Virology Exam 2

What is the function of DBP protein during Adenovirus replication?

Adenovirus ssDNA binding protein (DBP) coats the displaced strands of the template DNA molecule. Essential for initiation and elongation, stimulates the activity of the viral DNA pol 100x. multimerizes on DNA template via high-affinity binding.

How do Adenoviruses replicate?

Adenoviruses use protein priming. They have Ori at both ends. The 5' ends are covalently linked to pTP protein (terminal protein) that serves as the primer for viral DNA synthesis. Strand displacement synthesis. Semiconservative DNA replication. Primer: polymerase is bound to pTP which links to the 3' OH through the serine residue of pTP. This starts the synthesis, displacing the top strand. the 3' OH group of the pTP primes synthesis of daughter viral DNA strands. Once the few nucleotides have been incorporated, the DNA pol dissociates from pre-TP to elongate the daughter strand. No end problem, because pTp protein bound to each end. dsDNA at the end with pol attached to it, so no need for primer! Adenovirus ssDNA binding protein (DBP) coats the displaced strands of the template DNA molecule. Essential for initiation and elongation, stimulates the activity of the viral DNA pol 100x. multimerizes on DNA template via high-affinity binding.

How do Parvoviruses replicate?

First the cellular enzymes "fix" the ssDNA. A nick is made on the other strand by exonuclease (Rep 78/68, viral protein), to expose new 3' OH group which serves as a primer for another strand. Hairpins (T-shaped) are formed at both strands. New strand is being synthesized and it displaces the top, previously synthesized strand. Continuous synthesis by strand displacement, genome primes itself, no end problems, no need for primers. this is all because of the palindromic sequences. CONTINUOUS SYNTHESIS BY STRAND DISPLACEMENT AND NO NEED FOR A PRIMER.

o What is a common test being used in the HIV epidemic?

o Antibody test- 3-12 weeks after infection. o Antigen/antibody test- lab test. Takes longer. o Nucleic Acid Test (NAT)- detects the viral load. Very expensive. Quickest test. o Can also do a CD4 T-cell count.

· Where does the transcription start?

o At a specific promoter sequence.

· What does it mean when someone is susceptible to an EBV infection? Primary infection? Past infection?

o Susceptible to infection- do not have EBV, have never had it, don't have antibodies. No anti VCA and no EBNA o Primary infection- anti VCA but no EBNA. o Past infection- anti VCA and EBNA.

· What are endogenous retroviruses and what is their function in human genome?

· Endogenous retroviruses are when a retrovirus gains access to the GERMLINE, passing on to the offspring. Passed through the germline.

What are the four main drivers of viral evolution?

· Large number of progeny, large number of mutants, quasi-species effects, and selection.

· What is spliceosome?

· Pre-mRNA:protein complex. RNA is the catalyst in the spliceosome.

· What is juxtaposition?

· eIR4G has all the domains that bind these specific things and has Pabp binding site. This binds poly(A) binding protein that binds to the poly A tail. This loops the 5' and 3' UTR. This looping process is referred to as juxtaposition. This happens when there is a cap structure...

Which direction is DNA replicates?

ALWAYS the 5'-3' direction

Why ORI sites are AT rich? Are they close to transcription promoters? Do they bind specific viral proteins? Why?

Because AT bonds are less strong and easier to break apart easier because they have less hydrogen bonds. They are located near transcriptional promoters. Assembly points for many proteins, and some viruses have more than 1 Ori site. They bind origin recognition proteins. For SV40, the recognition protein is LT. for parvovirus it is Rep68/78 which nicks the genome to make a 3' OH. For adenovirus, it is pTP. For herpesvirus it is UL9 protein that recruits all the viral proteins to the origin site to bind.

Why do viruses replicate in actively dividing cells?

Because only during activation do they have enough dNTPs for replication and all the proteins they need for replication.

o True/False: HIV can be transmitted through kissing?

False

· Does Hepadnaviruses DNA integrate with the host genome?

NO

· How is viral DNA organized inside the cell?

Nucleosomes?

· Does transcription require primer?

o No. DdRp can carry out de novo synthesis which can lead to abortive initiation (short sequences).

· What kind of virus is WNV is the Baltimore system?

o Positive ssRNA

· What membranes can assist viral budding?

· Any membrane.

· How is budding influencing Dengue virus maturation?

· As the Dengue virus particle goes through the secretory pathway... ER to Golgi, the pH changes and affects the conformation of the glycoprotein. · Furin expressed by the cell in the Golgi cleaves off a protein that allows the glycoproteins to be functional.

· What is the side-effect on genetic bottleneck?

· By restricting population growth to serial single founders (the bottleneck) not many mutations accumulate, which decreases the fitness of the population.

· What is adaptive evolution?

· Evolutionary changes in an organism that makes it suitable to its habitat.

· Does diffusion take place during virus assembly and movement within the host cell?

· No, the cell is really crowded so viruses attach to motor proteins. Not a random movement, much faster than diffusion!

· What is nuclear localization signal

· Nuclear proteins go to the nucleus

What are the two main mechanisms of dsDNA synthesis?

-Replication fork- copying of both DNA strands: leading and lagging strands. Always initiated from RNA primer. Associated with 5' end problem. -Strand displacement- unique to viruses. Copying of only one DNA strand while its complement is displaced. Never RNA primed, but instead primed by DNA structure or a protein.

· What is the function of adenoviral E1A protein?

-binds Rb which stimulates transcription- leading to oncogenesis. Rb releases E2f which initiates transcription.

How do herpesviruses replicate?

2 OriS and 1 unique OriL sequence- total of 3 Ori. Has 3 Ori because it is a larger genome and would take too long with one. Enters the cell as a linear molecule but becomes circularization- allowing it to replicate as a "rolling circle." Larger genome= more proteins in the genome! Host proteins responsible for circularization- DNA ligase Rolling circle replication: initiated by introduction of a nick that creates a 3' OH end in one strand of a ds circular DNA. One strand is copied discontinuously. This mechanism produces head-to-tail concatemers- a long continuous DNA molecule that contains multiple copies of the same DNA sequence linked in series. Concatemers are subsequently cleaved into monomeric units. Like toilet paper. NO 5' END PROBLEM.

What biosynthetic/biochemical mechanism is used by all DNA polymerases?

2 metal catalysis mechanism. 2 Asp coordinate Mg metals, nucleophilic attack on the alpha-phosphate

What does it mean that SV40 replication is semi-discontinuous and bidirectional?

2 replication forks in the replication bubble. Not continuous strand and continuous strand.

How is large T antigen regulating/disregulating cell cycle?

