and protein synthesis begins.
Attachment inhibitor of HIV
Maraviroc (Selzentry) -CCR5 inhibitor, blocks ability of R5 strains of HIV-1 to attach to T cells/macrophages using CCR5 co-receptor -approved in 2007
How do non-enveloped viruses bind their receptor?
Non enveloped viruses- have protrusions/ cavities in the capsid surface that bind to cell surface proteins on the host cell. for both enveloped and non enveloped Interaction between receptor and virus - changes in virus particle and cell. e.g. binding of HIV-1 envelope glycoprotein (gp121) to CD4 triggers conformational change in viral glycoprotein that allows co-receptor binding e.g. attachment of adenovirus to integrin activates signalling pathways in the cell that increases endocytic activity, promotes virus uptake.
Transcriptase
Replicase-> RNA-dependent RNA polymerase it is called transcriptase when it is used to generate mRNA and called replicase when it is used to replicate the genome.
Know the typical differences between small and large DNA viruses.
Small DNA viruses(nuclear replicating) -parvoviruses- smallest DNA virus ssDNA linear -polymaviruses- circular dsDNA, this includes SV40. - papilloma viruses --> these include HPV dsDNA circulae small viruses use almost exclusively the host cellular machinery for transcription and replication- Use host DNA and RNA polymerase - poly and papi viruses encode proteins that trick the cell into entering S phase Larger DNA viruses(nuclear replicating) Still require host cellular machinery for transcription but encode their own DNA polymerase. E.g. adenovirus and herpees Large cytoplasmic replicating viruses - encode their own DNA and RNA polymerases e.g. smallpox
How is poliovirus spread?
spread by fecal contamination of food/water - "fecal-oral" route
How does poliovirus cause paralytic poliomyelitis?
• motor neurons - have receptors for poliovirus - if polio gets into bloodstream it can enter motor neurons and destroy them causing acute flaccid paralysis (AFP) = paralytic poliomyelitis
Where in the body does poliovirus normally replicate?
• receptors for virus (PVR) on intestinal epithelium - virus replicates in gut, virus causes cell lysis upon release, local tissue destruction can cause diarrhea, leads to further spread of disease
Where does Influenza A replicate? Why does it need to replicate there?
• viral transcriptional machinery can not make mRNAs on its own - can't cap its own mRNAs • uses cellular mRNAs as primers to initiate viral mRNA synthesis - process is known as "cap stealing" • PB2 - recognizes 5' cap on all cellular pre-mRNAs (in the nucleus - before splicing and polyadenylation and transport to the cytoplasm as mature mRNA) • PB1 - has exonuclease activity (in addition to its polymerase activity!!) - cleaves the cellular pre-mRNA just 3' of an A residue • the 3' A on the cleaved cellular pre-mRNA base pairs with a conserved U at the 3' end of the viral genome RNA • PB1 uses capped fragment as primer for copying viral genome - starts elongation
The Poliovirus RNA-dependent RNA pol (3D) requires a primer. What is the primer used by poliovirus?
•2 uridine residues attach to VPg/3B - hybridize to poly(A) tail at 3' end of viral RNA = primer to initiate RNA synthesis
What are the major categories of viral vaccines?2
(2) Live attenuated virus: • generated by serial passage of pathogenic virus in other animals, tissue culture - leads to acquisition of a variety of mutations that reduce the pathogenic properties of virus in its natural host, attenuated = weakened - often little knowledge of mechanism of attenuation • new attenuated virus called vaccine-strain virus • still replicates in host - induces effective immune response • can lead to viral-induced disease in some cases (immunosuppressed) or if attenuated virus undergoes a mutation that allows it to revert to a more virulent form • Ex. Edmonston B vaccine strain Measles virus (used in Measles-MumpsRubella (MMR) vaccine), also Oka strain vaccine strain chicken pox virus
Understand the basics of how you use a viral vector to express a gene of interest in a target cell.
- A virus has sequence elements for replication (Ori) and packaging - Replication and structural proteins are deleted and replaced with the transgene in the interest of safety and room for the transgene. -A packaging cell line is engineered to express the viral replication and structural proteins -transfect the virus vector into the packaging cell line (use physical/chemical methods)
Understand the basics of assembly of a helical capsid ?
1) Capsids of helical viruses assemble around the genome. e.g. tobacco mosaic virus 2) ss or ds RNA/DNA can readily form a helical structure. 3) capsomers self-assemble into disk 4) the disk interacts with specific sequence in the viral genome - pac (packaging signal) 5) subsequent capsomer proteins thread on in a helical array.
Know the 4 functional units that make up the SV40 genome.
1) Control region Contains the Ori (origin of replication) Early promoter/enhancer (Pe) has a TATA box binds the TBP of the TFIID complex- recruits cellular RNA pol II and additional transcription factors. Also the Late promoter (PL) Drives transcription of late genes No TATA box, has a binding site for LArge-T-Ag 2) Early transcription region Encodes the small and large T-antigens early genes 3) Late transcription region Encodes the VP1,2 and 3 (structural proteins) and the Agnoprotein (function unknown) 4) Polyadenylation region Early and late transcripts terminate in this region. Early and late transcripts have a region of 3' overlap which can lead to the generation of dsRNA during replication cycles- triggers an interferon response.
