BIOL 4190 Rui Lu Exam 2

What are the structure features of reovirus genome ?

Hybrid (+/-) RNA genome with a double capsid

exopeptidase vs. endopeptidase

Exopeptidases cleave peptide bonds at the ends of protein chains, whereas endopeptidases cleave peptide bonds within the chain.

What’s the major disease caused by rotavirus infection?

Diarrhea/ gastroenteritis

What’s the major function of rotavirus  VP1 protein? Is it considered as structure protein?

RNA-dependent RNA polymerase

No

What’s a viroplasm?

discrete regions of the host cytoplasm in which viral proteins accumulate in early rotavirus replication

• site of replication and assembly

Are there viroplasms formed during reovirus infection?

yes

How does rotovirus with intact VP4 proteins (the surface spike) enter host cells?

direct penetration across the plasma membrane

Where does rotavirus assembly occur?

vesicles within the endoplasmic reticulum

Describe how rotavirus virion with intact spike proteins (VP4) enters host cell

Virions with uncleaved spike proteins VP5 hidden by VP4. The hydrophobic regions of VP5 mediate direct penetration across the plasma membrane

How does rotavirus exit host cells?

after forming a temporary envelope derived from the membrane of the endoplasmic reticulum (acquired through budding) the virion exits via lysis of the host cell or endocytosis

What’s the major function of rotavirus VP3 protein?

guanyl transferase and methyl transferase activity. During replication, VP3 is used to cap the 5' end of the new RNA. In the viroplasm, VP3 is used alongside other viral proteins to assemble the virion core (inner capsid layer)

Is rotavirus VP3 considered as structure protein?

yes

What are the terminal structure feature (cap and polyA tail) of rotavirus produced early transcripts.

5' cap, no poly-A tail

What are the terminal structure feature (cap and polyA tail) of rotavirus produced late transcripts.

uncapped and no poly-A tail

What's the cell receptor for poliovirus?

Glycoprotein CD155

Can poliovirus genomic RNA alone be used to initiate viral infection?

Yes

Poliovirus is a Class IV (+) ssRNA virus, so its genome can be directly used in translation

Why is a 5' end cap structure not required for picornavirus genomic RNA to serve as mRNA for protein translation?

In place of a cap structure, picornaviruses have a VPg protein on the 5' end. This structure is cleaved at the beginning of replication and used as primer.

Which viral protein is released when a poliovirus is bound to the cell receptors?

VP4 is release so that the hydrophobic n-terminus of VP1 is displaced to virion surface, forming a pore in the cell membrane

Describe the structure features of a picornavirus genome.

7-8kb (+)ssRNA with a VPg protein covalently linked at the 5' end and a polyadenylated 3' end

Do picornaviruses always carry their replicase (RNA-dependent RNA polymerase) inside mature viral particles?

No, only their genome is enters the cytoplasm

Describe the important features for plus-stranded RNA viruses in Class IV.

1. Translation occurs before transcription

2. RNA replication take place on membranes

3. transcription and genome replication are the same process

4. (+) RNA serves as:

a) templates for (-) RNA

b) as mRNA

c) as genomes of progeny

What are the major translation Strategies used by Plus-stranded RNA Viruses in Class IV.

1. Polyprotein: All genetic information is encoded in one ORF to produce a poly protein which is then cleaved to produce individual proteins

2. Subgenomic mRNAs: genome has 2 or more ORFs. RNA-dep. RNA-pol. is encoded at the 5' end in the first ORF so it can be translated from the infecting genome. The other ORFs are then transcribed into subgenomic mRNAs with identical 3' ends to the original strand

3. Segmented Genome: one ORF per segment

Do picornaviruses need a primer to replicate their genomes?

yes, the VPg protein functions as a primer after cleavage from the 5' end

Describe the structure feature and genome constitution of rhabdoviruses.

enveloped, helical nucleocapsid composed of (-) ssRNA

Describe the major functions conferred by  rhabdovirus M protein

forms a layer between the nucleocapsid and the envelope, condenses the nucleocapsid into a tight helix and binds it to a region of the plasma membrane, and binds cell proteins involved in budding with its late domain

Describe all enzymatic activities of the L protein produced by rhabdoviruses

RNA-dependent RNA pol.

