Virology Exam 2

Cleavage of the HA protein of influenza by cellular proteases is important for

Activation of the fusion peptide.

Discontinuous transcription of genome mRNA of coronavirus

Adaptability and evolution of coronaviruses

the process of RNA editing used by the paramyxoviruses?

Addition of one or two non-template nucleotides to the mRNA.

Most animal influenza viruses do not infect humans. Why

Animal viruses bind to a different form of sialic acid than human viruses. The HA proteins of animal viruses are activated by the protease furin.

What describes the phenomenon of antibody-dependent enhancement?

Antibodies bound to a flavivirus particle allows entry into a cell via the Fc receptor.

How does Antibody-dependent enhancement cause more severe disease in flaviviral infection?

Antibody-dependent enhancement occurs when a patient previously infected is subsequently infected with a second serotype. The first infection is believed to expand the population of cells that can be infected by the second serotype. After the first infection, there is a higher population of igFC receptors available for attachment during the second infection.

Influenza virus is one of the only RNA viruses that replicates in the nucleus of the host cell. How does this virus regulate transport of viral nucleocapsids into and out of the nucleus at various times during the infection?

At the start of an infection with influenza, virions are dissembled due to the low pH in the endosome. This releases the nucleocapsids into the cytoplasm where the exposed nuclear localization signal on the NP protein causes the import of the nucleocapsids into the nucleus. In addition, most of the viral proteins contain a nuclear localization signal which allows them to be transported back into the nucleus after they are synthesized in the cytoplasm. As nucleocapsids form in the nucleus of the infected cell, they must be transported out of the nucleus so that they can bud from the plasma membrane and become enveloped. The newly made nucleocapsids interact with the M1 protein. Next, the NS2 protein, which contains a nuclear export signal, binds to the M1 protein and allows the nucleocapsids to be exported out of the nucleus and into the cytoplasm for final assembly and release.

conditions during infection with a paramyxovirus causes the RNA-dependent RNA polymerase to switch from start-stop transcription to production of full-length positive-strand antigenomes?

Concentration of the N protein.

The yellow fever vaccine strain has 32 amino acid mutations in comparison to the wildtype pathogenic virus strain. Which genes contains the majority of these amino acid substitutions?

Envelope glycoprotein

What cellular proteases help to process the polyprotein of flaviviruses?

Furin

What describes the structure of the coronavirus virion?

Helical nucleocapsid surrounded by an envelope

The NS2 protein from influenza can bind and inhibit cellular proteins involved in polyadenlyation of mRNAs. Why doesn't this affect the virus?

Influenza virus uses a stuttering mechanism to add the poly(A) tail.

the function of the NS1 protein made during an influenza virus infection?

Inhibition of cellular antiviral defenses.

The abundance of which of the following proteins from influenza virus controls whether the virus is producing mRNAs or full-length positive antigenomes.

Nucleocapsid protein.

How does a togavirus ensure that only full length genomes are packaged into the virions?

Only full length genomes contain the packaging signal.

Why do many positive-strand RNA viruses form their genome replication complexes on the surface of cytoplasmic membranes?

The membrane acts as a nucleation sites to bring all of the proteins together.

The open reading frame that takes up the 5' end of the togavirus genome encodes what?

The nonstructural proteins.

There are two sequence elements at the 3' end of the genomes of paramyxoviruses that are required for mRNA transcription and genome replication. These two sequences are 78 nucleotides apart. How can the polymerase bind to both of these sequence elements at the same time?

The two sequences are both on the same side of the helical nucleocapsid.

How are the structural proteins in togaviruses expressed?

They are translated from a subgenomic RNA.

Many of the picornaviruses use host cell receptors that have what characteristic?

They belong to the immunoglobulin superfamily.

Compare flaviviruses with picornaviruses

They both use an IRES to initiation translation of the viral genome, They both encode the structural proteins at the 5' end of the genome, They both produce their proteins from a single open-reading frame, They both produce viral proteases to cleave the polyprotein.

Since translation of mRNAs in eukaryotic cells produces only a single polypeptide, how do picornaviruses produce multiple proteins from their RNA genome?

They cleave a single polypeptide into individual proteins

Why are reassorted influenza viruses usually more likely to cause a severe worldwide pandemic?

They have acquired new glycoproteins for which there is no prior immunity.

How do coronavirus enveloped virions get released from the host cell?

Virus containing vesicles fuse with the plasma membrane, releasing virions.