Binding of LT is within the promoter. Has a helicase domain that unwinds the domain, and an origin DNA binding domain. Rb domain binds Rb- keeps transcription at base so it doesn't go crazy. P53 also regulates cell cycle. Binds to very important regulators of the cell cycle! Can manipulate these regulators and the cell cycle. Manipulate our cell cycle because most cells do not divide or synthesize, so viruses cannot use the stuff inside the cell. So the viruses stimulate replication because only then will they have enough stuff for DNA replication. Sequesters Rb and p53 to stimulate replication.

What does it mean that the replication is semiconservative?

Both parent strands of a duplex DNA template are copied by base pairing to produce two daughter molecules identical to one another and to their parent.

IS poxvirus replication continuous or discontinuous?

Continuous

o Where does HIV undergo reverse transcription?

Cytoplasm

What enzyme guides viral DNA replication?

DNA-dependent DNA polymerase (DdDp) + accessory proteins.

What does it mean that cells are quiescent?

Do not divide

What is the function of large T antigen in SV40 replication?

It binds to the Ori site and unwinds it. It is like a donut that fits perfectly into the DNA and creates a replication bubble when it unwinds it. has 3'-5' helicase activity.

How is the 5' end problem solved by SV40?

No 5' end problem because it is a circle!

Are there viruses that code for entire replication machinery?

No, cannot replicate without host cells. At least 1 viral protein is needed before replication, so when viruses enter the cell- ECLIPSE period! Complex viruses do have more replication proteins. Large DNA viruses encode most of their own replication systems, but still they don't encode it all!

What is the function of inverted terminal repeats in Parvoviruses?

Parvovirus is a ssDNA genome. Inverted terminal repeats (TR) are fully complementary palindromic hairpins that allow free 3'OH- so it primes itself! TRs base pair to form T-shape structures- primer for DNA synthesis. The 3' end of the T-shape structure serves as a primer.

What is the 5'-end problem and how is it solved by viruses? Humans?

Pieces where RNA from primer is removed and ligase glues internal pieces together. The discontinuous mechanism of DNA synthesis creates a problem at the ends of linear DNAs, where excision of the terminal primer creates a gap at the 5' end of the daughter strand. In chromosomal DNA, telomeres at the ends of each chromosome prevent loss of terminal sequences (simple repeated sequences maintained by reverse transcription of an RNA template). Viruses deal with this problem in other ways (ex- circular genomes have no ends so not a problem).

What type of polymerases guide lagging strand synthesis? What is their function?

Poly ⍺- primase binds at the Ori and synthesizes RNA primers on the leading strand. Possessive DNA pol 𝜹 is recruited and produces a lagging strand segment. Multiple DNA fragments produced during discontinuous synthesis are sealed by: - Removal of the RNA primers by RNase H and the 5'-3' exonuclease (Fen1) - DNA pol 𝜹 repairs resulting gaps - DNA ligase I joins the DNA fragments

What type of polymerases guide leading strand synthesis? What is their function?

Poly ⍺- primase binds at the Ori and synthesizes RNA primers on the leading strand. Replicative polymerase 𝝴 then binds to Pcna/Rf-C complex and is loaded at the replication forks for continuous DNA synthesis.

What viral proteins bind Rb protein and why?

Rb- protein that checks where the cell is and makes sure everything is in order. Tumor gene suppressor. Checkpoint. If you lose this checkpoint, there will be proliferation that leads to tumor development. Rb interacts with 2 adenoviral proteins- E1A and E7. E1A regulates transcription and is bound to E2f in G1, sequestering E1A and stopping transcription. But... when adenovirus replicates, E1A outcompetes E2f, binds Rb protein, and stimulates transcription, translation, and viral replication.

What is replicon? What is Ori? What are Okazaki fragments?

Replicons- place where replication occur. Replication bubble. Ori site- origin at which replication begins. Each replicon has a ori. Okazaki fragments: synthesis of the lagging strand. Short segments synthesized via discontinuous mechanism of synthesis.

· What is the first biochemical reaction that occurs in cells infected by dsDNA viruses?

Transcription

What is a function of topoisomerase?

Type I topoisomerases- break one of the strands and pass the other strand through the gap, followed by resealing the nick. This introduces negative supercoils. Breaks ONE strand. Type II topoisomerases- break both strands and pass another duplex region through the gap, which generally relaxes DNA by removing negative supercoiling. After replication. Breaks BOTH strands.

· Does poliovirus enter the nucleus?

Yes

Where do poxviruses replicate?

cytoplasm. only virus we learn about that does not replicate in the nucleus. It is too large to go to the nucleus!

· What type of viral DNA genome can be immediately transcribed?

dsDNA only!

What are concatemers?

head-to-tail concatemers- a long continuous DNA molecule that contains multiple copies of the same DNA sequence linked in series. Concatemers are subsequently cleaved into monomeric units.

How was splicing discovered?

in viruses. virologists notices adenoviral DNA is longer than its mRNA. extracted mRNA. introns were looped out.

· What are early and late genes?

mostly DNA viruses do this o Early genes are transcribed first, mRNAs coding for enzymes, replication factors, regulatory proteins. Needed at the beginning, support replication. o Late genes are transcribed later, as they are not needed immediately- mRNA coding for structural proteins ex: capsid. Structural proteins!

Do viruses have telomeres?

no

· Where does the transcription occur?

nucleus

· What type of genome makes up poliovirus?

o (+) ssRNA genome

o What is the genomic structure of RSV?

o (-) sense ssRNA

· What type of genome does MuV have?

o (-) sense ssRNA genome, enveloped

o Where was RSV first isolated from human subjects?

o 1957- john Hopkins med school.

o How long after infection of RSV does it take for symptoms to present?

o 4-6 days after exposure.

· What are transcriptional activators and repressors? What is their function?

o Activators- bind to the DNA promoter and facilitate transcription. Can bind from local or distant sequences. Discreet functional domains can be distinguished in all transcriptional activators: 1. DNA binding domain 2. Dimer formation domain- these activators bind to DNA as dimers 3. Activation domain o Repressors- bind to DNA sequence and halt transcription. Less common. Repressor competes with activator for binding. Repressor binds DNA and inhibits transcription by protein-protein interactions. Either don't allow activator to bind or block the assembly of activation domain.

