(-) ssRNA viruses (rabies, influenza, ebola)

Question 1

All negative-strand RNA viruses must package a _______ in their _______.

Why?

Answer 1

RdRp; capsid

The first step of the replication cycle involves producing positive-sense RNAs for protein translation and to serve as a template for genomic negative-sense RNA production.

Question 2

Rabies is an _______, __________ (-) stranded RNA virus.

Its order is ________, while the family it belongs to is ___________.

Answer 2

enveloped, non-segmented

Mononegavirales; rhabdoviridae

Question 3

Briefly answer the following questions regarding rabies virus.

1) What is the tropism?

2) What are the symptoms?

3) What is the disease it is associated with?

4) How is it transmitted?

5) What receptors does it interact with?

Answer 3

1) Neurons

2) Insomnia, anxiety, confusion, hallucinations, slight or partial paralysis, hydrophobia, and then death. It takes weeks to years for symptoms to manifest themselves and then it’s too late!

3) Rabies fatal encephalitis

4) It is transmitted by bites of infected animals along axons, across synaptic junctions to CNS.

5) NCAM1, NGFR, nAChR

Question 4

Briefly describe the steps leading to the release of ss(-)RNA into the cytoplasm of rabies virus.

Answer 4

Attachment of the viral G glycoproteins to host receptors (neural cell adhesion molecules 1 - NCAM1) mediates CLATHRIN-MEDIATED ENDOCYTOSIS of the virus into the host cell.

Fusion of virus membrane with the vesicle membrane; ribonucleocapsid is released into the cytoplasm.

Then, there is the release of ss(-)RNA into the cytoplasm.

Question 5

What are the 5 proteins encoded by the rabies virus?

Answer 5

N: nucleocapsid

P: Phosphoprotein (transcription cofactor)

M: Matrix protein

G: Envelope glycoprotein

L: RdRp

*SYNTHESIS OF THE 5 PROTEINS NEEDED TO MAKE THE RABIES VIRUS

Question 6

Transcription always starts at the ____’ end of the (-) strand RNA.

Answer 6

3’

(goes in a 5’–>3’ direction)

Question 7

(T/F) For rabies virus, RdRp binds to the leader sequence and there is a gradient accumulation of mRNAs by sequential transcription.

Answer 7

True!

Question 8

There is _______ _______ at the intergenic regions, where the polymerase either falls off (frequent) or continues (less frequent).

Polymerase doesn’t always reinitiate transcription at ________ sequences.

Answer 8

Polymerase stuttering

Intergenic

Question 9

(T/F) mRNAs from the 3’ end of the genome are less abundant than those from 5’ end.

Answer 9

False!

mRNAs from the 3’ end of the genome are MORE abundant than those from 5’ end.

Question 10

Briefly describe Polymerase suttering.

Answer 10

When RdRp hits a sequence of 7 U residues, it starts to stutter. This leads to the addition of approx 200 A’s (poly A tail) and termination then occurs.

The RdRp can either fall off or initiate transcription of the following ORF immediately after the NA nucleotide (which are not transcribed into the mRNA sequencE).

Question 11

Which one of the statements regarding rabies virus transcription is true?

1) Transcription generates a polyA tail on the mRNAs

2) RdRP has proofreading abilities

3) mRNAs are not capped

Answer 11

1!

For 2: RdRp does NOT HAVE proofreading abilities.

For 3: mRNAs contain a m7G cap.

Question 12

When there is an abundance of ___ and ___ proteins, nascent mRNAs get rapidly coated.

__ proteins maintain ___ proteins soluble and amenable to binding mRNAs.

Coating of the (+) stranded RNA enables transcription through the _______ regions without _________.

Answer 12

N (nucleocapsid) and P (phosphoprotein)

P; N

Intergenic; stuttering

*coating allows synthesis of full (+) strand, which is then used to generate a full (-) strand genome!

Question 13

At the final stages of the rabies virus replication cycle, what is the role of glycoprotein (G), phosphoprotein (P), RdRp (L), and matrix protein (M)?

Answer 13

Glycoprotein (G) enters secretory pathway and is embedded in plasma membrane.

Nucleocapsid with associated phosphoprotein (P) and RdRp (L) are organized into BULLET-SHAPED core by matrix protein (M).

Question 14

The rabies virus core buds through membrane at sites of ?

Answer 14

G-protein accumulation

Question 15

What is the role of matrix protein (M) in the infectious cycle?

Answer 15

Blocks nuclear pores and prevents cellular molecules from exiting nucleus.

Question 16

What happens around ~1 hour and ~8 hours after rabies virus infection?