Replication of Picornavirus RNA

-Carry out replication on the cytoplasmic membranes because of the nucleation site which brings all the enzymes together -RNA synthesis is primed by VPg protein which is covalently bound to uridine residues. uridylation of VPg allows VPg to hybridize to the poly(A) tail and serve as a primer for (-) strand synthesis. -RNA dependent RNA polymerase is first translated when the genome enters the cell. then the viral RdRP then makes the complementary negative strand, then uses that as a template to make multiple copies of the positive strand genome. The positive strand and the negative strand are produced together, because the mRNA is circularized during synthesis. Membrane-bound protein-primed circular RNA-based replication system.

Describe the Replication and transcription: synthesis of genome and subgenomic RNAs of Togavirus.

-first viral RdRP is translated from P1234 immediately -transcleavage of p1234 generates nsP1, nsP2, and nsP3. with nsP4, the four proteins form complexes to initiate synthesis of positive stand RNA using negative stand as template. -then uses a sequence in the negative strand to induce subgenomic RNA synthesis, which then is translated and replicates the positive strand full strand

assembly of the picornavirus virion

1. Precursor polypeptide containing VP0, VP3, and VP1 are cleaved by 3C and 3CD proteinases. 2. VP0, VP1, and VP3 assemble into protomers. 3. Five protomers self-assemble into the 14S pentamer. 4. The pentamer and RNA assemble into provirion. 5. VP0 cleaved to VP2 and VP4, forming mature virion.

Explain the role that antigenic drift and shift play in the ability of influenza to cause new outbreaks. Which one is thought to have caused the 1918 strain of influenza to emerge?

Antigenic drift consists of the gradual changes of amino acids due to lack of fidelity of the RNA-dependent RNA polymerase. These amino acid changes, particularly in the HA and NA protein, make these external protein unrecognizable by the immune response to previous influenza viruses. This explains why infection with influenza does not lead to a life long immunity to the virus. Antigenic shift is a larger change in the HA and NA proteins. This involves the swapping of two or more genome segments when two viruses infect the same cell. These reassorted viruses have completely different HA and NA proteins. Since few people have any prior immunity to these reassorted viruses, they are usually the cause of worldwide pandemics. While it was thought that the deadly 1918 strain was due to antigenic shift, after the genome was recovered and sequenced, it was actually due to antigenic drift, since only a few amino acids in several crucial proteins were changed.

The majority of known coronaviruses have been isolated from which vertebrates?

Bats

What mechanism do the paramyxoviruses use to produce 5 different proteins from a single gene on their genome?

Both RNA editing and alternative start codons are used.

In the mature flavivirus virion, the fusion peptide on the E protein is buried in the dimer interface. Which conditions causes the fusion peptide to become exposed?

Drop in pH in the endosome.

Translation of the subgenomic mRNA from togaviruses occurs on ribosomes that are located on or in what cellular compartments?

Endoplasmic reticulum

In the paramyxoviruses, cleavage of the F0 protein into two subunits F1 and F2 is important for what

Exposing the fusion peptide.

Compare flaviviruses with togaviruses

Flaviviruses have a T=3 capsid while togaviruses have a T=4 capsid, Flaviviruses have smaller virions than togaviruses, Flaviviruses do not produce subgenomic RNAs like togaviruses, Flaviviruses produce a single-polyprotein while togaviruses produce more than one.

Translation initiation of the genomes of picornaviruses require what

IRES at the 5' end of the genome, pyrimidine-rich sequence upstream of an AUG, Small subunit of ribosome, Host cell translation initiation factors

How does the the P/C/V gene code for several proteins?

In Paramyxovirus, using alternative translational starts and mRNA editing. Translation from multiple start sites create a nested set of proteins and editing occurs when RNA polymerase "stutters," adding an extra G residue.

Unlike the other positive-strand RNA viruses that have simpler genomes, coronaviruses produce a nested set of sub-genomic mRNAs, each of which is translated into a single protein. Explain why the virus needs to produce so many subgenomic mRNAs.

In eukaryotic cells, almost all mRNAs are monocistronic and only the first open reading frame at the 5' end of the mRNA will be translated. When the ribosome reaches the first stop codon, it falls off of the mRNA and will not translate a downstream open reading frame. This means that for each of the coronavirus structural proteins to be produced, it must be at the 5' end of one of the subgenomic RNAs. The mechanism that coronaviruses use to ensure this is to have a nested set of subgenomic RNA that all have the same 3' end but have their 5' ends at a different position. The results is that each structural protein open reading frame is at the 5' end of one of the nested set of subgenomic mRNAs, which will allow it to be translated.