· Provide an example of cascade regulation during viral transcription.

o Adenovirus transcription regulation- cascade. Three phases of infection: immediate early (IE), early (E), and late (L). o E1A is the first protein synthesized after virus entry, binds to Rb and releases E2f (factor necessary for global transcription). o Rb is normally bound to E2f and that complex inhibits transcription- stimulates histone deacetylase. E1A binds to Rb, so E2f is free and binds transcription promoter so transcription happens. o First thing adenovirus does after infection is it produces E1A protein that binds Rb and stimulates transcription (leading to oncogenesis). -E1A stimulates E2 which stimulates IVa2. cascade event

· What is TAR and Tat? And how do they guide HIV transcription? Where does that process occur?

o Before TAT is present, a small number of RNA transcripts is made, which allows the Tat protein to be produced. Tat then binds to TAR (apical loop and budge) and mediates phosphorylation of cellular factors, resulting in increased transcription of all HIV genes. o TAR without TAT- short transcripts. Not efficient. o Tat comes and associate with polymerase.

· Why is there a WNV vaccine for horse, but not humans?

o Cannot use a vaccine to prevent WNV for us. The USDA has said they need a vaccine and the FDA has not yet. The horses have more severe cases.

o How does HIV kill people?

o Causing AIDS- which attacks your immune system. Death within 3 years. Does not directly kill you, works through breaking down the human immune system.

o What happens in the second stage of HIV symptoms?

o Chronic HIV infection. Asymptomatic HIV infection or clinical latency. Lasts about 10 years. Can still transmit the virus.

· What is the arrangement of DNA genome in SV40?

o Circular dsDNA genome with Ori as the origin of replication. o Has 2 phases of transcription... Early (counterclockwise) and late (clockwise)

· What sequences regulate transcription?

o Core promoter- the starting point of mRNA synthesis, includes TATA sequences and initiation sequence (+1). o Termination site- the point on the DNA template at which the synthesis of mRNA stops. o Distant regulatory sequences- enhancers/silencers far from the core promoter. o Local regulatory sequences- enhancers/silencers close to the core promoter.

· To what genera do the mosquito vectors for WNV belong, and how many species live in the United States?

o Culex spp are vectors for WNV- 3 common to the US (pipiens, tarsalis, quinquefasciatus).

· What two cellular receptors with which WNV can interact?

o DC-SIGN on dendritic cells. o DC-SIGNR on endothelial cells in the placenta, liver, and lymph nodes.

· What is the main enzyme catalyzing viral transcription?

o DNA-dependent RNA polymerase II is the main one. Cellular or viral.

· Where is the main enzyme catalyzing viral transcription coming from? Virus? Cell?

o DdRp II is cellular or viral. Viruses that spend their entire life cycle in the cytoplasm do not have access to host polymerases, which are located in the nucleus. These viruses need to encode their own DdRp for transcription and replication (big viruses!). in contrast, some small DNA viruses that spend their life cycle in the nucleus do NOT need to encode their own polymerase, and instead rely entirely on host DdRp for genome replication and mRNA synthesis.

· What happens during SV40 early and late phases of infection?

o EARLY PHASE OF INFECTION: late promoter is inactive, because there is a steady concentration of Ibp vs low concentration of viral DNA. Ibp binds all promoter sites of viral DNA resulting in total inhibition of late transcription. oLATE PHASE OF INFECTION: steady concentration of Ibp, but much higher concentration of viral DNA (stimulation by T antigen), not all sites are occupied by Ibp, so late transcription commences.

· Which gene distinguishes the difference between the two types of EBV?

o EBNA-2 gene. EBV 1 transforms B lymphocytes more.

· What is a function of large T antigen during SV40 transcription?

o First protein transcribed and translated. Large T antigen binds near Ori and blocks early mRNA transcription, supporting late transcription. Large T antigen binds to Enhancer region and turns-on late promoter. Looks like a ring, can unwind DNA (helicase activity), binds and degrades/sequesters cellular tumor suppressors- promotes S-phase entry. Binds viral replication origin, regulatory elements and cellular replication machinery- perpetuates synthesis of large number of progeny and induces cell lysis. o Stimulates replication and late transcription

o Where is HIV believed to originate from?

o From a type of chimpanzee in Central Africa. From the Simian Immunodeficiency Virus. They are eating meat from Chimpanzee (Africa).

· What is the function of Ibp protein during SV40 transcription?

o Ibp- initiator binding protein. cellular protin present on a steady level. Stimulates early transcription and stops late transcription by binding to DNA.

· Who discovered the etiology of MuV and how?

o Johnson and Goodpasture - infected monkeys with human sample and the virus was able to replicate

· What replicates EBV during the lytic cycle? Latent cycle?

o Lytic replication: production of infectious virions. Only happens in B cells after reactivation from latency. Epithelial cells go into lytic replication after viral replication. Produces immediate early, early and late gene products. o Latent cycle: persistent viral infection without active viral production. Mostly in memory B-cell compartments. Host DNA polymerase replicates episomal replication. Not integrated into the host DNA, just closely associated. Three different latency programs. Goes through latency I, latency II and latency III.

· What is epigenetic regulation?

o Methyl groups are + and DNA is negative so histone methylases tightenes the DNA. Demethylation loosens. Acetyl groups are negative so they unwind when added. o Gene switched ON: unmethylation, acetylation o Gene switched OFF: methylation, deacetylation

· Which cell receptor does MuV bind to? Which MuV protein mediates this?

o MuV binds to sialic acid via HN (hemagglutinin-neuraminidase).

· What is positive, negative and cascade regulation?

o Positive autoregulation- viral protein can stimulate transcription of the same transcription unit from which it came. Product of transcription stimulates transcription. o Negative: product of transcription stops further transcription. oCascade regulation: viral protein can stimulate transcription of the different transcription units (gene y), stepwise regulation. Herpes. Best way to regulate timing.

o Who are the "at risk" populations to severe RSV infections?

o Premature infants, young children/adults with compromised/weakened immune systems or lung/heart issues.

· What is a brief overview of EBV viral cycle?

o Primary infection in tonsil by exposure to infected saliva. Virus enters cell and alters B cells. Causes activation of B cells, beginning of latency. Full latency in resting B cells. Then reactivation of viral shedding.

· How is poliovirus transcribed?

o RdRp hijacks transcription machinery, makes RdRp. RdRp translates (+) to (-) then (+) again. o VPg is cleaved. RdRp binds to Cre, then VPg binds to Cre. Adenosine on the loop of Cre is the template for RdRp to add to uridine nucleotides. o Poly A binding protein bound to the 3' group creates circulated genome. PCbp bound to the membrane via a 3AB receptor.

· Why was the presence of dead birds a valuable clue in diagnosis WNV?

o Returned from tropical forest (rash around mosquito bite), death of local birds (can have vectors of human or birds.

