Virology Exam 3

How are complex retroviruses different from simple retroviruses?

Complex retroviruses encode regulatory (ex. tat and rev) and accessory genes (ex. vif, vpr, vpu, nef) while simple retroviruses do not

What is the major purpose of SV40 early proteins? What is the major purpose of late proteins?

Early - induce cells to enter S phase of cell cycle and to increase availability of components that it needs for viral DNA replication Late - capsid proteins

What is the role of the viral ion channel in influenza replication?

Facilitates the uncoating/penetration of the virus into the cell, causes conformational change. If you block M2, the pH won't be lowered in the vesicle for entry --> conformational change cannot occur --> M1 won't come off --> NLS won't be exposed --> RNPs can't get into the nucleus

Why does SV40 need to replicate in the nucleus? Give two reasons.

1) access to cellular DNA polymerases and other components, such as DNA ligase, gyrases, and topoisomerases, to replicate its genome 2) host RNA pol II and associated enzymes to transcribe, cap, and polyadenylate its mRNA 3) the cellular splicing machinery to make all viral mRNAs 4) cellular histones found in the nucleus to package its genome

Name at least two differences between the transcription of rhabdoviruses and orthomyxoviruses

1) rhabdovirus transcription occurs from a single negative sense RNA, whereas orthomyxovirus transcription occurs from each one of its 7 or 8 segments 2) rhabdovirus transcription occurs by the "start-stop" mechanism, leading to a gradient of transcription from the 3' end to the 5' end of the viral genome template, whereas orthomyxoviruses do not use "start-stop" transcription 3) orthomyxoviruses make capped mRNA by stealing the caps from newly synthesized cellular RNAs, whereas rhabdoviruses have an RNA-dep RNA poly with capping activity 4) some orthomyxovirus transcripts are spliced during transcription in the nucleus, whereas rhabdovirus transcription occurs in the cytosol, where they cannot be spliced.

What is an advantage of a segmented viral genome?

Ability to generate recombinants after co-infection of a host cell, would provide a source of genetic diversity under selective conditions, each segment can be transcribed as a monocistronic RNA

Why are retroviruses and orthomyxoviruses sensitive to the presence of Actinomycin D?

Actinomycin D prevents any new CELL mRNA from being made, given that orthomyxo and retroviruses need cell mRNA in order for transcription and furthermore the making of all its needed viral protein, actinomycin D prevents them from doing this. It inhibits DNA dependent RNA synthesis, so it affects the host.

The 1918 pandemic strain of influenza had mutations in the NA, HA, and PB2 genes. Explain how each of these genes could contribute to pathogenicity of the virus.

HA - anti-receptor, without the anti-receptor, the virus particles will never enter the cell for infection PB2 protein - part of RNA-dep RNA polymerase, without which, the virus cannot replicate NA - ???

What happens to the infectivity of picornavirus RNA when you remove VPg? Give your reasoning.

Removal of the VPg will not affect the infectivity of picornavirus RNA. The viral RNA binds to the ribosomes through an IRES, whereas VPg acts at later stage in viral life cycle (primes RNA synthesis).

What is the function of ribonuclease H in retrovirus replication?

-degrades RNA from the RNA/DNA hybrid during DNA synthesis using reverse transcriptase -does this 3 times during replication: 1) after synthesis of minus-strand strong STOP DNA 2) after synthesis of minus-strand genome DNA 3) after synthesis of plus-strand strong STOP DNA

Influenzaviruses have helical capsids. What advantages are shared between helical and icosahedral capsids?

-extremely stable (multiple protein interactions/subunit) -composed of repeating subunits that require small amount of genetic coding capacity -can self-assemble with little external input of energy

Explain two ways in which some animal viruses can be transmitted, but HIV cannot

-mosquitoes -coughing or sneezing

Name at least three functions for the influenza viral polymerase

1. RNA synthesis 2. Cap-stealing 3. Stutters at U's at the end to make a polyA tail 4. exonucleause activity (endo??)

Describe briefly one mechanism that the innate immune system may use to detect the presence of the UT2 virus or its associated DI particles.

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SV40 encodes at least two early proteins. What is an early protein? Expression of SV40 large T antigen leads to the onset of cellular DNA synthesis. Why is stimulation of cellular DNA synthesis and advantage for SV40?