Influenza virus is unusual for an RNA virus because it replicates in the nucleus of the host cell. Describe the two processes that influenza uses the nucleus to accomplish. For which of these two is replication in the nucleus essential?

Influenza replicates in the nucleus of the host cell for two reasons: 1) it snatches caps from cellular pre-mRNAs to use as primers for synthesis of viral mRNAs and 2) it uses the host cell mRNA splicing enzymes to carry out alternative splicing of mRNAs produced from two genome segments, 7 and 8. The splicing enzymes only exist in the nucleus of the host cell, so this is the process that is absolutely dependent on the virus replicating in the nucleus. However, the virus could snatch caps from mature cellular mRNAs in the cytoplasm, similar to bunyaviruses, if it replicated in the cytoplasm instead.

What are the difference between Replication and transcription of influenza virus.

Influenza replication results in mRNA that do not have a 5' cap or 3' poly(A) tail. Replication of mRNA uses a nascent positive strand as a template. Influenza translation results in mRNA that have a 5' cap and a 3' poly(A) tail. Transcription of mRNA uses the original viral genome to make the new strand that will be transcribed.

Instead of a 5' cap on the viral genomic RNA, genomes of the picornaviruses have a covalently bound protein. In addition, the viral 3C protease cleaves a major component of the cap binding complex. This situation should inhibit translation initiation of the picornaviral RNA into protein. Explain how the virus "solves" this problem.

Instead of using cap-dependent translation, which requires the 5' cap on the RNA and the cellular cap binding complex, picornaviruses use a cap-independent process for initiating translation of the viral genome. They have a very long 5' noncoding sequence that has a high degree of secondary structure which functions as an internal ribosome entry site (IRES). This IRES can bind to cellular proteins, some of which are involved in translation initiation, which then bring in the small subunit of the ribosome. This allows the ribosome to be deposited near the correct AUG to begin translation of the picornavirus genome, bypassing the need for the 5' cap and the cap binding complex.

The VPg protein from picornaviruses has what function?

It acts as a primer for RNA synthesis.

the function of the M2 protein from influenza virus?

It acts as an ion channel to allow H+ ions to enter the virion.

Mutation of the RNA editing site in the genome of a paramyxovirus will have which of the following effects?

It eliminates the production of the V protein.

What function does the neuraminidase protein of influenza virus carry out?

It helps release virions from virus producing cells.

What describes the source of the protease that cleaves the picornavirus polyprotein into individual functional proteins?

It is part of the polyprotein.

Several of the protein from influenza virus interact with the cellular protein α-importin. Why is this necessary for virus infection?

It transports the viral proteins into the nucleus.

What protein from paramyxovirus virions interacts with the tails of the envelope glycoproteins during assembly of virions?

M protein

The nonstructural proteins of flaviviruses are produced on the rough ER and have membrane spanning domains. What explains why the nonstructural proteins are localized to cellular membranes?

Membranes serve to localize the viral replication complex.

Synthesis of mRNAs in paramyxoviruses occurs through a single-entry mechanism, which is where the RNA-dependent RNA polymerase can only start transcription from the 3' end of the genome. a consequence of this mechanism?

More mRNA is produced from the coding regions at the 3' end of the genome.

Which influenza virus protein is involved in suppressing host cell antiviral response pathways and how does it suppress these pathways?

NS1 protein inhibits RIG-1, activates P12K/Akt pathway, inhibits double-stranded RNA-dependent protein kinase (PKR), and inhibits 2',5'-oligo(A) synthetase/Ribonuclease L. PB1-F2 induces increased apoptosis in host immune cells responding to influenza virus infection.

What are some of the specific functions of flavivirus nonstructural proteins (NS1 through NS5)?

NS1: RNA replicase component, possibly involved in replication complex formation NS2: RNA replicase component, also part of viral proteinase that cleaves viral polyprotein NS3: Viral serine proteinase involved in polyprotein cleavage; RNA replicase component; nucleoside triphosphatase and helicase activities NS4: RNA replicase component, may recruit NS1 and other proteins into replicase complex NS5: RNA-dependent RNA polymerase; methyltransferase (methylates cap structure)

The 5' two-thirds of the coronavirus genome contains what?

Non-structural protein coding sequences

How did a bat coronavirus mutate and enter humans to become SARS coronavirus?

One possibility is that the virus mutated in other host species before it spread to humans. Another possibility is that the nonpathogenic virus passed from bats to humans, then to animals where it mutated to become pathogenic.

major difference between the positive-strand RNA viruses and negative-strand RNA viruses?