· What makes up the virion of EBV?

o Single linear molecule of dsDNA o Capsid- icosahedral capsid o Tegument- space between capsid and envelope o Envelope- altered host membrane and viral glycoproteins

· Why does a gradient of mRNA abundance occur during transcription?

o Start-stop transcription regulated by cis-acting elements. Lot of copies of genes closer to 3' end than 5' because RdRp has to reinitiate at the LE promoter when it dissociates.

o What is the cytopathic effect of RSV?

o Syncytium. Cells fuse together.

· Why timing of viral transcription is important?

o The expression of viral genes is strictly coordinated by transcriptional regulation: sequence and proteins... timing: o Early genes are transcribed first, mRNAs coding for enzymes, replication factors, regulatory proteins. Needed at the beginning, support replication. o Late genes are transcribed later, as they are not needed immediately- mRNA coding for structural proteins ex: capsid. Structural proteins!

· Explain the Rule of 6.

o The number of nucleotides is always a number of six because these viruses are dependent on nucleoprotein molecules that each bind to 6 nucleotides. o All paramyxovirus.

· What is VPg and why is it important?

o Transcription is dependent on VPg. Pg is cleaved by cellular enzymes, then RdRp binds to Cre, then Vpg binds to Cre. RdRp adds 2 U nucleotides to the end of VPg which makes it the primer for RNA synthesis!! o VPg= primer for RNA synthesis.

· How is transcription regulate in retroviruses?

o Transcription occurs after retroviral genome reverse transcription. o In HIV, transcription is regulated by the first transcript. TAR (5' UTR that forms a stable stem loop structure. Releases short RNA). TAT protein goes back to the nucleus and stimulates efficient transcription. o Before TAT is present, a small number of RNA transcripts is made, which allows the Tat protein to be produced. Tat then binds to TAR (apical loop and budge) and mediates phosphorylation of cellular factors, resulting in increased transcription of all HIV genes. o TAR without TAT- short transcripts. Not efficient. o Tat comes and associate with polymerase.

How is poliovirus translated?

o Type I IRES with domain structures 5 and 6 where the 40S subunit binds to the genome. The VPg protein is removed, then eIF4G binds 40S and stabilizes it. Cleaves it, does not need eIF4G. Proteolytically processed. One large polypeptide cleaved by many proteases

· What modifications occur to VPg?

o Uridylation.

· What is VP16 protein?

o Viral regulatory factor of herpesvirus. Already present in the viral particle. Associates with immediate early promoters and recruits cellular demethylases to remove heterochromatic marks along with histone acetyltransferase that promotes hyperacetylation of the viral chromatin. Tegument protein (part of the particle). o Transcription is initiated by VP16, a virion associated protein. Has no sequence-specific DNA binding site. Instead it has acidic activation domain (lots of negative charges) that stimulates transcription of promoters. VP16 activity is also regulated by numerous cellular proteins.

· What is the DNA looping model? What is its function in transcription?

oThe DNA looping model postulates that proteins bound to a distant enhancer interact directly with the transcription initiation complex looping out the DNA template. Such interactions stabilize the initiation complex. formed by transcription factors

· What is a phylogenetic tree and what does it measure?

· Measures the genetic distance between organisms and identifies the nearest relatives. Each division in a tree is a node (blue dots). The "root," at the extreme left, is the assumed common ancestor of all organisms in the tree.

· What is lariat?

· The 2' hydroxyl group of the A residue in the intronic branch point makes a nucleophilic attack on the phosphodiester bond at the 5' side of the GU dinucleotide at the 5' splice site to produce the intron-3' exon lariat and the 5' exon (circular RNA).

· What molecules are used to prime (-) sense and (+) sense DNA synthesis during RT?

· (-) sense strand uses tRNA as the primer. This is the (-) strand because it copies the mRNA (+) strand). The first piece of DNA that is synthesized is short because RT reaches the 5' end of the mRNA. RNase H domain of RT degrades the RNA part of the RNA:DNA hybrid molecule. The short DNA product is called the minus-strand strong-stop DNA. It has a R sequence complementary to the R sequence on (+) strand RNA. · Then first strand transfer- changes the template and (-) DNA strand is extended and cleaves off all the RNA except for PPT. · Then PPT is used as a primer for the (+) strand. At the end, tRNA is cleaved by RNase H. template switch.

· How many subunits build reverse transcriptase?

· 2 subunits- p66 and p51 · 3 domains... DNA polymerization domain (synthesis), DNA unwinding domain (helicase), and the hydrolysis of RNA-DNA hybrids (RNase H activity).

· How many template exchanges occur during HIV RT?

· 2. · One template exchange when the R on the minus strand strong stop DNA is complementary to the r on the (+) sense RNA. · Another template exchange mediated by PBS:PBS complementarity. The PBS region of the (+) strand is base paired to its complement on the (-) strand.

· How is envelope acquired by viruses? What proteins assist that acquisition in the case of Influenza? HIV?

· 4 ways that envelopes are acquired by viruses: · Type I budding: interactions among the envelope heterodimers and capsid drive budding · Type II budding: Gag-dependent budding, internal matrix or capsid proteins drive budding (retroviruses) · Type III budding: envelope protein driven (influenza:HA) · Type IV budding: driven by matrix proteins, its proper functioning requires additional components. · Influenza: type III budding. HA drives the budding, M1 brings in influenza virus genome, forms RNP complex and targets it to the membrane. · HIV/retroviruses: Gag alone can lead to the formation of viral particles. Myristylation (addition of fatty acid tail) to Gag is required for virus particle budding. If not myristoylated, Gag will not interact with the plasma membrane and virus assembly and budding are inhibited.

· What RNA structural elements regulate translation?

· 5' cap (most eukaryotic mRNAs have this too), UTRs · 5' cap- m7G (7-methlguanosine) joined to second nucleotide of mRNA by 5'-5' phosphodiester linkage. Protects from cleavage from cellular 5'-3' exonucleases. Base 1 and base 2 can be methylated at position 2. · 5' and 3' UTR. Surround an ORF that codes for a reading fram3 · 5' UTR often has complex RNA structures that act as a docking place for ribosome or regulate when translation happen. · 3' UTR has a poly A tail. Needed for efficient translation.

· Do all viral RNAs need to have 5' cap? What are the alternatives?

· 5' caps are present in almost all mRNA and some non-coding RNAs... EXCEPT picornaviruses and flaviviruses which have IRES structure.

· What is ADAR and what is its function?

· ADAR: Adenosine deaminase. A → I. changes coding info. Protein expressed with this has different amino acids. · A→I RNA editing of protein-coding sequences results in recoding and subsequent alterations of their functions. It enhances viral replication and inhibits dsRNA-activated protein kinase.