Know what each of these 4 functional units contains, and what each does
1) Control region Contains the Ori (origin of replication) Early promoter/enhancer (Pe) has a TATA box binds the TBP of the TFIID complex- recruits cellular RNA pol II and additional transcription factors. Also the Late promoter (PL) Drives transcription of late genes No TATA box, has a binding site for LArge-T-Ag 2) Early transcription region Encodes the small and large T-antigens early genes 3) Late transcription region Encodes the VP1,2 and 3 (structural proteins) and the Agnoprotein (function unknown) 4) Polyadenylation region Early and late transcripts terminate in this region. Early and late transcripts have a region of 3' overlap which can lead to the generation of dsRNA during replication cycles- triggers an interferon response.
(1) Know the general characteristics of DNA viruses that set them apart from the other virus groups we have been talking about.
1) DNA virus use a significant portion of cellular machinery to transcribe and replicate their genomes 2) In general , the larger the size of the virus the less they depend on cellular machinery. Since they have more DNA to encode the proteins they need e.g. RNA polymerase III 3) DNA viruses that replicate in the nucleus use the cellular transcription machinery 4) DNA viruses that replicate in the cytoplasm, must encode their own RNA polymerase 5) DNA replication requires a primer. Viral primers are not always RNA. 6) Replication of the discontinuous/lagging strand always requires a primer leading to a loss of DNA with each cycle of replication. To prevent this end problem , viruses circularize their genome. 7) A common feature of many nuclear-replicating DNA viruses - life long association with the host.
Know the basic characteristics of HIV-1 - ie. Genome, capsid shape, whether or not enveloped.
1) Lipid envelope derived from the plasma membrane 2) 2 identical copies of RNA genome covered with nucleoplasmid. (positive sense RNA 7 to 10 kb) 3) Conical capsid
How does poliovirus get into the cell?
1) Poliovirus binds to CD155 at the cell surface and this causes a major conformational change in the capsid structure. 2) At the start VP4 is inside the capsid, N terminus of VP1 is inside the capsid. 3) After conformational change --> VP4 is externalized and hydrophobic N-terminus of VP1 is extruded to the surface of the virus particle. 4) The hydrophobic N-termini of VP1 inserts into the membrane of the host cell to form a channel through which viral RNA can pass directly into the cytoplasm(channel formation is hypothesized) 5) VP4 is myristolated and inserted into the membrane as well. 6) Poliovirus can infect dynamin mutant cells which suggests that receptor mediated endocytosis is not required for poliovirus entry in contrast to Rhinovirus.
What effects does poliovirus infection have on host cell functions?
1) Shutoff cap-dependent translation of host cell mRNA - Encodes a protease(2A) that cleaves eIF-4G. -eIF-4G is essential for recognition of 5' end of capped cellular mRNAs - infected cells can't translate cellular mRNAs and devote themselves to cap-independent translation of viral RNA 2) Shutoff host cell RNA synthesis - RNA synthesis by all 3 classes of mammalian RNA polymerases is inhibited -3C viral protease cleaves RNA polymerase subunits. 3) Induction of cytoplasmic vesicles - nucleate RNA replication 4) Alteration of intracellular transport pathways between the ER and the golgi apparatus- proteins 2B and 3A inhibit membrane trafficking from the ER to Golgi to pm- build up of vesicles that can be used by virus for replication.
What are the characteristics of + sense viruses?
1) The first step in the infectious cycle of + sense RNA viruses is the translation of viral protein- do not need a transcription step prior to expression of viral protein. 2) The genome is infectious: Transfect the virion RNA into a cell without any other viral protein and new virus will be produced. 3) Incubate genomic RNA from virion with ribosomes, tRNA,amino acids and other components of an in vitro translation system - you will get viral protein synthesis 4) Life cycle does not require a host cell nucleus. -> Entire replicative cycle occurs in the cytoplasm -> experiment: Treat cells with cytochalasin B to break down actin cytoskeleton that anchors nucleus inside the cell. Gently centrifuge the cell and the nucleus will pop out. The enucleated cell can be infected with positive sense RNA virus and will produce new virus particles just like a normal nucleated cell.
optional info? about receptor?
1) The host cell receptor for poliovirus is CD155(or poliovirus receptor - PVR) 2) It is a member of the immunoglobin superfamily, many picoviruses bind to Ig superfamily proteins. Function of CD155 in host is unknown 3) CD155 binds to depressions on the surface of the poliovirus virion called canyons/pits --> unusual since most naked viruses bind to receptors via surface exposed loop regions of capsid proteins.
What are the 4 main steps of the basic replication process of any virus?
1) Virus receptor recognition, attachment, and entry into host. - viruses must use specific features on host cell to introduce their genome into the cell. 2) Viral gene expression-> viral genes must be decoded from nucleic acid (mRNA production) and translated into viral proteins. 3) Viral genome replication -->viruses must be able to replicate their genome to produce new virus particles. 4) Viral capsid formation, virion assembly and exit from host --> new virus particles must assemble and be released.
What are the 6 different classifications of viruses in the Baltimore scheme?