Answer 16

Rapid shut down of host cell RNA, DNA and protein synthesis beginning ~1hour after infection.

Cell death ~8 hours after infection.

Question 17

The infleunza A virus is an ________, ________ (-) stranded RNA virus.

There are three members at the genus level: ____, _____ and _____.

Subtypes refer to the __________ and ________ proteins.

Answer 17

enveloped, segmented

A, B, and C

Hemmaglutinin (HA) and Neuraminidase (N)

Question 18

Briefly answer the following questions of influenza A virus:

1) What is its family?

2) Who are its hosts (reservoirs)?

3) What is its tropism?

4) Which cell receptors does it interact with?

5) How is it transmitted?

Answer 18

1) Orthomyxoviridae

2) Aquatic birds, human, pig, horse, seal

3) Epithelial respiratory cells

4) Sialic acids

5) Mammals: respiratory zoonosis, animal contact. Birds: fecal-oral route from contaminated water.

Question 19

(T/F) While hemmaglutinin is important for the attachment of the virus, neuraminidase is important for the release of virus.

Answer 19

True!

Question 20

Briefly describe the first 3 steps of influenza A virus replication cycle (entry –> production of mRNAs).

Answer 20

1) Virus attaches to SIALIC ACID through the HA protein and penetrates the host cell by CLATHRIN-MEDIATED ENDOCYTOSIS

2) Endosome ACIDIFICATION induces fusion of virus membrane with the vesicle membrane and encapsidated RNA segments migrate to and PENETRATE THE NUCLEUS.

3) Viral RdRp transcribes all the genomic genes to produce mRNAs and express viral proteins using CELLULAR mRNAs as primers (CAP-SNATCHING) in the nucleus. Once produced, these mRNAs are transported into the CYTOPLASM.

Question 21

There are ____ segments that encode for ____ proteins in Influenza A virus.

Answer 21

8; 11

Question 22

What are the five molecular tricks used by Influenza A?

Answer 22

1) Cap snatching
2) Polymerase stuttering
3) Ribosomal frameshifting
4) Alternative initiation (leaky scanning)
5) Alternative splicing

Question 23

What is Cap Snatching? How is it used in influenza?

Answer 23

The cap snatching is a transcription initiation process during which a nucleotide sequence between 10 and 20nt in size is cleaved by an endonuclease encompassed with in the RdRp.

In influenza, the capped leader obtained is subsequently used to prime transcription on the viral genome, which ultimately leads to the synthesis of capped, translated viral mRNAs.

Question 24

Cap snatching occurs in the ______ for influenza.

Answer 24

Nucleus

Question 25

(T/F) The influenza virus uses cellular mRNAs that synthesize proteins for the interferon pathway to prime its transcription. The virus steals mRNAs that raise a defence against the virus!

Answer 25

True!

Another way the virus protects itself!

Question 26

What is alternative splicing?

Answer 26

Alternative splicing occurs when several splice donors and acceptors are eligible. This leads to transcripts having different splicing outcome (or not).

It is regulated by CELLULAR and VIRAL proteins which modulates locally the activity of splicing factors U1 and U2.

Question 27

What are the two pros of alternative splicing?

Answer 27

1) It offers the opportunity to encode several proteins with less messenger RNAs

2) It is a way to regulate the expression of proteins during the early/late phase of the LYTIC cycle

Question 28

Influenza RdRp needs a ________ for transcription and replication where their origins are _________.

RdRp binds a __________ sequence in the (-) RNA to generate a (+) RNA strand.

It binds a _______ sequence in the (+) RNA strand to generate a (-) RNA strand.

Answer 28

Primer; different

Leader

Trailer

Question 29

Fill in the blanks regarding influenza A viral proteins:

1) Viral proteins _____, ____ and _____ accumulate at the ER and are ________. They are secreted to the cell surface where they accumulate in the _______.

2) ____, _____, _____ and ____ are imported to the _______ and participate in the synthesis in full strand (+) strand RNAs and then (-) strand genomic RNAs.

3) ____ and ____ proteins are imported to the ______. ____ binds to the (-) RNAs (to form the nucleocapsid) and shuts down host mRNA synthesis.

4) ____ and ____ then induce ______ of the nucleocapsid attached to the (-) RNA to the site of viral budding (HA, NA, M1 and M2 accumulation)

Answer 29

1) M2, HA, NA; glycosylated; membrane

2) PA, PB1, PB2, NP; nucleus

3) M1, NS1; nucleus; M1

4) M1; NEP; export

Question 30

Influenza virus ____ can bind to CPSF30 and PABII and this inhibits pre-mRNA processing.

