(+) ssRNA viruses (poliovirus, coronavirus, sindbis, and norwalk)

Question 1

Which one of the statement regarding poliovirus is true?

1) It contains an enveloped spherical capsid.

2) The capsid has icosahedral arrangement made of 60 promoters, each consisting of 8 proteins.

3) It is present in the feces of infected animals and normally infects the cells of the GI tract.

4) Complications of the virus result in the infection of sensory neurons leading to paralysis and death (poliomyelitis)

Answer 1

3!

1) It contains a non-enveloped spherical capsid

2) The capsid has icosahedral arrangement made of 60 promoters, each consisting of 4 proteins (VP1-4)

4) Complications of the virus result in the infection of MOTOR neurons leading to paralysis and death (poliomyelitis) (due to virus changing its tropism and attacking neural cells).

Question 2

(T/F) Poliovirus virion-packaged RNA serves as both a genome and a mRNA.

Answer 2

True!

Question 3

Fill in the blanks of the poliovirus genome:

It is __________ and has a ______ (+) ssRNA genome of 8kb.

It is composed of a single ____________ that encodes a single ________.

A viral protein (VPg) located at its ___ end instead of a methylated cap structure.

The ______ contains a type of internal ribosome entry site.

Answer 3

Monopartite; linear

Open reading frame; polyprotein

5’

UTR

Question 4

Briefly answer the following questions regarding Poliovirus:

1) What is its family and genus?

2) What is its tropism?

3) What is the tropism of rhinovirus which is in the same family as poliovirus?

4) What receptor does it interact with?

5) What disease is it associated with?

Answer 4

1) Family: Picornaviridae. Genus: Enterovirus

2) The gastrointestinal tract in the primary site of infection

3) Upper respiratory tract is the primary site of infection

4) PVR (poliovirus receptor)

5) Polyomyelitis (paralysis, summer cold, meningitis, diarrhea)
*due to virus changing its tropism; instead of attacking the GI tract, it enters neural cells (full body paralysis + death)

*rhinovirus; common cold

Question 5

What are the two key characteristics of the polyprotein encoded by the poliovirus?

Answer 5

1) The P1 region encodes structural polypeptides (forms capsid proteins)

2) The P2 and P3 regions encode non-structural proteins linked to REPLICATION (impt 4 infection)

Question 6

What does IRES stand for?

Answer 6

Internal Ribosome Entry Sites

Question 7

What is the function of IRES?

Answer 7

IRES are structures of RNA that fold onto itself, which allows for a CAP-INDEPENDENT initiation of translation of UNCONVENTIONAL RNAs (no Cap or poly A).

After being recognized by the translational machinery, it allows a virus to SHUT OFF the cell’s translation machinery and produce its own proteins!

Question 8

Briefly describe the Canonical Cap-DEPENDENT initiation of translation.

Answer 8

1) elF4E binds to the CAP and contributes to the recruitment of the 40s ribosome

2) Recruitment of the other eukaryotic initiation factors including eIF2 (holds the tRNAmet)

3) Scanning in the 5’ to 3’ direction for the first AUG with a Kozak sequence

4) Once the AUG is identified, the 60s ribosomal unit binds to the complex to form the 80s ribosome.

5) eIF2 mediates the binding of tRNAmet to the ribosome in a GTP-dependent manner.

Question 9

(T/F) In the canonical cap-dependent initiation of translation, the mRNA loops back until it reaches the polyA tail bound by protein PABP. In this setup, the same pool of ribosome can go through the mRNA and come back for additional protein synthesis.

Answer 9

True!

Question 10

The poliovirus encodes 2 proteases; _____ causes the proteolytic cleavage of cellular eIF4G, while ____ cleaves the poliovirus peptide.

Answer 10

2A^pro, 3C

*2A^pro prevents eIF4G to bind other cellular mRNAs (cuts off section that binds the cap-binding protein 4E)

Question 11

Fill in the blanks regarding cap-independent translation:

Most viral IRES sequences recruit the 40s ribosome through ________.

Repression of Cap-dependent translation ________ IRES-dependent translation.

The 40s ribosome binds downstream of __________ tract.