1) dsDNA --> mRNA e.g. Polyomaviruses, Herpes virus 2) ssDNA-->dsDNA--> mRNA e.g. parvovirus, B19 virus 3) dsRNA -->mRNA--> rotavirus 4) + sense ssRNA --> mRNA 5) -ve sense ssRNA --> + sense ssRNA--> mRNA 6)+ sense ssRNA --> dsDNA (reverse transcriptase)-->mRNA
Understand how a virus acquires a lipid envelope
1) lipid bilayer of a virus is derived from the infected host cell. 2) Envelope is made virus specific by the insertion of one or several virus-encoded membrane proteins. 3) Formation of viral glycoproteins parallels formation of cellular glycoproteins. 4) Viral glycoproteins contain trafficking signals that direct the protein to its destination - plasma membrane, Golgi, nuclear membrane, apical or basolateral plasma membrane. - glycoproteins directed to region of the cell from which virus will bud. 5) viral envelope glycoproteins that have been transported to the pm in vesicles form a patch in the membrane 6) the naked capsid forms and migrates to the virus modified path on the plasma membrane 7) Nucleocapsid and membrane patch associate- often through virus-encoded matrix proteins that serve as adaptors. The matrix serves as the glue between the capsid and the envelope. 8) Bud forms 9) cellular proteins involved in pinching off the bud (dynamins) are recruited. 10) free infectious virion is released. 11) Not all viruses bud at the plasma membrane- some bud from nuclear membrane into exocytic vesicles i.e. herpes virus
(9) If a virus needs to traffic to a particular location within a cell how do they do that?
1) the herpes simplex virus and rabies virus traffic along the nerve axon of peripheral neuron to reach the cell body. 2) Virus uses host microtubule cytoskeleton to traffic to location in the host cell (normal host system) motor molecule used: dynein mediates retrograde microtubule transport. -herpes virus/adenovirus can interact with dynein. 3) Virions can be transported as free capsids or in endocytic vessicles
Describe 2 mechanisms of retrovirus transformation/cancer induction.
1)Acute transforming retroviruses: •transform cultured cell rapidly, induce cancer within a short time after infection •encode a viral protein that is not required for replication, but directs tumour formation -called a viraloncogene •this protein is a mutated cellular proteinincorporated into retroviral genome •Ex. -Rous sarcoma virus-viral Src -v-Src is a mutated copy of the c-Src gene (cellular proto-oncogene) -mutation has rendered it constitutively active other acute transforming retroviruses -oncogenes that derive from cellular genes that code for growth factors, growth factor receptors, kinases, TFs that regulate cell growth and division -possess mutations that deregulate them -cause the cell to undergo uncontrolled growth and division
How does HIV-1 enter the cell? Be able to describe the actions of SU and TM in virus entry.
1)gp120(SU) binds to CD4on T cells or macrophage 2)gp120 undergoes 1st confirmational changethat exposes the co-receptor binding site, co-receptor= chemokine receptors CCR5or CXCR4 -HIV "R5"strains -use CCR5, HIV "X4"strains -use CXCR4 -only R5 strains are sexually transmitted, so natural resistance to HIV infection is associated with mutations in CCR5 that cause loss of cell surface expression 3)co-receptor binding -triggers 2nd confirmational change in gp120 4)triggers gp41 (TM) to mediate fusionof viral and cytoplasmic membranes
What anti-viral drugs have been developed for treating Influenza?
2 neuraminidase inhibitorshave been developed as influenza anti-virals -Zanamivir (Relenza) -Oseltamivir (Tamiflu)
Understand the basics of how a retroviral/lentiviral vector works. What is the advantage of a lentiviral vector over a retroviral vector?
A retroviral vector can only enter the nucleus when the nuclear membrane breaks down during cellular division. The retroviruses then generate dsDNA copy of their RNA genome and integrate this in the DNA. The packaging cell line expresses - gag, pol and env genes. The transfer construct - viral promoter, packaging signal and transgene. The lentiviruses are better since the can transport the pre-integration complex into the nucleus without the need for the cells to be dividing. Lentiviruses express genes in terminally differentiated cells.
Compare and contrast retroviral/lentiviral, adenoviral and AAV vectors.
AAV vectors are small , non enveloped ssDNA genome viruses with only 2 genes. Requires the presence of a helper virus in the same cell for replication and transcription of the AAV genomes and production of new AAV virions - either adenovirus or herpes virus Can actually integrate into chromosomes unlike adenoviruses which exist as episomes. But can also exist as stable episomes for long periods. -no strong immune response like in adenoviruses. however small size limits the size of the transgene.
How do enveloped viruses enter the cell`?
After attachment the virus must get past the plasma membrane to enter the cell: For enveloped virus, fusion of the viral envelope with a host membrane (either the plasma membrane or a vesicle membrane) must happen. Once it has entered the cell it must uncoat (shed its capsid) in order to reveal its nucleic acid genome. Enveloped viruses can enter the cell either by endocytosis or fusion(which involves both processes.
Be able to describe the SV40 life cycle. Know which genes are expressed first ie. the difference between early and late genes. Understand the splicing patterns that give rise to these different genes.