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The c-Src and v-Src proteins are approximately 60,000 daltons in molecular mass. What is the functional difference between these two proteins?

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The retroviruses have a complex scheme of replication. During integration, 2 bp are lost from the ends of the linear provirus. Why are these sequences retained in the newly replicated genome?

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Why do avian sarcoma viruses transform every cell that they infect whereas avian leukosis viruses do not?

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What is HAART therapy? Briefly explain the rationale for this therapy.

A cocktail of anti-HIV drugs in order to suppress the disease. This drug cocktail contains several anti-RT or proteases that keep replication of HIV under control. Not necessarily a treatment, more about managing the symptoms.

Which virus families have genomes that also act as mRNAs? Explain brieftly whether or not these genomes are infectious.

Any (+) sense genomic RNA have genomes that can act as mRNAs, such as Picornaviridae, Togaviridae, and Flaviviridae. These genomes are infectious because they can enter the cell and immediately be translated into all the proteins the virus needs to replicate.

We have discussed several virus families that express mRNAs that lack m7G-containing caps. How can these mRNAs be recognized and translated by the ribosome?

Can use an IRES to be recognized and translated by the ribosome. Example: Picornaviridae

You isolate temperature-sensitive mutants of V2013 and subject them to complementation assays. Briefly explain what happens inside virus-infected cells when complementation occurs.

Complementation occurs when two viruses infect the same cell, and the titer is two or more times greater than when each virus infects alone under the same condition. The viruses are able to grow better together because they are able to provide gene products (for which one of the viruses is defective) by diffusion inside the cell. Together, the two viruses provide a complete set of wild-type gene products.

How is complementation related to DI particles?

DI particles are defective in one or more gene products. To produce new virus particles, the DIs must co-infect a cell containing a wild-type virus that can provide a complete set of diffusible viral gene products needed for DI replication. This process is a type of one-way complementation, where the DI benefits from the presence of wild-type virus, but the wild-type virus does not benefit from the co-infection.

What sequences are duplicated in retroviral DNA (the provirus) compared to retroviral RNA? Why is the sequence duplication necessary for the expression of the provirus?

DNA - LTR (long terminal repeat) RNA - R (direct repeat) These sequences are needed for integration into the host DNA.

What is the first step of retroviral replication after uncoating?

DNA synthesis using reverse transcription.

Why do HIV patients often develop cancers and many other viral and bacterial infections?

HIV depletes the number of CD4 cells in the body, and CD4 cells are needed in order for our body to defend itself. Since their numbers decrease, opportunistic infections occur since there's no methods of defense.

Name at least two features that distinguish HIV-1 from other replciation-competent retroviruses, such as the avian sarcoma viruse (ASV).

It is able to infiltrate the nucleus with the help of an integron at any time, the cell doesn't need to be undergoing replication in order for the nuclear envelope to degrade in order to attack. pH independent entry, it uses the CD-4 receptor found in helper T-cells.

Why do certain viruses shut down host macromolecular synthesis?

Mammalian viruses often inhibit host translation, transcription, and DNA replication - allows easier access of viral mRNAs to the ribosomes and also prevents the synthesis of cellular proteins, such as interferon, Toll-like receptors, major histocompatibility complex proteins that serve in both innate and adaptive immunity.

What step of viral replication is blocked by retroviral protease inhibitors?

Maturation (???)

The orthomyxoviruses and rhabdoviruses need continuous protein synthesis during their replication. Why?

Need protein synthesis of their nucleocapsid protein to encapsidate the newly replicated RNAs.

Is naked rhabdovirus RNA (no associated proteins) infectious? Why or why not?

No, it is negative sense or complementary to viral mRNA. Without RNA-dep RNA poly, the mRNA can't be transcribed.

Retroviruses and orthomyxoviruses both have presented obstacles for the preparation of single vaccines. Why?

Orthomyxoviruses have segmented genomes that can reassort or cause changes in their proteins (via drift/shift). Retroviruses use RT, which is highly error prone, so mutates very often.

Name three virus families that encode polyproteins.

Picornaviridae, Flaviviridae, and Togaviridae.

In addition to retroviridae, we have discussed another virus family that encodes an oncogene. Name the family and the oncogene.