Answer 11

ITAF (expressed by host)

Increases

Polypyrimidine

Question 12

Match the terms to their definitions:

1) ITAF
2) VPg

a) Viral Protein Genome-linked. Acts as a 5’ CAP for translation initiation and as a PRIMER during RNA synthesis. Present in many negative-sense RNA viruses.

b) IRES trans-acting factor (host-encoded protein)

Answer 12

ITAF: IRES trans-acting factor (host-encoded protein)

VPg: Viral Protein Genome-linked. Acts as a 5’ CAP for translation initiation and as a PRIMER during RNA synthesis. Present in many negative-sense RNA viruses.

Question 13

(T/F) An mRNA without a cap structure can synthesize proteins using an IRES. It leads to polycistronic vectors that produce very large amount of proteins.

Answer 13

True!

Question 14

Briefly describe the first stages of poliovirus replication cycle.

Answer 14

First, the poliovirus attaches to cells that is mediated by the poliovirus receptor (Ig like protein) and internalized using a pore-mediated penetration mechanism, where there is the injection of the RNA into the cytoplasm.

This is followed by mRNA being translated in an IRES dependent manner to create the proteins.

Question 15

(T/F) The RdRP is encoded by the poliovirus genome.

Answer 15

True!

*not needed for protein production, needed for genome replication

Question 16

What happens after the translation of the first polyprotein of poliovirus?

*mention proteases involved and how cellular protein translation is arrested

Answer 16

After the translation of the first polyprotein, PROTEOLYTIC CLEAVAGE of the polyprotein into constituents by the 3C PROTEASE occurs.

The 2A^pro protein causes PROTEOLYTIC CLEAVAGE of cellular eIF4G.

2A^pro with 3C^pro causes the degradation of PABP, reducing the stability of endogenous mRNAs.

Now, there is an arrest of cellular protein translation.

Question 17

What is PABP?

Answer 17

Poly(A)-binding protein (PABP) protects the poly(A) tail of cellular mRNAs from nuclease degradation.

It is degraded by the polio proteases, resulting in the host polyA tail no longer being protected.

Question 18

Besides cleaving cellular eIF4G and PABP, what else does protease 2A cleave?

How is this beneficial for the virus?

Answer 18

It cleaves nuclear pore proteins Nup214 and Nup62.

This prevents the exports of cellular mRNAs.

This gives viral mRNAs a competitive edge to recruit translation factors (increases IRES translation).

Question 19

Poliovirus replicates its RNA in ________ _____, which helps to hide _________ ______ (dsRNA from innate sensors).

Answer 19

double-membrane vesicles; replication intermediates

*all replication intermediates are dsRNA

Question 20

Host-encoded _______ (aka unlinkase) cleaves off the 5’ _____ creating an RNA with a 5’ ____ used for replication in poliovirus replication cycle.

Answer 20

TBP2; VPg; pUp

*a viral protein (VPg) located at its 5’ end instead of a methylated cap structure in the mRNAs of polioviruses

Question 21

What happens to the cleaved VPg?

Answer 21

Its tyrosine residues become linked to URACIL nucleotides that will serve as PRIMERS to replicate the genomic RNA.

Uracil base-pairs with the sequence of the CRE element.

Question 22

Along with VPg, CRE and Cloverleaf are critical elements in the poliovirus replication cycle. What are their functions?

Answer 22

CRE is also known as Cis-active Replication Element.

TFs and the polymerase assemble at CRE and Cloverleaf.

Question 23

Replication of poliovirus genome is cap _____ and primer _______.

Answer 23

Independent; dependent

Question 24

(T/F) In poliovirus replication, the (+) strand genomic RNA is used to generate (-) strand using strand displacement, and the (-) strands are then used to generate (+) strand genomes using strand displacement.

Answer 24

True!

Question 25

There are a series of ____ at the 3’ OH of the poliovirus genome.

Answer 25

A’s

Question 26

Briefly answer the following questions regarding the late stages of the poliovirus life cycle:

1) Where do the poliovirus capsids assemble?

2) What does poliovirus protein 2B do?

3) How are the virions released?

Answer 26

1) They assemble at the vesicle surface where the (+) ssRNA is released

2) 2B induces vesicle membrane PERMEABILITY ‘PORES’ to enable the release of genomic (+) ssRNA (some are translated, others serve for gRNA)

3) Cell lysis!

Question 27

Coronavirus is a _________ ss (+) RNA virus, with a _________ and ____________ genome.

Its (+) ss RNA genome is encapsidated by __________ protein.

Answer 27

Enveloped; large; non-segmented

Nucleocapsid protein (N)

Question 28

Answer the following questions regarding coronavirus:

1) Which diseases is it associated with?