Attachment -> SV40 attaches to sialic acid residues on glycoproteins Entry Enters via receptor mediated endocytosis via caveolae(specialized vesicles on the cell surface) Entry into the nucleus - VP1 has a nuclear localization signal The Uncoated viral genome in the nucleus remains associated with histones as a mini chromosome. Transcription of Early genes Large and Small T-ag. The mRNA is exported to the cytoplasm where translation occurs. Large -T ag inactivates Rb(releasing E2F)--> promotes cell cycle progression as well as p53 another tumor suppressor. Large-Tag also prevents transcription of early genes and promotes transcription of late genes. There is also a dilution of a cellular repressor that binds to the alte promoter . It acts as a helicase and assembles as a hexamer at the Ori where it unwinds the DNA. The single stranded region is ready for replication. Large T-ag also recruits important proteins like DNA polymerase, Replication protein A, topoisomerase etc. and this leads to recuitment of other important proteins like Replication protein C and DNA pol delta. 2 late mRNAs generated by splicing encode VP1,2 or 3 which enter the nucleus for capsid assembly. Viral progeny is then released by lysis.
What is the Baltimore scheme of virus classification based on?
Based on the way that viruses produce mRNA during infection. All viruses must express mRNA for translation into viral proteins. How this is done is determined by the type of viral genome.
What are the major categories of viral vaccines?4
Component/Subunit Vaccines: • consist of purified viral proteins that are known to be immunogenic - some self-assemble into "virus-like particles" • do not contain other components of virus - no nucleic acid genome • no danger of inadvertent infection with live virulent strain that hasn't been inactivated properly • Ex. Hepatitis B vaccine, Human papilloma virus (HPV) vaccine (Gardasil; prevent development of cervical cancer), new one! - Flublok (approved in 2013 by FDA) protects against 2 Influenza A strains (H1N1 and H3N2)
New approaches to viral vaccine development
DNA vaccines: • inject DNA encoding viral proteins intra-muscularly - can be taken up (probably by dendritic cells), expressed and can trigger immune response • developed from failed gene therapy trials!! • DNA vaccine for West Nile virus developed for use in horses - human trials have not been as promising • DNA vaccines for avian influenza, herpes simplex virus, others in current clinical trials (Pfizer)
What is the difference between an enveloped and a non-enveloped virus? Where does the envelope come from?
Enveloped viruses are surrounded by a lipid bilayer. The lipid bilayer is acquired from the host cell since viruses do not have the ability to perform lipid biosynthesis. Viruses insert virus encoded membrane proteins into the lipid bilayer.
Describe the process of assembly of a new retroviral particle? When are the viral proteases activated?
Envprotein-transported through Golgi apparatusto p.m.,cleavedby host cell enzymes into SU and TM •SU and TM-modify the p.mthat will be the viral envelope •Gag and Gag/Pol polyproteins-released into cytosolafter translation, associate with each other, not yet cleaved into final, active proteins •Capsid assembly: -Packaging signal on genome RNA binds to Gag -tRNAprimer incorporated into assembling virus particle -Gag-targeted to p.m.by myristatetag added to polyproteinafter translation (brings assembling particle to membrane) •Virus particle buds from plasma membrane: -viral proteasebecomes activated -cleavesGag and Gag/Pol polyproteinsinto structural and enzymatic proteins -virus core rearranges into its mature form, becomes infectious
Why is HIV-1 protease a good target for an anti-viral drug?
HIV-1 proteaserequired for final maturation of virion(occurs after budding) -cleavage of polyproteins into active proteins -proteases become active, capsid assembles around RNA genome -without final cleavage progeny virions are not infectious •HIV-1 protease cleaves between phenylalanine and proline residues-site very rarely cleaved by human proteases-possible to develop HIV-1 specific protease inhibitor, with hopefully fewer side effect
Be able to describe the differences between a positive sense and negative sense RNA virus.
In order to replicate all viruses must express mRNA for translation into proteins. 1) + sense RNA viruses- genome can be directly utilized as mRNA 2) negative sense RNA--> viral genome is opposite sense from mRNA and must be transcribed into mRNA by viral-encoded transcriptase(Carried in virion). ds RNA viruses behave the same way as -ve sense RNA viruses
What are the major categories of viral vaccines?3
Inactivated virus: • Chemical (formalin or betapropiolactone), heat or UV is used to make purified virus non-infectious - can't replicate in host • control of inactivation treatment needed to retain immunogenicity of viral proteins so that immune response is generated • usually not as robust an immune response as with live virus - immunity may not be maintained for as long, may require more booster shots, adjuvant must be used to stimulate innate immune response • but can't revert to virulence! • Ex. Rabies, Hepatitis A, most common Influenza A vaccines (not FluMist)
How does the cellular interferon response induce resistance to viral replication?
Interferoninduces expression of the dsRNA-dependent protein kinase (PKR): -PKR = serine-threonine protein kinase -2 molecules of PKR bind dsRNA, cross-phosphorylate each other and become highly activated -activated PKR -phosphorylates eIF-2 -blocks exchange of GDP for GTP on eIF-2 that is catalyzed by eIF-2B -if eIF-2 doesn't cycle between GDP and GTP it can't facilitate the formation of a translation initiation complex -PKR blocks viral (and cellular!) protein synthesis and may induce apoptosisof infected cell
What Influenza vaccines exist? Be able to compare and contrast them.