· What are viral subassemblies?

· ALL viruses go through subassemblies. Like quality control. Each subassembly must be created correctly before final structure is made. Assembled step by step so the final structure can be made correctly.

· What are accessory proteins?

· Accessory proteins orchestrate the host molecular machinery. Nonstructural proteins encoded by complex genomes.

· What are the four main strategies for making subassemblies?

· Assembly from folded protein monomers: the assembly reaction is the result of specific interactions among individual protein subunits that form larger structural units · Assembly from a polyprotein precursor: polyprotein contains 4 proteins and is processed by proteolytic cleavage. All at the same time, cleavage allows assembly into 1 subunit. · Viral chaperone assisted assembly: viral protein assists proper folding of hexon trimer from the monomer. Virus codes for chaperones. · Cellular chaperones assisted assembly: heat shock proteins (expressed in cells under stress). LT together with heat shock chaperone 70 assists correct foling and assembly of SV40.

· What are the main decay pathways for RNA degradation?

· Basal Decay Pathway: deadenylation (rate limiting), decapping, then exonucleolytic degradation (rapid) · QC Decay Pathway (NMD): translation linked endonucleolytic cleavage, exonucleolytic degradation (rapid) · Virus-Induced Decay Pathway: endonucleolytic cleavage of translatable mRNA, exonucleolytic degradation (rapid). MAIN: Deadenylation then decapping then exonucleases to degrade.

· How is Cap 0, different from Cap 1 and Cap 2?

· Cap 0- not methylated · Cap 1- 1st nucleotide is methylated at 2' position of the ribose · Cap 2- 2nd nucleotide is also methylated at the 2' position of the ribose.

· What is the first mechanism of RNA processing that occurs co-transcriptionally?

· Capping- the addition of m7GpppN to the 5' end, ensures efficient translation (supports circularization), protects RNA from exonucleases. G is linked to the first nucleotide by 5' 5' triphosphate linkage. G is methylated and the two bases may be methylated

· How are non-enveloped viruses are released from cells?

· Cell lysis- apoptosis or necroptosis. may code viroporins that form pores in the membrane. Loss of membrane integrity with inhibition of protein synthesis. · Can also be non-lytic release. Uses exocytosis.

· What molecular mimicry did we discuss today (3 examples)?

· Cellular mimicry- eRF mimics the structure of tRNA and stops translation. Recognition of stop codons. · Viral mimicry- starting protein without Met. IRES mimics the structure of tRNA, so initiation takes place at the A site on the ribosome. · Viral mimicry- The 3' end of viral RNA mimics tRNA in turnip yellow mosaic virus. 3' end folds and circularizes the DNA.

· What is central dogma and how do virus's reverse the flow of genetic information?

· Central dogma- DNA → RNA → Proteins · But retroviruses have RNA → DNA → RNA → protein. They reverse the flow of genetic information.

· How is sequential assembly different from concerted assembly?

· Concerted assembly- individual elements form all at the same time creating subassemblies, then they come together by association with the genomic nucleic acid. · Sequential assembly- occurs step by step.

· What are stress granules?

· Created by cells when under stress. Specks in the cytoplasm where translation complexes that are stalled accumulate.

· Describe the conformation of Hepadnaviruses DNA genome?

· DNA virus with RNA intermediate during replication. One full (-) strand and one incomplete (+) strand of DNA. The longer (-) stand is covalently linked to a protein. Direct repeats at the 5' ends facilitate template switch during reverse transcription.

· What mechanisms regulate 5' capping?

· De novo synthesis by cellular enzymes · Synthesis by viral enzymes · Acquisition of pre-formed 5' cap snatched from cellular pre-mRNA or mRNA (influenza)

· What translational factors regulate the process?

· Due to a specific sequence in between. Has a slippery site where the ribosome slips and a pseudoknot that slows down the ribosome, so it slips and studders. Frameshifting can be backwards (-1) or forwards (+1).

· What is ESCORT complex?

· Endosomal Sorting Complex. Cellular complex that supports export of proteins from a cell. Regular cellular process that is used by virus. Used during cellular division when cells are dividing. · Single amino acid change in the p6 domain of Gag can stop HIV from budding. Mutations in p6 stops a virus from being released and gets them stuck on the surface of the cell.

· What is the purpose of the 5'-5' linkage in the cap structure?

· Enzymes cannot cut a 5' 5' linkage, protecting it from exonucelytic attack. Also essential for the efficient translation, it is recognized by translation initiation complex, facilitates "circularization" of the genome. Makes the host think it's its own.

· What is internal initiation?

· Facilitated by IRES- Internal Ribosome Entry Site. Discovered by poliovirus. Very long 5' UTR (~750 nucleotides). Poliovirus. The UTR codes a huge mRNA that is cleaved into multiple proteins. Makes structure proteins and polymerases. There is no 5' cap, but there is a VPg protein.

· What is genetic bottleneck? Can you find examples of genetic bottleneck in the typical laboratory experiments? What about real life?

· Genetic bottleneck: viruses are less fit due to genetic bottleneck- extreme selective pressures on small populations that result in loss of diversity, accumulation of non=selected mutations, or both. · The bottleneck arises by restricting further viral replication to the progeny found in a single plaque. By restricting population growth to serial single founders (the bottleneck) not many mutations accumulate, which decreases the fitness of the population. · Bottlenecks in the real world- infection by a limited virus population and subsequent amplification are often found in nature. Small droplets of suspend virus during aerosol transmission. Activation of a latent virus from a limited population of cells. Small volume of inoculum introduced in infection by insect bites.

· What is genetic drift and shift?

· Genetic shift and genetic drift are evidence of the quasispecies effect. · Genetic Drift: diversity arising from copying errors and immune selection. May occur each time the genome replicates. Example: existing antigens, HA, are subtly altered. · Genetic Shift: diversity arising after recombination or reassortment. Relatively rare. Example: emerging new strains of Influenza virus.

· What is the packaging signal of HSV? HIV? Influenza?

· HSV: pac1 and pac2 (near regions that are cleavage sites). Pore complex has motor proteins that reel it in. · HIV: Psi, ѱ. Near the tRNA binding site (PBS) at the 5' UTR. 4 hairpins; SL 1, SL 2, SL3 & SL4. SL1 creates a kissing loop interaction with another SL1. G4/quadruplexes are formed between G rich sequences. · Influenza: 8 segments, all 8 segments have packaging signals.

· What is homologous and non-homologous recombination?

· Homologous: occurs between regions of high sequence similarity. More frequent. · Non-homologous: exchange between dissimilar genomic regions or between non-related RNA molecules. Often produces deleterious genomes.