Answer 30

NS1

Question 31

Match influenza proteins to their definitions:

1) PB2
2) PB1
3) PA
4) NP
5) M1
6) M2
7) NS1
8) NS2

A) matrix protein 1 - major component of virion & shuts down host mRNA synthesis

B) transcriptase - elongation (of + RNA)

C) non-structural protein 2/NEP - vRNA nuclear export

D) non-structural protein 1 - RNA transport, translation, inhibits antiviral response, polyadenylation of cell mRNA

E) matrix protein 2 - integral membrane protein (ion channel)

F) transcriptase - cap binding

G) nucleoprotein - RNA binding; transport of vRNA

H) transcriptase - protease activity

Answer 31

PB2: transcriptase - cap binding

PB1: transcriptase - elongation (of + RNA)

PA: transcriptase - protease activity

NP: nucleoprotein - RNA binding; transport of vRNA

M1: matrix protein 1 - major component of virion & shuts down host mRNA synthesis

M2: matrix protein 2 - integral membrane protein (ion channel)

NS1: non-structural protein 1 - RNA transport, translation, inhibits antiviral response, polyadenylation of cell mRNA

NS2: non-structural protein 2/NEP - vRNA nuclear export

Question 32

Which one of the statements regarding Hemmaglutinin is true?

1) It is a GLYCOPROTEIN responsible for binding to SIALIC ACID on the host cell (lower respiratory tract). It is a hidden antigen.

2) It leads to the fusion of the viral envelope with the endosome for cytoplasmic release of the capsid.

3) There are at least 15 HA antigens in nature (H1, H2, H3 and H6 are found in human strains). H6 is mainly an avian antigen.

Answer 32

2!

1) It is a GLYCOPROTEIN responsible for binding to SIALIC ACID on the host cell (upper respiratory tract). It is an exposed antigen.

3) There are at least 18 HA antigens in nature (H1, H2, H3 and H5 are found in human strains). H5 is mainly an avian antigen.

Question 33

Which one of the statements regarding Neuraminidase (NA) is false?

1) It is an enzyme with glycoside hydrolase activity. It is an exposed antigen.

2) It hydrolases sialic acid

3) It is required for entry in the cell.

4) There are 9 NA antigens with 2 in humans (N1 and N2)

Answer 33

3! It is required for detaching from the cell when budding off.

Question 34

Match the terms to their definition:

1) Antigenic drift

2) Antigenic shift

A) It is a result of HETEROGENEOUS PACKAGING OF THE RNAs and/or RECOMBINATION OF RNAs. Multiple strains of viruses can be present in a host at the same time and there can be genetic recombination and reassortment of packaged viral RNAs yielding new strains.

B) It is a result of POOR FIDELITY OF ALL RdRp. It yields in variation in HA and NA sequences due to copying errors and selection (including immune selection). Errors occur every time the genome replicates. This results in single amino acid changes that accumulate over the course of time.

Answer 34

Antigenic drift: It is a result of POOR FIDELITY OF ALL RdRp. It yields in variation in HA and NA sequences due to copying errors and selection (including immune selection). Errors occur every time the genome replicates. This results in single amino acid changes that accumulate over the course of time.

Antigenic shift: It is a result of HETEROGENEOUS PACKAGING OF THE RNAs and/or RECOMBINATION OF RNAs. Multiple strains of viruses can be present in a host at the same time and there can be genetic recombination and reassortment of packaged viral RNAs yielding new strains.

Question 35

(T/F) Viruses are constantly “drifting” in humans and thus there is a requirement of new formulation fo vaccine every few years.

Answer 35

True!

Question 36

Some species, such as ______, act as INCUBATORS that foster genetic reassortment because they can get __________ by influenza strains from different animal species. These are generally ________ to this.

Answer 36

Swine; co-infected; asymptomatic

Question 37

Influenza type ____ is almost only found in humans and is less severe.

Influenza type ___ causes the most severe cases of flu.

Answer 37

B; A

Question 38

Which kinds of viruses cause deadly pandemics?

Answer 38

Viruses that display genetic shift.

We have little immunity to the antigens of these viruses as they normally do not infect humans.

Question 39

Which one of these statements is false?

1) Influenza A viruses are found in many different animals, including ducks, chickens and pigs

2) In birds, there are 16 different HA subtypes and 9 different NA subtypes.

3) Wild birds are the primary natural reservoirs for all subtypes of Influenza B viruses and are thought to be the sources of influenza B viruses in all other animals.

4) Most influenza viruses cause asymptomatic or mild infection in birds, but the range of symptoms vary greatly depending on the strain of viruses. H5 and H7 can cause death in chicken and turkeys.

5) Pigs are susceptible to avian, human and swine influenza viruses and thus can create a new virus when infected at the same time.