Killed vaccineagainst the predicted annual strains of influenza A and B viruses: -production of influenza vaccine -relies on growth of virus strains in embryonated chicken eggs -problem: highly pathogenic H5N1 strain -kills embryos before sufficient virus produced •Live, attenuated vaccine (FluMist): -cold-sensitive strain-it can replicate in the cooler nasal passages but not in the bronchi/lungs -combine the HA and NA genes of targeted virus strain with 6 other gene segments from mutant viruses that can't grow at 37⁰C -stays in upper respiratory tract-causes few/no disease symptoms! -administered by nasal spray -approved for use in humans 2-59years old -not in use for those most susceptible to serious influenza disease (very young and very old) •Component vaccine (Flublok): -approved by FDA January 2013 -composed of 3 Influenza HA proteins (H1N1, H3N2, and 1 from Influenza B) -proteins produced in Insect cells -doesn't use chicken eggs!
How do viruses gain access to the nucleus (both large and small viruses)?
Many animal viruses(particularly DNA viruses) use the nucleus as the site of replication. Viral proteins contain nuclear localization signals. Some small DNA viruses- Hep B, parvovirus, SV40 can squeeze through the nuclear pore complex, the capsids interact with importins that are nuclear targeting receptors. Larger DNA viruses- Adenovirus, herpesvirus, 1)associate with cytoplasmic face of nuclear pore complex,capsid stays in the cytoplasm while DNA and associated proteins enter the nucleus. 2) In some viruses, there is dissassembly of the capsid in the cytoplasm and entry of genome into the nucleus through the nuclear pore i.e. HIV, influenza.
Describe the mechanism of anti-viral drugs for treating Influenza?
NA inhibitorsblock the spreadof influenza by preventing its release from the cell surf. where it becomes stuck following buddingdetermined structure of NA bound to sialic acidby x-ray crystallography -sialic acid occupies 2 pockets on enzyme -designed small molecule inhibitorsthat would fit in these pockets on NA
How does Influenza A inhibit the cellular interferon response?
NS1 -binds to RIG-I and prevents it from signalling -binds to dsRNA and reduces amount of dsRNA available to bind to PKR -activates the PI3K/Akt signalling pathway which suppresses apoptosis -NS1 mutant viruses replicate fine in cells with defective interferon response
What does the Influenza A NS1 protein do?
NS1is the most abundant viral proteinin influenza infected cells •involved in host suppression -Influenza viruses with mutated NS1 proteins replicate poorly •for example -inhibits formation of maturecellular mRNA it inhibits CPSF:Cleavage of cellular pre-mRNAs downstream of polyadenylation signal and PABII(Facilitates production of poly(A) tails on 3'terminus created by cleavage) NS1 also inhibit the cellular interferon response.
Nef functions
Nef is Negative effector --> 210 aa protein localized at the inner face of the pm via myristylation. 1)Nef decreases CD4 and MHC class I and II surface expression by increasing cycling from cell surface to endosomal comp. This is a type of immune evasion, disabling T-cells 2) Nef alters signalling pathways in T cells. It causes cell activation which promotes viral replication. But T cells activated by Nef cannot mount an immune response. Nef suppresses apoptosis in infected T-cells but induces apoptosis of uninfected T-cells around infected cell. - Increased expression of inflammatory cytokines IL-6 , TNF-alpha, IL-1beta which cause cell activation.
- What does the Influenza A NA protein do?
Neuraminidase (NA)= Type II integral membrane protein •envelope glycoprotein-major antigenic determinant of influenza virus (along with HA) •cleaves terminal N-acetyl neuraminic acid (sialic acid)from sugar residues on mucoproteins, cell-surface glycoproteins and glycolipids -can reverse binding of HA to sialic acid residues •as influenza virus buds from the surface of infected cells -binds to HA •NA is thought to cleave sialic acid residuesand release bound influenza particles from the cell surface -may also release influenza particles that get stuck on mucoproteins in the respiratory tract
Characteristics of poliovirus Capsid structure?
Nonenveloped, icosahedral capsid(around 30 nm in diameter) Icosahedral capsids-20 sided figure with 12 vertices and 20 faces. In a mature poliovirus virion--> 60 copies of 4 proteins: VP1,2,3 and 4 arranged with icosahedral symmetry. VP1,2 and 3 combine to form the capsid shell. VP4 is buried beneath the shell-not exposed on surface.
What are the 2 major groups that ss RNA viruses can be divided into? Explain the difference between them.
Positive(+) sense/strand RNA viruses- RNA genomes are the same sense as host cell mRNA (look like mRNA to host) - genome can be directly utilized as mRNA by translation machinery. Negative(-) sense/strand RNA viruses-- RNA genome is antisense(complementary) to host cell mRNA (can't be used as mRNA to host) - genome must be transcribed into mRNA (uses viral enzymes).
Which Influenza A mRNAs undergo alternative splicing? What proteins do these mRNAs encode?
RNAs from segment 7 (encodes M1/M2)and segment 8 (encodes NS1/NS2) contain cellular splicing sequences-recognized by cellular splicing machineryin the nucleus -not all viral mRNA #7 and viral mRNA #8 gets spliced -more unspliced than spliced mRNAs means greater abundance of M1 and NS1 proteins, which is what virus wants! -unspliced = M1 and NS1 -spliced = M2 and NS2
What are the 2 activities of the RT? Why are both of these activities important?