· What is IRES? Can IRES support translation of circular mRNAs?

· IRES- Internal Ribosome Entry Site. IRES helps the ribosome to associate, and allows efficient translation of 5' ORF and upstream ORF. IRES can support circular mRNA, because they do not have a 5' end! So IRES allow the ribosome to bind better. There are many IRES'. · 5' ORF is expressed, lots of proteins made. Binding of ribosome further from the 5' end makes less proteins because the binding of the ribosome is less efficient. Sooo. If you put IRES there, the ORF further from the 5' end will make more proteins!

· What is the function of eIF4G?

· Important! Works like a bridge to keep everything together. Bridges eIF4e (binds to cap), eIF3 and rRNA 40S together. · Has all the domains that bind these specific things and has Pabp binding site. This binds poly(A) binding protein that binds to the poly A tail. This loops the 5' and 3' UTR. This looping process is referred to as juxtaposition. · Has a squiggly line, a potential cut site. Viruses can cut it to disfavor cellular translation and favor viral translation. Has a · Remember- 5' cap brings the ribosome to mRNA through a series of protein-protein interactions. · eIF4e: binds the cap directly, but also can bind to other things. Some viruses replace their cap structures with other things.

· What proteins are involved in the assembly and release of Influenza virus? HIV? Adenovirus?

· Influenza: concerted assembly. (-) sense RNA. NP expressed, goes to nucleus, builds NEP (nuclear export protein) and M1, NEP has a nuclear localization signal so goes back to the nucleus to bind RNA and facilitate its export to the cytoplasm. HA, NA, and M2 expressed and go to the membrane to begin expression. Assembled at the same time. · HIV: Psi, Ψ · Adenovirus: viral protein IVa2

· What is integrase and what enzymatic activities does it possess?

· Integrase is the enzyme responsible for integrating proviral DNA into the host cell DNA. It catalyzes specific and efficient insertion of the proviral DNA product into host cell DNA. · Binds to the ends of the retroviral dsDNA and forms tetramers. Carries out 3 enzymatic activities- exonuclease that cleaves 2-4 nts from the 3' ends of the dsDNA molecule, endonuclease that makes a staggered cut in the host cell DNA, newly formed 3' OH groups attack the host target DNA, and ligase activity that joins DNA to the host cell DNA.

· How does cell-to-cell spread occur in Measles virus? Hiv virus?

· Measles: induces formation of syncytia- fusion of many cells. multinuclear structure. · HIV: virological synapses: highways that allow one cell to infect another cell.

· What is the difference between mono- and polycistronic mRNAs?

· Monocistronic- each ORF codes for 1 protein, most eukaryotic mRNAs. Only 1 ORF. · Polycistronic- multiple ORF that can code for multiple proteins. Most bacterial, archaeal, and a lot of viral mRNAs

· What is error threshold? RNA vs DNA viruses.

· Mutation is a powerful advantage, but many mutations are not compatible with viral replication. Consequently, if mutation rates are too high, at some point accumulating base changes leads to lethal mutagenesis. This limit is called the error threshold (how much viral genomes can take). · Exceed it- loss of infectivity; below- cannot produce enough mutations to survive selection. · RNA viruses: evolve closer to their error threshold (living on the edge) · DNA viruses: evolve far below their error threshold

· What are mutations? Are they all deleterious?

· Mutations are changes in nucleic acid and are produced during copying of any nucleic acid molecule. · Some mutations are deleterious, others are beneficial, and they are maintained in the population. Viral genomes are always mutating!

· Why mutations arise? What viruses have highest mutation rates?

· Mutations arise during copying of any nucleic acid molecule. RNA viruses are mutating the most because RdRp lacks proofreading activity.

· Is this enzyme specific only for retroviruses?

· No!! It is in bacteria, archaea, and in our telomerases.

· Is 5' capping always catalyzed by cellular enzymes?

· No, it can be done by viral enzymes or de novo. very large or RNA viruses stay inside the cytoplasm so they must have their own capping machinery.

· Do all proteins start with Methionine?

· No, some structural viral proteins do not start with Met. Instead, they start with glutamine, proline, or alanine. Methionine-independent initiation. Also has IRES element that mimics the structure of tRNA, so the initiation takes place within the A site on ribosome. This is an example of molecular mimicry. · Cricket paralysis virus. Turnip yellow mosaic virus (3' end folds into tRNA structure)

· How is termination of translation suppressed?

· Occurs during translation of many mRNAs as a means of producing a second protein with an extended C terminus. There is normally a structure around these or sequences upstream and downstream of the codon regulate the termination of translation.

· How does splicing mark RNA for export in different viruses? Simple retroviruses vs. complex retroviruses (HIV)?

· Only spliced RNA is ready to go into the cytoplasm and is exported from the nucleus with proteins. Viral mRNAs are exported from the nucleus in association with proteins- RNPs. · CTE- constitutive transport element- sequence at 3' end that marks unspliced retroviral RNA as suitable for export (simple retroviruses). -HIV/complex= Rev protein associates with RE element on spliced RNA and facilitates transport.

· What is selective packaging?

· Packaging always goes in order: S-M-L. · S enters alone. Entry of M depends on presence of S. Entry of L depends on presence of S+M. serial dependence of packaging! Very effective, pretty much every particle is effective.

· Does polyadenylation happen before or after the cleavage of RNA?

· Polyadenylation occurs after cleavage of mRNA when using cellular enzymes. - polyadenylation that adds A's due to slippage happens during mRNA synthesis

· Where and how does the polyadenylation happen?

· Polyadenylation occurs in the nucleus. · Cleavage and polyadenylation sites are identified by specific sequences. Polyadenylation signal: AAUAAA. Cleavage signal: U or GU sequence located 5-20 nucleotides downstream of polyadenylation signal. Separate protein CPSF (cleavage and polyadenylation signal and induces cleavage). Cleavage happens, then polyadenylation. -Poly(A) polymerase then adds As to the end of the template. -Cpsf binds the poly A signal and recruits cleavage proteins. Cstf interacts with the downstream U/GU sequence to stabilize the complex that also contains the two cleavage proteins CfI and CfII. Binding of poly(A) polymerase is followed by cleavage at the poly(A) site by a subunit of Cpsf, and CfI and CfII, Cstf, and the downstream RNA product is released and degraded. Polymerase slowly adds 10-15 A residues to the 3' OH terminus. Poly(A)-binding protein II (PabII) binds to this short A sequence and tethers poly(A) polymerase to the poly(A) tail for subsequent synthesis.

· What is PPT and why is it resistant to RNase H cleavage?