2) What is its tropism?

3) How is it transmitted?

4) What does SARS-CoV interact with on host cells?

Answer 28

1) Mainly respiratory diseases and gastroenteritis: Severe Acute Respiratory Syndrome (SARS)

2) Primary site of infection is the epithelial cells of respiratory or enteric tracts. NEUROLOGICAL TISSUES are also frequently infected.

3) Zoonosis (needs to get into animals and then to humans), fomite. Respiratory or fecal-oral in humans.

4) ACE2

Question 29

The order of coronavirus is ________, the family is __________, the subfamily is ________, while the genus are:

Answer 29

Nidovirales; coronaviridae; coronavirinae; alpha/beta/gamma/deltacoronavirus

Question 30

(T/F) The spike proteins of coronavirus are heavily glycosylated, which causes immune system to recognize it easily.

Answer 30

False. The spike proteins of coronavirus are heavily glycosylated, which HELPS EVADE THE IMMUNE SYSTEM.

Question 31

Spike protein interactions with the cellular receptors (ACE2) initiate _________.

Spike, in combination with host factors (what kind?), promote ______ ______ and ________ at the cellular or endosomal membrane.

Then, there is a release of the ss(+)RNA in the _________ of the infected cell.

Answer 31

Contact

Viral uptake; fusion

*host factors such as cell surface serine protease TMPRSS2 which cleaves S2’ of the spike protein.

Cytoplasm

Question 32

Fill in the blanks regarding the coronavirus genomic organization:

1) There are ____ open reading frames in the viral genome that encode for 2 proteins: ________ and ________.

2) __________ encodes for polyprotein Pp1a, while _________ encodes for Pp1ab.

3) Pp1ab polyprotein include _________.

4) __________ is generated by ribosomal frameshifting.

Answer 32

1) 2; ORF1a; ORF1ab

2) ORF1a; ORF1ab

3) RNA dependent RNA polymerase

4) ORF1ab

Question 33

Which one of the statements regarding coronavirus genomic organization is true?

1) All other proteins besides Pp1a and Pp1ab are encoded on subgenomic RNAs and they all have a 5’7mG cap and no polyA tail

2) All the subgenomic RNAs are synthesized immediately

3) Nsp14 has an exonuclease activity and therefore participates in the proofreading activity of the polymerase

4) Most drugs target PL proteinase (Ns3) which cleaves the 1st parts of the coronavirus mRNA

Answer 33

3!

1) All other proteins besides Pp1a and Pp1ab are encoded on subgenomic RNAs and they all have a 5’7mG cap and A polyA tail

2) All the subgenomic RNAs are NOT synthesized immediately

4) Most drugs target 3CL protease (Ns5) which cleaves the later parts of the coronavirus mRNA

Question 34

What is ribosomal frameshifting?

Answer 34

It is an alternative mechanism to merge proteins encoded by two overlapping ORF. Ribosome travelling down the mRNA will hit something (pseudoknot) that slows it down. Then, it goes back and tries again.

Going back and forth, it will slip one base toward the 5’ (referred to as -1 frameshifting) or toward the 3’ (referred to as +1 frameshifting), changing the ORF, introducing new sets of aas.

The frameshift occurs at low frequency.

It is commonly used by viruses.

Question 35

(T/F) All cis-acting frameshift signals encoded in mRNAs are minimally composed of two functional elements: a hepta nucleotide “slippery sequence” and a pseudoknot that is 5-9 nucleotides downstream.

Answer 35

True!

Question 36

What happens to polyproteins pp1a and pp1ab after being produced?

Answer 36

They are co-translationally and post-translationally processed into the individual non-structural proteins (nsps) that form the viral replication and transcription complex.

Question 37

Double-membrane vesicles (DMVs), convoluted membranes (CMs) and small open double-membrane spherules (DMSs) create a protective environment for (replication/transcription complex):

Answer 37

1) viral genomic RNA replication

2) transcription of subgenomic mRNAs which are a series of nested sets mRNAs

Question 38

Briefly answer the following questions regarding coronavirus genomic organization:

1) What is a subgenomic RNA?

2) How are subgenomic RNAs produced?

3) How many subgenomic RNAs are there in coronavirus and what do they code for?

4) What similarities do subgenomic RNAs share + how are they different?