RTgenerates a dsDNA copy of the RNA genome = provirus -RT of retroviruses is error prone, and has no proofreading activity -1-10 nucleotide errors/proviral DNA molecule made! 2 activities: -RNA andDNA-dependent DNA pol. -RNAse H activity-destroys the RNA part of an RNA-DNA hybrid both activities are important for the conversion of RNA to DNA.
Integration inhibitor
Raltegravir (MK-0518, Isentress) -inhibits HIV-1 integrase, prevents incorporation of provirus into cellular DNA -used in combination with RT inhibitors and protease inhibitors -used when other treatments not effective -results of Phase III clinical trial very promising -but resistance can be acquired by single point mutation in HIV-1 integrase!
What is the function of REV
Rev(regulator of expression of viral proteins) mediates nuclear export of unspliced and singly-spliced HIV-1 mRNAs.
2 types of Polio vaccine -salk vaccine
Salk vaccine (IPV): • formalin-inactivated preparation of 3 virus serotypes grown in monkey cells • developed by Dr. Jonas Salk - 1955 • no chance of reverting to virulence • but chance that it isn't inactivated properly: - "Cutter incident" April 1955- • stored more cheaply - doesn't need to be refrigerated • but more expensive to make, and needs to be injected • prevents spread of Polio in the bloodstream (which is what is problematic for causing paralysis) BUT doesn't provide good mucosal immunity on its own (allows replication in intestinal epithelial cells if infected with Polio)
second type of transformation
Slow transforming retroviruses: do not encode an oncogene, usually long latency period •transformation due to where the provirus integratesin the genome •integration is random-if insertion occurs in/adjacent to cellular proto-oncogene, cellular gene may come under control of viral LTR(acts as promoter/enhancer) -viral LTR inserted upstream of cellular gene-transcription starts at LTR, proceeds through adjacent cellular gene -can happen if lefthand LTR gets inactivatedor if righthand LTR is deleted •deregulation of cellular gene -inappropriate cell growth and division •Ex. Murine Leukemia Virus
Be able to describe the role of the SV40 Small t antigen in cell cycle regulation. Be able to describe the roles of the SV40 Large T antigen - both in cell cycle regulation and in DNA replication. Be able to explain why nuclear replicating DNA viruses often encode proteins that regulate the cell cycle.
Small-Tag inhibits PP2A (protein phosphatase that inactivates Map Kinase which is a negative regulator of the MAPK pathway) The MAPK pathway is upregulated and more AP-1 transcription factor is assembled. AP-1 results in greater cyclin D1 production which drives the cell into S phase.
How do enveloped viruses bind their receptor?
Specific viral glycoproteins embedded in the viral envelope serve as binding sites for receptors. The same glycoprotein may mediate attachment and membrane fusion. e.g. Influenza virus hemagglutinin (HA)- HA1 domain contains sialic acid binding site, HA2 domain mediates membrane fusion.
What are the 2 basic shapes of virus capsid?
Spherical (icosahedron) --> capsomers form a regular solid structure encasing the viral genome. Helix-- capsomers associate with helical nucleic acid as a nucleoprotein.
- How do you get production of mRNA that encodes the Env protein?
Splicing of the primary transcript- All retroviruses make at least 2 mRNAs and a singly spliced mRNA - synthesis of Env protein (gag and pol regions get spliced out) also Vpr, Vif and Vpu
Understand and be able to outline the process of RNA-directed RNA replication and know what enzyme is required(happens only in ss-ve RNA and dsRNA
Step 1: A virus encoded replicase mediates replication of the viral RNA genome. 2) moves from the 3' to 5' along the genomic RNA 3) proceeds through the production of 2 replication intermediate complexes RI-1 and RI-2. 4) RI-1 and 2 often have a multi-branched structure. Multiple replicases initiating replication on one strand of viral genome RNA. 5) RI-1 results in the production of RNA with opposite sense to genomic RNA 6) RI-2 results in new genomic positive sense RNA
Receptor mediated fusion of enveloped virus with plasma membrane
Step 1: attachment of virus to receptor on cell via proteins in the envelope. Step 2: fusion of viral envelope with cell envelope Step 3: release of nucleocapsid into the cell Step 4 : the viral envelope forms a patch on the plasma membranes.
Assembly of a spherical capsid
Step 1: preassembly of procapsids ( immature capsid). The procapsid often assembles around scaffold proteins. Step 2: maturation of the procapsid to mature capsid involves proteolytic cleavage by maturational proteases-removal of scaffold proteins. Step 3: capsid assembly occurs before entry of viral genome - usually.
Be able to describe the process of receptor-mediated endocytosis of a non-enveloped virus.
Step 1: virion binds to cell surface receptor Step 2: virion receptor interaction triggers the formation of a clathrin coated pit Step 3: acidic environment of endocytic vesicle triggers changes in capsid that causes the release of the viral genome occurs at pH 6.5 to 5.3 --> the pH of early/late endosomes. Step4: induces the formation of pores or disrupts the endosomal membrane- release of viral genome into cytoplasm.
Which HIV-1 regulatory proteins are essential for viral replication?
Tat and rev genes are essential for virus replication in cell culture--> greatly increase the level of HIV-1 proteins produced by regulating synthesis and transport of HIV-1 RNAs
What are the functions of Tat?