Methyltransferase

Guanylyltransferase

Poly-A synthase activity

Kinase

How do rhabdoviruses enter host cell?

Clathrin-mediated endocytosis

Can RNA dependent RNA  polymerase or its activity be found in mature virion of rhabdoviruses?

yes

Describe the terminal structure features (cap and polyA tail) of rhabdovirus genomic RNA and mRNAs.

Genomic RNA: no cap or poly-A tail

mRNA: capped and polyadenylated

Do rhabdoviruses require a primer for their genome replication?

No

Retrovirus genomic RNAs are plus-stranded RNAs with cap at the 5’ end and polyA at the 3’ end but do not function as mRNA, why?

The genomic mRNA has no chance to be bound by ribosomes until after reverse transcription

The transcription of retrovirus genome is carried out by which cellular enzyme?

reverse transcriptase

What's the function of the primer binding site in retrovirus genome?

binds a tRNA primer

Describe where the reverse transcription occurs during retrovirus genome replication.

within the reverse transcription complex

Describe the terminal structure features (such as cap and polyA tail) of retrovirus genome and mRNA transcripts

Both are capped and polyadenylated

Describe how each of the three classes of retrovirus proteins (env, gag and gag-pol) is produced.

env: translated from spliced mRNAs in rER

gag: produced from unspliced mRNAs without a frameshift

gag-pol: produced from unspliced mRNAs with a frameshift

Describe how HIV genome is replicated

Reverse transcription (within the reverse transcription complex). it is then integrated into the host genome and the host machinery transcribes new RNA genomes

The transcription of HIV is carried out by which cellular enzyme?

RNA polymerase II

What a function does the Tat protein of HIV confers during HIV infection?

It binds to TAR, increasing the processivity of the RNA polymerase alon the proviral template. It is a transcription factor that binds to RNA but not DNA

Describe how the rev protein of HIV regulate HIV gene expression

It binds to the Rev response element (RRE) in the virus RNA, shifting the virus from early to late protein synthesis

Describe how HIV Gag-Pol polyprotein is produced.

During RNA genome replication, 5% of cases in unspliced transcripts undergo a frameshift to produce the Gag-pol protein

What's the cell receptor for HIV?

CD4

What's the function of HIV vif protein?

it hinders host immune system by targeting APOBEC3 cell proteins for degredation, preventing them from being incorporated into the virus

HIV is resistant to host immune systems, why?

First. HIV targets and destroys CD4 T cells, which play pivotal roles as helpers for several cell types including B cells, cytotoxic T cell precursors, natural killer cells and macrophages, hence immune responses are impaired. Second, the virus evolves as the infection proceeds, producing new antigenic variants that may not be recognized by the antibodies and T cells present. The third, in latently infected cells the virus is shielded from the immune system.

Which HBV protein contains the virus attachment site?

L protein

Describe how HBV enters host cell.

endocytosis

In HBV virion, a P protein is often covalently linked to which strand of viral genomic DNA?

the (-) strand aka the long strand

Describe how the P protein of HBV is translated.

the ribosome bypasses the upstream start codons by Leaky scanning of the long mRNA to produce the P protein

Does HBV genome replication require a primer? If yes, what's the nature and identity of the primer?

yes, the P protein

Which HBV proteins can not be found in the non‐infectious HBV particles?

P protein (HBV polymerase)

Does the formation of covalently closed circular DNA (cccDNA) of HBV occur in cytoplasm?

no, the nucleus

Describe how HBV genes are expressed? (hint: what's the transcription template? which host enzyme perform transcription to produce viral mRNAs? How many types of mRNAs produced? Do all viral mRNAs have the same 3' ends? How many types of proteins are produced by HBV?)

As the host RNA polymerase II transcribes viral RNA, 4 types of RNA are produced which in turn produce 7 different proteins. They all have coterminal 3' ends

Describe the structure feature of influenza viruses (hint: envelop, segmented genome, genome constitution, matrix protein, nucleocpasid symmetry etc.)