Answer 38

1) A subgenomic RNA is a product transcribed from a (-) ssRNA intermediate

2) They are produced by DISCONTINUOUS TRANSCRIPTION of the (+) genomic RNA

3) There are 8 subgenomic RNAs (+) that code for STRUCTURAL PROTEINS

4) Subgenomic RNAs all contain the same 5’ leader sequence and 3’ sequence, but contain a unique AUG sequence.

Question 39

(T/F) The (+) genome of coronavirus can either be transcribed as a full (-) strand or as truncated (-) strands that can be served as templates for subgenomic mRNAs.

Answer 39

True!

Question 40

What is discontinuous transcription? How does it occur? What does it produce?

Answer 40

Discontinuous transcription occurs, presumably due to looping of the positive strand RNA during transcription and “jumping” of the RdRp.

When RdRp reaches a Transcription Regulatory Sequence (TRS), it disengages until it meets another TRS.

It generates truncated (-) stranded RNAs that serve as templates for (+) SUBGENOMIC mRNAS.

Question 41

Subgenomic mRNAs are ______ more abundant than the full-length (-) strand RNA.

The transcription process and efficiency is much ______ than the full length (+) RNA. This helps produce more _______ ________ _____.

Answer 41

70x

higher; structural capsid proteins

Question 42

What happens when structural proteins are translated during coronavirus replication cycle? How does the virion exit the host cell?

Answer 42

Translated structural proteins TRANSLOCATE into the ER membranes and TRANSIT through the ER-to-Golgi intermediate compartment (ERGIC), where interactions with N-encapsidated, newly produced genomic RNA results in budding into the lumen of secretory vesicular compartments.

Virions are secreted from the infected cells by EXOCYTOSIS.

Question 43

Sindbis virus is an _______ (+) ssRNA virus of the ________ family and ________ genus.

It is transmitted by ___________ and zoonosis.

It interacts using ______ ______ on ______ receptors.

Answer 43

Enveloped; togaviridae; alphavirus

Arthropods (mosquitoes)

Heparan sulfate; laminin

Question 44

What are the four spectrum of disease symptoms caused by Sindbis?

Answer 44

1) mild, unapparent infections (typical)
2) febrile illness (with/ without rash)
3) arthritic manifestations
4) encephalitis

*not lethal!

Question 45

The initial events of the Sindbis virus lytic cycle starts with the binding of _________ RECEPTOR and _________.

Following entry, __________ of the vesicules contributes to the _______ of the virus and thereby release of its _________, which can be READILY TRANSLATED.

Answer 45

LAMININ; ENDOCYTOSIS

ACIDIFICATION; uncoating; (+) ssRNA

Question 46

Non-structural proteins (including RdRp) are translated from the _____ RNA in the ______ stage.

Structural proteins are translated from a _______ mRNA in the ______ stage.

The RdRp (_____) is expressed by suppression of translation _________.

Answer 46

genomic; early

subgenomic; late

nsP4; termination

Question 47

(T/F) Translation termination is the same as translation reinitiation.

Answer 47

False! They are not the same.

Question 48

What are the three stop codons? What are they normally recognized by?

Answer 48

UGA, UAA and UAG

They are normally recognized by a RIBOSOMAL RELEASE FACTOR (eRF) rather than a tRNA.

Question 49

What is a readthrough? What induces readthroughs?

Answer 49

Readthrough is caused by a pausing of the translation that is long enough to allow for insertion of NON-OPTIMAL amino acid (anticodon mispairing).

Structural factors such as RNA pseudoknot or specific downstream nucleotides sequences can induce a readthrough.

*impt for supression of translation termination

Question 50

(T/F) Efficiency of the readthrough is between 5% to 30% depending on the virus. In the case of the Sindbis virus the stop codon is UAA and the readthrough is 10%

Answer 50

False!

Efficiency of the readthrough is between 5% to 30% depending on the virus. In the case of the Sindbis virus the stop codon is UGA and the readthrough is 10%

Question 51

During sindbis virus transcription, the non-structual proteins are translated first from the genomic RNA into a polyprotein called _____ and _______.

Then in later stages, the _______ protease sequentially processes the polypeptide into its basic components.

Answer 51

P123; P1234

nsP2

Question 52

Match the following steps of sindbis upon infection:

1) Step 1
2) Step 2
3) Step 3
4) Step 4

A) It is then transcribed to produce a full length (-) strand RNA

B) The subgenomic RNA contains a DOWNSTREAM HAIRPIN LOOP (DLP) that enables cap-dependent translation to produce the 26s mRNA.