Only negative-strand viruses must package their polymerase in their virion.

viruses that are members of the order Mononegavirales

Paramyxoviruses: Rabies, Measles, Ebola, Sendai Virus

What is the major difference between flavivirus and picoronavirus? (Hint: Genomes, Proteases etc.)

Picornavirus: non-enveloped, 5' protein cap and 3' poly(A) tail, uses 3CD and 3D to cleave structural proteins, only uses viral proteins Flavivirus: enveloped, 5' cap but no 3' poly(A) tail, uses furin to cleave structural proteins, uses both host and viral proteins

The killed Salk and live Sabin vaccines have been used successfully to nearly eliminate which virus from the human population?

Polio

Poliovirus virions enter cells through a pH independent mechanism at the cell surface, which is an unusual pathway for a naked virion. Describe the entry mechanism and explain why this is this important for a virus that uses the oral-fecal transmission route?

Poliovirus capsids bind to the host cell receptor on the surface of the cell. This binding induces a conformational change in the capsid that includes the release of the VP4 protein and the extrusion of a hydrophobic tail of the VP1 protein. It is thought that the hydrophobic tail of VP1 forms a channel through the cell membrane which allows viral RNA to pass through the membrane and into the cytoplasm of the host cell. The reason that poliovirus must use a pH independent pathway is that when it infects a new host it enters through the gastrointestinal tract. If the virion used a pH dependent pathway, then it would be affected by the low pH in the stomach acid. This would cause the capsid to change conformation prematurely and prevent it from entering the correct host cells.

What allows the production of the P1234 nonstructural protein in a togavirus

Readthrough of a stop codon.

What describes the mechanism used by coronaviruses to produce the ORF1b protein?

Ribosomal frameshifting

The complex RNA structure called a pseudoknot in the genome of coronaviruses is involved in what processes?

Ribosomal frameshifting from ORF1a to ORF1b.

What is a coronavirus that emerged as a serious human pathogen in Asia in 2003?

SARS

What coronavirus proteins is responsible for virus entry and is the primary determinant of host range?

Spike (S) protein

Describe the Assembly of Togavirus virions.

Structural proteins are cleaved by host signal peptidase and furin protease during translation and sent to different locations. Capsid proteins interact with the cytoplasmic tails of envelope proteins studding the plasma membrane.

Enveloped virus-like particles can be formed in cells when only the M and E protein from a coronavirus are expressed. What does this indicate?

That these proteins are sufficient for assembly and budding of virions.

During infection with a togavirus, the subgenomic mRNAs represent the complementary strand of what

The 5' half of the negative-strand antigenome.

What do you know about a coronavirus variant of concern?

The delta variant is more pathogenic than other variants

Many of the symptoms caused by infection with the influenza virus, including fever, headache and malaise, are actually the result of what

The effect of cytokines induced by the virus.

Describe the two models that explain how the monopartite negative-strand RNA viruses produces individual mRNAs. Which of the two models does the experimental evidence support?

The multiple-promoter model states that the viral RNA-dependent RNA polymerase can begin synthesis of the viral mRNAs at any of the intergenic sequences. This would predict that transcription of each mRNA is independent of any of the upstream or downstream coding regions. The single-entry model states that the viral RNA-dependent RNA polymerase would always start RNA synthesis at the 3' end of the negative-strand genome. It would then pause at each intergenic region and either disengage from the template to restart synthesis at the 3' end or continue on to transcribe the next protein coding region. To demonstrate which of these two mechanisms is correct, where a thymine dimer was introduced into a gene (caused by UV radiation) and it inhibited transcription of all the downstream genes, which supports the single-entry model.

Describe the final processing steps that the flavivirus virion goes through in order to be converted from an immature virion into a mature infectious virion.

The nucleocapsid buds into the lumen of the ER to obtain the envelope. The immature virion then progresses through the exocytosis pathway to eventually be released from the host cell. Movement of the virion through the low pH environment of the trans Golgi network causes the surface proteins of the virion to fold down into the flattened structure and exposes the furin cleavage site. This allows furin to cleave the prM protein into the final M protein. However, the "pr" peptide is not released from the virion until after the virion leaves the cell and is exposed to the neutral pH outside the host cell.

How is the poly(A) tail added to the 3' ends of the mRNAs produced during infection with a influenza virus?

The viral polymerase stutters at a poly(U) sequence.

How is the poly(A) tail added to the 3' ends of the mRNAs produced during infection with a paramyxovirus?

The viral polymerase stutters at a poly(U) sequence.

If you add togavirus virions to host cells and then lower the pH of the medium, what will happen?

The virions will fuse to the plasma membrane of the cell.