Envelope: hemagglutinin and neuraminidase surface proteins on a lipid bilayer comprised from host cells

Genome: (-)RNA with 7-8 segments

Matrix protein: M1 forms a rigid structural layer just under the envelope while M2 is crucial for uncoating

Nucleocapsid symmetry: helical rod-shaped

Which enzyme replicates the genome of influenza viruses?

PB1/ viral RNA dependent RNA polymerase

Can Tamiflu/oseltamivir be used to treat influenza virus infection?

yes

How do influenza viruses enter host cell?

endocytosis

cell receptor: sialic acids

Where do Influenza A viruses replicate their genomic RNAs within host cells (hint: cytoplasm or nucleus)?

nucleus

Transcription of Influenza A virus mRNAs needs primers. Where do the primers come from?

they are derived from host cell pre-mRNAs within the host nucleus

PB2 and PA perform "Cap Snatching" to prepare a primer

Describe the difference between antigenic drift and antigenic shift.

Drift: occurs over time due to the natural variation of the viral genome across generations

Shift: occurs due to reassortment of multiple strains present within a single animal reservoir

What amino residues does trypsin cleave after?

lysine and arginine

What amino acid residues do chymotrypsin cleave after?

phenylalanine, tyrosine, tryptophan

common features of dsRNA

•Replicate in cytoplasm

•Viral genomes can not be directly translated

•Always carry RdRP required for genome replication and mRNA production

•Have to use their enzyme to add cap structure to their own mRNAs, if such a structure is required for translation

•The genome is a potent trigger of RNAi-based antiviral mechanism

Icosahedral viruses with dsRNA genomes isolated from the _____and _____ tracts of humans and animals, and with which no disease could be associated (____), became known as reoviruses

respiratory, enteric, orphan

haw does orbivirus replicate?

replicates in vectors

Rotavirus

member of Reovirus family

• triple layers

  • Inner layer = VP2

  • middle layer = VP6

  • outer layer = glycosylated VP7

• VP1 AND VP3 in core

What do VP1 and VP3 protein do for rotavirus

the RNA-dependent RNA polymerase

What does VP3 do for rotavirus

provides guanylyl transferase and methyl transferase activities

Each RNA segment in rotavirus encodes ___ protein

1

How many proteins are encoded by rotavirus?

12 proteins

• 6 VP

• 6 NSP

(+) ssRNA (Positive-Sense)

Genome Status: Acts directly as mRNA. Immediate translation by host ribosomes. Enzymes: Encodes RdRp in its genome but does not need to carry the protein in the virion. Sub-Groups: Unenveloped: Picornaviruses (Polio, Rhino), Caliciviruses (Norovirus). (Resistant to detergents/acid). Enveloped: Coronaviruses, Flaviviruses (Hepatitis C, West Nile), Togaviruses.

(-) ssRNA (Negative-Sense)

Genome Status: Complementary to mRNA. Must be transcribed into (+) RNA before translation. Enzymes: MUST carry RdRp protein inside the viral particle (otherwise it's dead on arrival). Structure: All are Enveloped. The 'S' List (Segmented): Orthomyxoviruses (Flu), Bunyaviruses, Arenaviruses. Note: Segmentation allows for Antigenic Shift. Non-Segmented: Rhabdoviruses (Rabies), Filoviruses (Ebola), Paramyxoviruses (Measles/Mumps).

(+/-) dsRNA (Double-Stranded)

Genome Status: Double-stranded; one strand is used as a template for mRNA. Enzymes: Must carry RdRp to transcribe the dsRNA. Key Feature: Double Capsid. Why? dsRNA is a potent trigger for the host's innate immune system (Interferon). The capsid stays intact during transcription to 'hide' the dsRNA. Example: Reoviruses (Rotavirus).

(+) RNA via DNA

Genome Status: Two identical copies of (+) ssRNA. Enzymes: Carries Reverse Transcriptase (RT) and Integrase. Process: RNA \rightarrow DNA \rightarrow Integrated into host genome (Provirus). Structure: Enveloped. Example: Retroviruses (HIV, HTLV).

Which viruses carry their own polymerase?