C) The (+) strand RNA is translated first.

D) The (-) strand genome serves as a template to produce a full length copy of the (+) strand viral genome and a subgenomic mRNA (26s) that codes for structural proteins.

Answer 52

Step 1: The (+) strand RNA is translated first.

Step 2: It is then transcribed to produce a full length (-) strand RNA

Step 3: The (-) strand genome serves as a template to produce a full length copy of the (+) strand viral genome and a subgenomic mRNA (26s) that codes for structural proteins.

Step 4: The subgenomic RNA contains a DOWNSTREAM HAIRPIN LOOP (DLP) that enables cap-dependent translation to produce the 26s mRNA.

Question 53

What is the function of Downstream Hairpin Loop (DLP)?

Answer 53

DLP are RNA structures that allow initiation of cap-dependent translation in the absence of eIF2 initiation factor, where the initiating methionine tRNA is placed by the DLP structure on the ribosome.

• They help avoid the Cap-dependent translational shutoff when dsRNAs are detected by PKR.
• PKR phosphorylates eIF2 which inhibits its RNA binding activity.
• DLP REPLACES THE FUNCTION OF eIF2

Question 54

The subgenomic RNA of sindbis is translated in a CAP-_______ but eIF2 ________ manner.

Answer 54

dependent; independent

Question 55

Norwalk virus is a ___________ virus composed of 180 _____ proteins.

It is the leading cause of _________ illness.

It has a (+) _______ ss RNA.

Answer 55

non-enveloped; VP1

foodborne

linear

Question 56

Briefly answer the following questions about Norwalk virus:

1) What is its family + genus?

2) What disease is it associated with?

3) How is it transmitted?

4) What is its tropism?

5) What are the cell receptors it interacts with?

6) What are its natural hosts?

Answer 56

1) Family: caliciviridae, Genus: norovirus

2) Gastroenteritis

3) Fecal-oral route from contaminated water and food

4) Intestinal epithelium

5) Histo-blood group antigens

6) Human and mammals

Question 57

(T/F) Norwalk is a very small and very infectious virus. It has relatively acute effects because host immune system is very reactive to non-enveloped viruses.

Answer 57

True!

*needs only 20 viral particles to get sick

Question 58

Fill in the blanks regarding first stages of Norwalk life cycle:

The capsid attaches to the cell surface through interactions between ______ and host _____-_____ group antigens (HBGAs) and is subsequently _______, _______ and ________.

The (+)ssRNA is then transcribed and translated in the ________ of the host cell.

Translation is mediated by ______ translation factors that are recruited by the non-structural virus protein ______, which ______ binds to the __‘end of the genome.

Answer 58

VP1; histo-blood; internalized; uncoated; disassembled

cytoplasm

host; Vpg; covalently; 5’

Question 59

(T/F) The norwalk RNA has a VPg (Viral protein genome linked) but is not polyadenylated.

Answer 59

False!

It has VPg and is ALSO polyadenylated.

Question 60

Fill in the blanks regarding the Norwalk virus genomic organization.

There are ______ open reading frames. _______ protein is made from the translation of a SUBGENOMIC RNA.

Both structural (capsid) proteins ______ (CP) and _______ are encoded by the SAME subgenomic RNA.

_______ is produced by ribosomal termination-reinitiation.

Answer 60

3; ORF2

VP1; VP2

VP2

Question 61

What is RNA termination-reinitiation?

Answer 61

Ribosomal termination-reinitiation is different from suppression of termination or frameshift.

It is a process which allows expression of a downstream ORF in a BICISTRONIC mRNA.

In this process, ribosomes translate the upstream ORF until it reaches a stop codon. By following termination, a proportion of 40S subunits remain tethered to mRNA while the 60s disengages.

Given that a start codon is close proximity, the 40S repositions and reinitiates translation using this new start codon and translates the NEW open reading frame.

This results in two SEPARATE proteins (ORF1 and ORF2)!

Question 62

Fill in the blanks regarding the last stages of Norwalk virus life cycle:

The polyprotein encoded by _______ is post-translationally cleaved by the virus-encoded protease, ______ (aka NS6 or 3C-like), into individual proteins.

Then, there is genome ______ and production of genomic and ________ ss(+)RNA.

Lastly, there is _________ where genomic and possibly subgenomic ss(+) RNAs are packaged into new virions.

Answer 62

ORF1, pro

replication; subgenomic

encapsidation