Tat(transactivator of transcription) expression-dramatically increases amount of viral RNA produced by increasing elongation efficiency of cellular RNA pol II(no Tat -HIV-1 transcripts short and HIV-1 genes therefore not highly expressed)
Viral vector approach
Viral vectors: • genes that encode viral antigens of interest can be inserted into non-pathogenic viral vectors - approach being taken for design of HIV vaccine, among others (Hepatitis C, Ebola virus - Canadian developed vaccine, now being developed by Merck, vesicular stomatitis virus vector engineered to express Ebola proteins) - STEP vaccine (Merck) - adenovirus engineered to express HIV proteins
What are the 2 basic components of all viruses?
Virus is composed of a nucleic acid genome (RNA or DNA) surrounded by a capsid made up of virus encoded proteins 1. Nucleic acid (viral genome) 2. Protein coat (viral capsid)
What are the functions of Vpu protein?
Vpu (viral protein U): Insets into membranes via N term domain, accumulates in ER and Golgi. -> Degrades the intracellular CD4 bound to gp 160 (SU and TM proteins) in the ER. -> This association would prevent gp 160(gp120+gp41) from reaching the cell surface to be packaged into new HIV-1 virions. Vpu binds to CD4 and cellular protein beta-TrCP to cause ubiquitination of CD4 and degradation by the proteasome. 2nd function: Vpu also enhances release from the pm - no Vpu, get accumulation of virions on the cell surface that are never released. Vpu may effect virion release by inhibiting the cellular protein Tetherin. In the absence of Vpu, HIV-1 particles are retained at the surface by cellular protein Tetherin. Tetherin is induced by interferon in the virus infected cells- part of the host cell anti-viral response.
What is the primer for RT?
cellular tRNA-binds PBS, acts as primer for RT
Sabin vaccine (OPV)
developed by Dr. Albert Sabin - 1961 • attenuated live vaccine • attenuated virus can attach to intestinal epithelial cells and replicate - but can't cause paralytic disease. • attenuated Sabin vaccine strains have pt. mutation in stem-loop V of IRES which causes defects in translation of poliovirus proteins in neuronal cells, but not in non-neuronal cells. • can revert to virulence - there are circulating vaccine-derived poliovirus strains - occurs by back mutation or by a recombination event with another virus infected individual • can cause disease in immunocompromised individuals • oral delivery - needs to be refrigerated, but doesn't need to be injected • Better mucosal immunity than IPV (but does need boosters to maintain this)
How is the Gag/Pol polyprotein produced? Is more Gag polyprotein produced than Gag/Pol? Why?
full-length RNA serves as mRNA for Gag and Gag/Pol polyproteins. how are these 2 polyproteins generated from a single mRNA? -ribosomal frameshifting-ribosome shifts its reading frame within the RNA prior to hitting termination codon. ribosome doesn't always frameshiftwhen translating unspliced, full-length retroviral RNA -result: Gag polyprotein > Gag/Pol polyprotein
What is the role of the Influenza M2 protein?
helical nucleocapsids containing viral genome are associated with M1 (matrix) protein which is associated with the viral envelope • following fusion, virus needs to dissociate the helical nucleocapsid from M1 so it can be released into the cytoplasm • major player = M2 (ion channel protein) - transmembrane regions of M2 form small pore in viral envelope - acts as H+ ion channel - during endosome acidification, allows H+ ions to enter the virion - low pH within virion weakens interaction of M1 with helical nucleocapsid (proposed - not entirely proven) - nucleocapsid can be released into cytoplasm following fusion
What is the difference between nucleoside and non-nucleoside RT inhbitors?
identified by high-throughput screeningof small molecules for inhibitors of reverse transcriptase •number of compounds identified -ex. Nevirapine -all compounds had similar structures -3 available currently for treatment of HIV-1 infection •bind to RTat site close to active site for DNA polymerase activity -drastically slow rate of DNA polymerization •both nucleosideand non-nucleosideRT inhibitors have problems with resistance -therefore used in combination (except for pre-exposure prophylaxis) -mutations in RT that allow resistance to nucleoside inhibitors are different from mutations that allow resistance to non- nucleoside inhibitors
What are the 3 phases of HIV infection? Be able to describe what is happening in terms of CD4+ T cells and viral load at each phase.
mucosal tissues-usual sites of primary infection, virus replicates in the gut associated lymphoid tissue (GALT) killing CD4 T cells (acute phase), spreads to all peripheral lymphoid tissues In the acute phase CD4+T cells drop sharpy and then recover slightly before dropping more gradually through the asymptomatic phase to a level where the patient dies from opportunistic infection. Viral load rises sharply in the acute phase and drops sharply at the start of the asymptomatic phase. Viral load gradually increases through the latency phase and rises to the highest point when the patient dies due to opportunistic infections.
How do non-enveloped viruses enter the cell?
naked or non enveloped virus particles are incorporated into the cell by translocation across lipid bilayer. The mechanisms of translocation in eukaryotes all (mostly) involve receptor-mediated endocytosis. e.g. clathrin-mediated endocytosis and caveolae-mediated endocytosis. Endocytosis involves the formation of an endosomal vesicles containing EC material (i.e. attached viral particle) that moves into cytoplasm of cell. - important for recycling of receptor-ligand complexes.