Do carry it: (-) ssRNA, dsRNA, and Retroviruses (Reverse Transcriptase). Do NOT carry it: (+) ssRNA (except Retroviruses). Logic: If the genome cannot be read directly by a ribosome (like (-) or dsRNA), the virus has to bring the 'machinery' to fix that problem immediately.

Translation of ____ involves the mechanism leaky scanning

NSP6

rotavirus targets _____ for replication

enterocyets

• cells on small intestine

How rotavirus enters a host

direct penetration from spikes or endocytosis

rotavirus proteins accumulate in cytoplasm regions known as ____

viroplasm

packaging signal in rotavirus

sequence that efficient rna synthesis and packaging of viral genomes into new virions.

dsRNA of infecting rotavirus is conservative/nonconservative

conservative

rotavirus late transcription occurs within the ____ layered particles

double

rotavirus transcription produces capped/uncapped transcripts by VP1?

uncapped

T/F the mRNA species are not made in equal numbers in rotavirus

True

what proteins help rotavirus bind and enter into the rough ER?

NSP4 with binding sites for VP4 and double layered particles

• vesicle membrane forms" “envelope” with VP7

• cleavage of VP7 releases them into the cell

What happens first to the virion after entry?

Outer Layer Removal: The outermost layer is stripped away. Result: Creates a Double-Layered Particle (DLP). Activation: This structural change 'turns on' the transcription machinery inside the particle.

How is new RNA made within the particle?

Entry: Free nucleosides enter the DLP through specialized perforated channels. Synthesis: VP1 (the polymerase) synthesizes new (+) RNA copies. Exit: These new mRNAs are 'extruded' (pushed out) through the channels into the host cytoplasm to be translated.

How is the new viral RNA modified?

5' End: VP3 adds a cap to the 5' end (helping it look like host mRNA to ribosomes). 3' End: These transcripts are NOT polyadenylated (they lack a polyA tail). Key Exam Fact: This distinguishes them from most host mRNAs and many other (+) RNA viruses.

Why keep the RNA inside the double-layered particle?

Immune Evasion: dsRNA is a 'Danger Signal.' By keeping the dsRNA genome locked inside the protein layers, the virus prevents host sensors (like TLR3 or RIG-I) from detecting it and triggering an interferon response.

T/F Rotavirus is released from the cell by lysis or exocytosis

True

is dsRNA directly translated into the host cell?

No, dsRNA is not directly translated. It must first be transcribed into mRNA by viral RNA-dependent RNA polymerase before being translated by the host's ribosomes.

• stays in capsid to avoid immune detection by host

Why do reovirus have a double capsid?

inner core = transcription factory

outer core = protection + entry

what is the activation step for rotavirus with VP4?

The activation step involves cleavage of the VP4 protein by trypsin (VP5 + VP8), which allows the virus to attach to the host cell and initiate infection.

What is the function of VPg protein?

• replaces 5’ cap

• used as primer for replication

• removed before translation

What is the mechanism for polio infection?

1. Poliovirus binds to the CD155 receptor on host cells.

2. VP4 is released, allowing VP1 to form a pore in the cell membrane.

3. Capsid rearrangement occurs, facilitating genome delivery into the host cell.

What is a key feature of -ssRNA viruses, such as rhabdoviruses, regarding their infectious ability?

-ssRNA is NOT infectious; it must be converted to +RNA first before it can be translated by host ribosomes.

Describe the transcription gradient of rhabdovirus.

Genes closer to the 3’ end of the genome are more expressed, leading to a gradient of protein abundance. The polymerase can fall off during transcription, resulting in varying levels of protein production along the genome.

Describe the multifunctional role of the L protein in -ssRNA viruses like rhabdoviruses.

The L protein functions as a 'mini nucleus' enzyme system handling multiple tasks including:

• Capping

• Methylation

• Polyadenylation

Describe the major functions conferred by rhabdovirus M protein.

The M protein forms a layer between the nucleocapsid and the envelope, condenses the nucleocapsid into a tight helix, binds it to a region of the plasma membrane, and drives virion release by linking the nucleocapsid to the membrane.

What is the first step for +ssRNA viruses after entry?

Translation occurs first.

What is the first step for -ssRNA viruses after entry?

Transcription occurs first; they must bring their own RNA-dependent RNA polymerase (RdRp).