· Polypurine tract- the only piece of RNA during RT that does not get cleaved. Unusual base pairing of RNA PPT and the DNA in a way that is twists it. Called an altered base pairing interaction and RT does not recognize it to cleave. · PPT is a primer for the (+) sense DNA

· What is the name of RNA that participates in Hepadnaviruses RT process?

· Pregenomic RNA

· What is a proofreading activity? Which enzymes have it and which don't?

· Proofreading as copying nucleic acid sequences. RdRp does NOT have proofreading activity, DNA polymerase does.

· What is viroporin?

· Protein that forms pores that allows the virus to leave the cell.

What is a provirus?

· Proviruses are viruses that are reverse transcribed into dsDNA that is integrated into the host chromosome.

difference between proviral DNA and RNA

· RNA does not have LTR but DNA does!

· What molecule catalyzes the splicing? Protein or RNA?

· RNA-RNA interactions organize substrates and catalysts during splicing. Splicing occurs in the spliceosome, which contains pre-mRNA, 5 snRNAs (U1, U2, U4, U5, U6), and associated proteins that form small nuclear ribonucleoproteins (snRNPs). · RNA is the catalyst in the spliceosome! proteins just hold the structure in the right place.

· What enzyme catalyzes reverse transcription?

· RNA-dependent DNA Polymerase (RdDp) · Requires RNA template, synthesizes DNA. In RNA tumor viruses. · Retroviruses and hepadnaviruses go through RT

· How do viruses avoid the ratchet?

· Reassortment and recombination

· What is reassortment? What viruses undergo reassortment? What genomic characteristics support reassortment?

· Reassortment is the mixing of the genetic material of a species into new combinations in different individuals. Viral progeny that contain genes that are derived from more than one parent. Influenza virus, animal, plant, and fungi viruses. New outbreaks= reassortment.

· What is recombination? What is the general mechanism of recombination?

· Recombination is the exchange of genetic information resulting in the production of offspring with combinations of traits that differ from those found in teither parent. · Copy choice mechanism is the most widely accepted model. Viral polymerase switches from one RN molecule (donor template) to another (acceptor template) during synthesis while remaining bound to the nascent nucleic acid chain, thereby generating an RNA molecule with mixed ancestry. RNA structure dictates recombination.

· How does Vaccinia virus uses cellular machineries for its release?

· Released from the cell by actin tails. Associates with kinesin and blasts out of the virus

· What is ribosomal frameshifting?

· Retroviruses. For HIV... there are 2 ORF that overlap. Frequently, they get expressed into one protein OR they can be expressed separately. Due to a specific sequence in between. Has a slippery site where the ribosome slips and a pseudoknot that slows down the ribosome, so it slips and studders. Frameshifting can be backwards (-1) or forwards (+1). This allows the production of 2 proteins from one mRNA. Also regulates the rate of production of 2 proteins.

· What is ribosomal shunting?

· Ribosome binds the cap, scans mRNA a bit, then it switches to AUG bypassing secondary structures. Shunting is predicted to decrease dependence of mRNAs for eIF4F (which includes eIF4A helicase) during initiation by reducing the need for mRNA unwinding. · Specific structures can create an obstacle for ribosomes that they cannot read through (at 5' end). Viruses overcome this using ribosomal shutting. If there is a structure that is a road blockage, the 40S ribosome jumps the structure, bypassing the structure. This releases the dependence of mRNA from helicase. · Adenovirus, sendai virus, and caulimovirus.

· What consist of translational machinery?

· Ribosomes (40S +60S = 80S), tRNA · Ribosomes- aminoacyl site, peptidyl site, and exit site. Catalytic site of the ribosome resides in RNA, not protein. · Initiation proteins (eIF), elongation proteins (eEF), termination proteins (eRF) · tRNA- 3' end with CCA (acceptor stem). Has anticodons flipped out that is complementary to the mRNA. Looks like a cloverleaf.

· What is the difference between complex and simple retroviruses?

· Simple: like alpha and gamma retroviruses. ASLC and RSV. Encode Gag (group antigen, structure protein), Pol (polymerase) and Env (envelope, SU and transmembrane proteins). Simple because this is all they have! Have long terminal repeats (LTR) as sticky sides, where the virus gets integrated. · Complex: HIV, HTLV. have gag, pol, env, and more! Are accessory and regulatory proteins. Tat protein (binds tar and supports transcription), rev (supports unspliced/single spliced HIV). The genes overlap and have tight packing. The expression of the genome leads to multiple mRNAs with many different splices.

· What is Muller's ratchet concept?

· Small, asexual populations decline in fitness over time if the mutation rate is high because they tend to incorporate mutations (the majority deleterious) in an irreversible (ratchet-type) mechanism, unless recombination or reassortment rescues the genome.

· What is cap-snatching?

· Some viruses are unable to synthesize their own cap structures so they steal it from their host. It is used by: Orthomyxoviridae (influenza virus), arenaviridae (lassa virus), and bunyaviridae (hantaan virus). The 5' caps of these viral mRNAs are acquired when viral cap-dependent endonuclease cleaves cellular transcripts to produce the primers needed for mRNA synthesis. The 5' cap of the influenza virus mRNAs are obtained from cellular pre-mRNA in the nucleus. The 5' cap of bunyaviral mRNA is cleaved from mature cellular mRNA in the cytoplasm. · No enzyme needed. Steals cap from the host. Steals what fits to the beginning of the viral message and steals what is abundant.

· What is leaky scanning? How is leaky scanning regulated? What is suboptimal sequence content?

· Specific for Paramyxovirus. Monocistronic mRNA can be processed as polycistronic. The 40S sununit binds to the 5' end and can bypass the first AUG codon and continue scanning to an alternate downstream start codon. This is caused by suboptimal sequence surrounding the first AUG codon. Can express multiple proteins!

· What are packaging signals? Are they present on genomes or proteins?

· Specific sequences that are recognized and packaged into particles. The cell is super crowded. But special sequences on RNA tells it where to go/how to find its proteins. Present in genomes.

· What is splicing?

· Splicing is editing of the nascent precursor messenger RNA (pre-mRNA) transcript into a mature messenger RNA (mRNA). · During splicing, introns (non-coding segments) are removed and exons (coding segments) are joined together (ligated) into a complete mRNA.

· What is the role of alternative splicing?

· Splicing of different combinations of exons, govern by regulatory sequences and arrangement of splicing proteins. Expands coding capacity. Creates different mRNAs and proteins. Leads to the synthesis of mRNAs that expand the limited coding capacity of viral genomes. Temporal regulation of viral gene expression. Control in production of spliced and unspliced mRNAs.