Polyomaviridae oncogene: large T Ag

Would you expect the simian virus 40 (SV40) (a member of the Polyomaviridae family) would induce tumors rapidly (in weeks) or slowly (a period of months)? Give your rationale.

Rapid, because this virus encodes the oncogene T antigen (a transforming protein). Therefore, any semi-permissive or non-permissive cell that expresses T antigen (and cannot make virions) will be transformed. SV40 is more similar to the retrovirus ASV, which encodes v-Src, and induces tumors rapidly. ALV, which lacks an encoded oncogene, induces tumors over a longer period of time because of relatively random integration.

Describe at least one other way besides ribosomal frameshifting that levels of viral proteins within a polyprotein can be controlled.

Read-through translation - togaviruses have a stop codon after nsp3 that sometimes causes the ribosome to stop, or sometimes it goes on. It makes nsp123 or nsp1234

Name at least three ways that HIV-1 is transmitted

Receiving blood from an infected person, being in contact with infected sexual fluids, from mother to child during pregnancy or delivery, by baby drinking infected mother's milk

Viruses that infect mammalian cells use both pH-dependent and pH-independent entry. Why are some viruses pH-dependent for entry?

Require low pH environment of the endosomes to allow conformational changes in one or more viral proteins needed for entry/fusion to the cytosol and uncoating. Often these conformational changes are in the viral anti-receptor.

Explain briefly a second shared feature of the replication phase of orthomyxoviruses and rhabdoviruses.

Rhabdovirus replication doesn't happen in nuclues, acquire their poly A tail by having the polymerase stutter at U residues. *ADD MORE*

Does SV40 virus package a polymerase? How is packaging of a polymerase related to the infectivity of SV40 DNA?

SV40 does not package its own polymerase. Early in the transcription they are transcribed by the cellular DNA dep RNA pol (RNA pol II)

The UT2 virus makes DI particles at high multiplicities of infection. These particles show cyclic production. There is a high production of DI particles for a few passages of the virus, and then low amounts of these particles are observed for the subsequent passages, and then the cycle repeats on subsequent passages of the virus. What is the reason for this observation?

The DI particles can out replicate the WT virus, so this leads to high production of DI particles. But because the DI particles rely on the WT virus for complementarity, the DI particles will die off to low amounts without the gene products from the WT. Then because the DI particles die off, the WT can increase again, providing more gene production for the DI particles to use, starting the cycle again.

HIV can use several different co-receptors. A change in co-receptor usage if often seen prior to development of AIDS. What is the relationship to disease onset?

The change in co-receptor usage is associated with a change from tropism and infection of macrophages to tropism/infection of helper (CD4+) T cells. Infection of helper T cells then leads to cell lysis and an altered immune response to multiple intracellular pathogens, including protozoa, bacteria, and other viruses.

You isolate a new virus from a patient with fever and a rash, and you decide to call it UT2. You titer this virus both before and after treatment with a non-ionic detergent. Why did you do this? Pt2: The titer does not change after treatment with detergent. What does this tell you?

To tell if the virus has an envelope. Pt2: The virus is not enveloped.

What virus families have we studies that makes multiple unspliced mRNAs from a non-segmented genome?

Togaviruses, rhabdoviruses

Give an example of a virally-encoded protease and a cellular protease that are involved in cleavage of viral polyproteins.

Virally encoded = NS2B/NS3A (in Flavi) and nsp2 (in Toga). Cellular proteases = furin and signal peptidase.

How can an animal virus make different amounts of some of the proteins encoded within a polyprotein? Give a specific example?

With the retroviruses, the ribosomes are able to do frame shifts as mRNA is being translated, it has certain shift codons that allow the ribosome to switch back and forth thus making different amounts of proteins from the same polyprotein. Togaviridae has multiple stop codons that are used to produce different amounts of nsp4, 3, 2, 1 (read through translation).

What will happen if you don't degrade the RNA during RT?

Won't generate ends of the.. and won't generate ends necessary for duplication and won't generate primers for the plus strand.. ppt preserved for... necessary for plus strand synthesis

What is the molecular difference in the activity of c-Src and v-Src?

c-Src has a regulatory tyrosine while v-Src does not, this allows v-Src to have kinase activity on all the time while c-Src does not