Influenza A Genome? Capsid shape? Enveloped or not?
negative-sense RNA genome of Influenza A = 8 genome segments • individually wrapped within helical nucleocapsids Enveloped
How is the initiation of translation of poliovirus mRNAs different from that of cellular mRNAs part 2?
poliovirus RNA not capped - use another mechanism to direct ribosomes to initiator AUG codon • poliovirus actually cleaves eIF-4G/ sequesters eIF-4E - stops capdependent translation in host cell! • all picornaviruses initiate protein synthesis at an IRES (internal ribosomal entry site) - lies in that highly structured 5' noncoding region • host cell factors bind IRES - help dock 40S ribosomal subunit onto RNA • IRES sequences - found in other virus mRNAs and in a number of cellular mRNAs • used in the construction of plasmids where internal ribosome initiation is needed
Ritonavir
protease inhibitordeveloped by process of rational drug design -solving the crystal structure of the HIV-1 protease bound to a target peptide led to design of Ritonavir -Ritonavir is a peptide -bound strongly by HIV-1 protease but cannot be cleaved -Ritonavir treated infected cells -bud viral particles from the cell surface -but they can't mature and are therefore non-infectious
Can HIV-1 integrate its provirus into a terminally-differentiated cell? Why? What proteins facilitate this process?
proviral DNAremains assoc. with virus capsid = preintegration complex (PIC) •too big to enter through nuclear pores! •many retroviruses -wait for breakdown of nuclear envelope that occurs during cell division -can't infect terminally diff. cells -must infect cells that undergo mitosis however lentiviruses like HIV-can mediate transport of the viral DNA complex through nuclear pore -MA, Vpr, integrase proteins impt. -MA has NLS •viral integrasemediates the integration of the proviral DNA into the host cell DNA -cleavage-ligation reaction
How does proviral DNA get access to cellular chromosomal DNA in a lentivirus? In a retrovirus that isn't a lentivirus?
proviral DNAremains assoc. with virus capsid = preintegration complex (PIC) •too big to enter through nuclear pores! •many retroviruses -wait for breakdown of nuclear envelope that occurs during cell division -can't infect terminally diff. cells -must infect cells that undergo mitosis •lentiviruses like HIV-can mediate transport of the viral DNA complex through nuclear pore -MA, Vpr, integrase proteins impt. -MA has NLS •viral
receptor-mediated endocytosis of an enveloped virus followed by fusion
step1:enveloped virus is taken up by receptor mediated endocytosis. step 2: Acidification of endosomal vesicle leads to conformational changes in viral glycoproteins/ modifications of viral envelope that occurs at pH6.5 to 5.3(pH of early endosomes) Step 3: fusion occurs between the viral envelope and endosome membrane--> mediated by specific viral glycoproteins that act as fusion proteins.
What is the function of the retroviral LTR in initiation of retroviral RNA synthesis?
the LTRs (U3-R-U5)act as promoters/enhancersfor the initiation of transcription
What stages of the HIV-1 life cycle are targeted?
there are multiple points in the HIV life cyclethat are targeted by anti-retroviral (ARV) drugs •inhibitors of reverse transcriptase and protease inhibitors are in widespread use -these are used in combination for anti-retroviral therapy (ART) •inhibitors of virus entry/fusion and integrase inhibitors have been developed and are being integrated into anti-retroviral therapy
Be able to describe the cellular interferon response. How is it triggered?
viral transcription and replication of RNA viruses -generate viral RNA complementary to viral genome -some formation of dsRNAis inevitable during RNA virus replication cycle •viral dsRNA triggers cellular interferon response -binds to TLR-3in endosomes or to RIG-I/MDA-5in cytosol, triggers Interferon and production
How does Influenza A produce mRNAs that are recognized by the host cell translation machinery?
viral transcriptional machinery can not make mRNAs on its own -can't cap its own mRNAs •uses cellular mRNAsas primers to initiate viral mRNA synthesis -process is known as "cap stealing" •PB2-recognizes 5'capon all cellular pre-mRNAs(in the nucleus -before splicing and polyadenylation and transport to the cytoplasm as mature mRNA) •PB1-has exonucleaseactivity (in addition to its polymerase activity!!) -cleaves the cellular pre-mRNA just 3'of an A residue •the 3'A on the cleaved cellular pre-mRNA base pairs with a conserved U at the 3'end of the viral genome RNA •PB1 uses capped fragment as primerfor copying viral genome - starts elongation
How does Influenza A enter the cell? (be able to describe the process of attachment and fusion and what proteins mediate it)
• HA1 binds to sialic acid residues on glycoproteins/glycolipids of many cell types Triggers receptor-mediated endocytosis • Influenza virions enter cell in endosomal vesicle • drop in pH (from 7 to 5.5) - conformational change in HA trimer • hydrophobic N-terminal portion of HA2 (fusion peptide) inserts into cell membrane • further conformational change brings N and C termini of HA2 towards each other (folds up like jackknife) • brings viral and cell membranes close together • outermost bilayer fuses first, inner bilayer fuses to complete fusion
What are the 2 groups that negative-sense RNA viruses can be divided into?
• can be divided into viruses with monopartite genomes (not segmented) and viruses with multipartite genomes (segmented)