· Explain the concepts of "survival of the fittest", "survival of the survivors"

· Survival of the fittest: a rare genome with a particular mutation may survive a selection event (drug treatment), and this mutation will be found in all progeny genomes. · Survival of the survivors: however, the linked, but unselected mutations get a free ride.

· What three HIV RNA structural motifs are important for HIV lifecycle?

· TAR- transactivation response element, hairpin that binds tat, regulates transcription of retroviral RNA is integrated in cellular DNA. · PBS- primer binding site- binds tRNA. Hairpin with open loop for tRNA to attach. Opens the loop more widely. · DIS- dimerization site, holds two RNAs together so that they can be packaged in a virion. 2 hairpins with complementary sequence (kissing hairpins).

· What is TAR/tat complex?

· TAR/tat complex directs efficient transcription.

· What is protein retention signal?

· Tells the membrane proteins to stay in the appropriate membrane.

· What is template switching?

· Template switching is guided by the sequence similarity between the nascent and the acceptor template. Changes from one template to another. Retroviruses switches the template.

· What two reactions are involved in splicing?

· The 2' hydroxyl group of the A residue in the intronic branch point makes a nucleophilic attack on the phosphodiester bond at the 5' side of the GU dinucleotide at the 5' splice site to produce the intron-3' exon lariat and the 5' exon (circular RNA). · A second nucleophilic attack by the newly formed 3' hydroxyl group of the 5' exon (1) on the phosphodiester bond at the 3' splice site then yields the spliced exons and the intron lariat.

· How is HIV transcription regulated?

· The LTR acts as a transcriptional promoter. TAR/tat complex directs efficient transcription.

· What is the path of envelope acquisition of Herpesviruses?

· The nucleocapsid assembled within the nucleus initially acquires an envelope from the nucleus, but when leaving through the ER it loses it and ends up in the cytoplasm naked. Then it goes to the Golgi and acquires the envelope at the entry site in Golgi, and once it is released it ends up being encapsulated TWICE in another vesicle derived from the Golgi. It then fuses with the plasma membrane to release singly-enveloped virion s from the cell.

· What is RNA editing?

· The process by which nucleotides not specified in the genome are introduced into mRNAs. · Viral mRNAs are edited by either insertion of nucleotides not directly specified in the template during synthesis or alternation of a base.

· How do viruses overcome the lack of 5' capsid?

· There are specific structures that facilitate 5' to 3' juxtaposition. This circulation will bring them closer. Still circularize just through different means! · Specific structures regulate this. Structures at the 3' end= 3' cap independent translational enhances (3' CITES). Very abundant in plant viruses.

· What is the function of p51 and p66 subunits in RT?

· These domains are identical except only p66 is catalytically active. P51 holds it, plays a structural role. · P51- structural. Closed conformation. · P66- catalytically active. Open conformation.

· How do viruses modify translational factors?

· Three types of translation factors- initiation proteins (eIF; rate limiting step), elongation proteins (eEF), and termination proteins (eRF). All have cellular origin. Viruses either shut off genes that code for these or modify via cleavage or phosphorylation. · eIF2/GTP is needed to initiate translation. eIF2 can be phosphorylated though, which binds irreversibly to eIF2B that makes it unavailable to create the initiation complex. Certain kinases leads to this, either triggered by stress or by infection by virus.

· What proteins recognize 5' cap structures during a) transport to the cytoplasm, b) translation?

· Transport to the cytoplasm: in the nucleus, the cap structure of pre-mRNAs is recognized by the cap binding proteins: CBP20 and CBP80. This cap binding complex (CBC) protects mRNA from degradation and assists RNA transport from the nucleus to the cytoplasm. Once in the cytoplasm, ribosomes and translation factors must be recruited for translation of mRNAs into proteins. · Translation: the eukaryotic translation initiation factor 4E (eIF4E) specifically binds to the RNA cap structure.

· What is nuclear export signal?

· Viral mRNA or RNP complexes move into the cytoplasm.

· What is a quasispecies concept?

· Viral population is in a dynamic equilibrium with viral mutants arising at a high rate on the one hand and being strongly selected against on the other. The viral genome cannot be described as a defined unique structure, but rather as a weighted average of a large number of different individual sequences.a quasispecies refers to a population of genetically related viruses that are closely distributed around a consensus sequence.

What is virulence and is it a positive or negative trait?

· Virulence is a pathogen's ability to infect or damage a host. Tradeoff between virulence and virus transmission.

· What are epitranscriptomic modifications and how viruses use them for their own benefit?

· Viruses develop strategies like a protein coat that exonucleases cannot degrade. Some destroy cell proteins involved in decay, some target all steps of RNA processing. · RNA has 140 modifications of AUGCs. · m6A- methyl group on A at the position 6- allows HIV to produce more viruses.

· Does viral assembly depend on cellular machinery? In what way?

· Yes! Assembly needs cellular chaperones (folding), transport systems (microtubules allow the building blocks of virus particles to be transported to the appropriate assembly site), secretory pathways (assembly and release of viral particles at the cellular membrane), and nuclear import and export machinery (dependent on a specific sequence; facilitate nuclear entry and attachment/integration of some viral genomes to the host DNA).

· Do plant viruses undergo reverse transcription? Which ones?

· Yes, cauliflower mosaic virus and hepadnaviruaes.

· Can recombination have positive effect? What kind?

· Yes, combos of mutations can increase the rate of adaptive evolution. Can also remove deleterious mutations and restore wild type genome AND can create a functional genome from damaged parental molecules.

· Is RNase H a part of reverse transcriptase? What is its function

· Yes. It is part of the p66 subunit. Allows cleaving of RNA when in duplex form with DNA.

· What is PKR? How do viruses counter its action? What does PKR sense?

· dsRNA- dependent protein kinase. Induced by interferon and the presence of foreign dsRNA. Begins forming dimers and phosphorylating itself, also phosphorylating eIF2 so it cannot form the translation initiation complex. Can happen as natural response to stress too. · Leads to inhibition of host translation, apoptosis. Different viral mechanisms have evolved to inactivate the PKR pathway. · Suppress phosphorylation of eIF2 or PKR directly. · Viruses mess with eIFs. Cleave eIF4G so cellular translation machinery cannot use it but the virus can. Also dephosphorylate eIF4E

· How viruses counter the mRNA decay system?

·Viruses can be decorated with different groups- pretends to be cellular. Uses cell machinery to decorate itself/stop decay. THEY MAY... -coated with proteins so exonucleases don't see the RNA -stabilize their RNAs by binding the cellular proteins -steal/stop proteins involved in decay. -target little steps of RNA processing