Answer 39

3!

Wild birds are the primary natural reservoirs for all subtypes of Influenza A viruses and are thought to be the sources of influenza A viruses in all other animals.

Question 40

What is the difference between the H1N1 and H5N1 strain of swine flu?

Answer 40

H1N1: affected the upper respiratory tract. It spread easily and was rarely fatal.

H5N1: affected the lower respiratory tract (lungs). It spread more slowly but was often fatal.

Question 41

Spanish flu: H__N__

Swine flu:

Avian influenza:

Answer 41

H1N1

H1N1/H5N1

H7N8

Question 42

Ebola is an _________ (-) ssRNA virus with a linear genome.

It is found in _________ and _________.

Fruit bats are believed to be an _______ reservoir.

Answer 42

enveloped

central Africa; South Asia

Asymtpmatic

Question 43

Briefly answer the following questions about Ebola:

1) What is its order, family and genus?

2) Who are its host?

3) What diseases are associated with it?

4) How is it transmitted?

Answer 43

1) Order: mononegavirales, Family: filoviridae, Genus: Ebolavirus

2) Bats are the reservoir, while humans and primates are occasional.

3) Hemorragic fever (fatality ranges from 50% –> 60%)

4) Through zoonosis (from infected animals) and contact with body fluids

Question 44

Fill in the blanks regarding Ebola virus genomic organization:

The viral RdRp binds the encapsidated genome at the ________ region and then sequentially transcribes each genes by recognizing start and stop signals ______ viral genes.

The mRNAs are _______ and ________ by the L proteins during synthesis (no Vpg).

The primary product of the unedited transcript of ______ gene yields a smaller non-structural glycoprotein _____ which is efficiently secreted from infected cells. _____ _______ allows expression of full length GP.

Answer 44

Leader; flanking

Capped; Polyadenylated

GP; sGP; RNA editing

Question 45

Which one of the statements is true regarding Ebola virus sequential transcription?

1) It is similar to several (-) ssRNA viruses, like deltavirus.

2) PolyA is produced by RdRp stuttering

3) There is a creating of a gradient of proteins (C being the most abundant).

4) There are subgenomic RNAs.

Answer 45

2!

1) It is similar to several (-) ssRNA viruses, WITH THE EXCEPTION TO deltavirus.

3) There is a creating of a gradient of proteins (N being the most abundant).

4) There are NO subgenomic RNAs.

Question 46

(T/F) Non-structural glycoprotein sGP are secreted to neutralize the antibodies of our immune system. They are not the glycoproteins found in the viral capsid and they are synthesized in large quantities.

Answer 46

True!

Question 47

What is RNA editing?

Answer 47

Some viruses encode genes that express multiple species of proteins by means of using overlapping open reading frames using RNA editing.

In these viruses, the RNA polymerase reads the same template base more than once, creating insertions (0,1,2, etc, base insertions), leading to different mRNAs and generating different types of proteins.

In ebola this allows for the production of ssGP, sGP and Gp1,2 using the same mRNA.

*not to be confused with ribosomal frameshift

Question 48

(T/F) Secreted GP (sGP) is the main product of RNA editing (71%). ssGP is expressed after a +1 editing (addition of 1 A, accounts for 24%), while GP1,2 is expressed after a +2 editing (5%).

Answer 48

False!

Secreted GP (sGP) is the main product of RNA editing (71%). However, GP1,2 is expressed after a +1 editing (addition of 1 A), while ssGP is expressed after a +2 editing (5%).

Question 49

Fill in the blanks regarding the infection cycle of Ebola.

1) There is an _______ to host receptors through ____ _________ like DC-SIGN and DC-SIGNR

2) Cellular receptors like _________ (TIM1) binds phosphatidyl ________ on virion membrane and a signal is transduced into the cell that trigger the macropinocytosis program by ________ _______.

3) The virion enters the cell by ___________. In some cells models, GP can be processed by host __________ L and B into GP1 (not always).

4) GP1 interacts with host _______-_____ disease, type ___, in late macropinosome and promotes _______ of virus membrane with the vesicle membrane.

5) Replication starts when enough nucleoprotein is present to ________ neo-synthesized ________ and genomes.

6) The ribonucleocapsid interacts with the matrix protein and buds via the ____ _______ complexed from the plasma membrane.

Answer 49

1) Attachment; GP glycoprotein

2) HAVCR1; serine; apoptotic mimicry

3) Macropinocytosis; Cathepsin

4) Niemann-Pick; C1; fusion

5) Encapsidate; Antigenomes

6) Host ESCRT

*Budding out involves our host export proteins!