The poliovirus protease cleaves the cellular eIF-4G protein. Why doesn't this affect the virus?

The virus does not use cap-dependent translation.

True statements about the proteinases from coronaviruses

They are encoded in the nonstructural region of the viral genome, They are cysteine proteinases, They first cleave themselves out of the polyprotein, They primarily cleave the non-structural polyprotein into mature proteins.

In the picornavirus genome, there are several A nucleotides found at the 3' end of both the positive and negative sense strands. How are these A nucleotides used in replication of the RNA genome?

They basepair with the U nucleotide bound to the VPg protein.

Alphaviruses, which are members of the togavirus family, have been developed as useful vectors for the expression of proteins in various human and insect cells. Describe two situations where alphaviruses could be used as vectors in biology or medicine.

They have been used to produce high levels of foreign proteins in various cell types. This is better than using bacterial cells because the proteins will receive their normal post-translational modifications (phosphorylation, glycosylation). One example of this is to produce proteins from unrelated viruses that could be used as vaccines. 2) Infection of insects with a virus that expresses an RNAi construct against other mosquito borne pathogens, such as yellow fever or West Nile Virus. 3) Alphaviruses could express a toxic protein to kill off mosquitoes or other insects. 4) Because alphaviruses infect neurons in their vertebrate hosts, they have been used to study live neurons in culture. These viruses could eventually be developed as gene therapy vectors for introducing genes into neurons

describes a difference between transcription and genome replication in the influenza virus?

Transcription requires the PB2 protein while genome replication does not. Transcription produces a shorter positive strand mRNA while replication produces a full-length antigenome. Transcription produces a poly(A) tail at the 3' end of the viral mRNA while genome replication does not. Transcription requires a primer derived from a cellular pre-mRNA while genome replication does not.

Describe two major differences between transcription and genome replication in paramyxoviruses.

When the levels of the nucleocapsid protein builds up in the infected cell, the viral polymerase switches from producing subgenomic mRNAs to producing full-length positive-strand antigenomes. One major difference is that the nucleocapsid protein only binds and encapsidates the full-length antigenome and genome copies. The subgenomic mRNAs are not bound by the nucleocapsid protein. The second major difference is that during genome replication the viral RNA polymerase does not pause at the intergenic regions and stutter to create the polyA tail. It continues through and produces an authentic full-length copy of the genome with no stops or reinitiation.

Which diseases is caused by a member of the flavivirus family?

Yellow Fever, Dengue Fever, West Nile Virus, Encephalitis, Hepatitis C

RNA dependent RNA polymerase

an enzyme that synthesizes RNA using an RNA template. Viruses with RNA genomes must code for such an enzyme, which is not available in the host cell. catalyzes both replication and transcription.

The 5' end of the poliovirus genomic RNA contains what characteristics

an unusually long noncoding region, a pyrimindine-rich tract, a high degree of RNA secondary structure, multiple AUG start codons

which diseases can be caused by members of the coronavirus family

bronchitis, hepatitis, gastroenteritis, encephalitis

Picornaviruses cause cytopathic effects in infected host cells by doing what?

inhibiting cap-dependent translation of mRNAs, Inhibiting intracellular transport from the ER to the Golgi, Cleaving histone H3 protein, Inhibiting host cell mRNA synthesis.

Which diseases is caused by a member of the picornavirus family?

meningitis, encephalitis, the common cold, foot-and-mouth disease, Polio, Coxsackie virus, myocarditis, and hepatitis

What is an enzymatic function found in the nonstructural proteins of a coronavirus

methyltransferase, helicase, RNA-dependent RNA polymerase, ribonuclease

assembly of Flavivirus virion

occurs at cytoplasmic membranes, where prM and E proteins are associated. C protein anchors to the membrane and penetrates the signal sequence into the lumen. C proteins condense the genome to form a nucleocapsid. C protein is cleaved by viral proteinase and released from the membrane.

replication of flavivirus RNAs

occurs on the cytoplasmic membranes and RNA replication complexes contain viral and host proteins. Similar to picornavirus, negative strand is formed. then, multiple positive strands are made from the negative strand.

internal ribosome entry site (IRES)

regions on viral RNAs (particularly picornaviruses and flaviviruses) that bind to ribosomes and lead to cap-independent initiation of protein synthesis at internal sites distant from the 5' end of the RNA. IRES contain extensive secondary and tertiary structures that interact with a variety of cellular proteins. Some cellular genes have been shown to contain IRES.

If the Spike protein from some coronaviruses is incorporated into the plasma membrane of host cells, it can cause cell fusion and the formation of syncytia. True or false?

true