Molecular Virology

Question 0

How does influenza virus enter the cell and release its genome?

Answer 0

Virus binds with receptor (Salic acid) at PM of the cell

Becomes endocytosed

Fusion of viral and endosomal membranes, release of capsid into the cytoplasm. Fusion occurs due to pH change

Capsid Uncoating then genome moves into the nucleus to replicate

Question 1

What does the 3D gene in picornaviruses encode for?

Answer 1

RNA dependant RNA polymerase (RdRp)

Question 2

Once capsid is in an endosome, what ways can the genome be released?

Answer 2

Uncoating then endosome disruption HRV14

Endosome disruption then Uncoating once capsid has left the endosome

Pore formation then genome is ejected (HRV2)

Question 3

How does polio virus Enter the cell and release its genome (2 methods)

Answer 3

Receptor binding then:

Membrane disruption and Uncoating or pore formation then genome ejected

Question 4

How does HIV enter the cell then enter the nucleus

Answer 4

Fusion of viral and PM = Release of capsid into cytoplasm

Reverse transcription within partially uncoated capsid

Capsid moves along microtubules then DNA enters the nucleus attached to integrase

Question 5

How does herpes virus enter the cell and move its genome into the nucleus?

Answer 5

Fusion of viral and PM release of capsid and tegument into cytoplasm

Moves along microtubules until it reaches the nuclear pores.

Capsid is too large to move through so DNA and associated proteins(tegument) and ejected into the nucleus

Question 6

Retroviruses basics: eg genome type, size and virion size?

Answer 6

Enveloped

Leaner, single stranded positive sense RNA genome

7-12kb

Virions = 80-100nm in diameter

Question 7

Examples of a simple and a complex retrovirus?

Answer 7

Simple = murine leukaemia virus

Complex = HIV-1

Question 8

Retrovirus lifecycle: basic outline of steps

Answer 8

Virus binding then fusion with PM releases capsid into cytoplasm
Reverse transcription of viral RNA into DNA in partially uncoated capsid
Virus moves into nucleus
Nuclear transport and integration 
Transcription and splicing 
Translation of viral proteins 
Virion assembly and release 
Maturation occurs outside of the cell

Question 9

HIV tRNA’s where do they come from and how many per virus

Answer 9

Virus particles contain 50-100 tRNA’s per virion

Recruited by RT during virus assembly

Different retrovirus utilise different tRNA’s

Question 10

HIV-1 reverse transcriptase

Answer 10

Is a heterodimer made up of p66 and p51

Has two enzymatic activities:

1) RNA dependant DNA polymerase
2) Ribonuclease H (RNaseH) = degrades RNA in an RNA:DNA hybrid

Question 11

What is the size of the pre-integration complex for HIV? and what does it contain?

Answer 11

30nm in diameter

Contains matrix, integrase, RT and Vpr (no capsid)

HIV can integrate into non diving cells

Nuclear localisation signal on matrix and Vpr- mediate energy dependant translocation through nuclear pore

Question 12

Endogenous retroviruses

Answer 12

Present in all vertebrate species: most examples come from mouse and chickens
Result from infection of germ line cells by exogenous, replication competent retroviruses
Generally defective-mutations or deletions so no infectious retroviruses are produces.

8% of human genome consists of endogenous retroviruses
Most recent= HERV-K113 (200k years)
Believed to be important in development of placenta, and ability to digest starch

Question 13

What does omina cellulae e cellulae man?

Answer 13

All cells from cells

Question 14

Poxviruses: genome and site of assembly

Answer 14

Large DNA viruses: vaccinia, smallpox

Replicate entirely in the cytoplasm
Assembly sites: virus factories which are discrete from cytoplasmic membrane

Question 15

Vaccinia: different types of virions and number of membranes

Answer 15

Intracellular mature virus (IMV) = 1membrane
Most abundant form

Intracellular enveloped virus (IEV) = 3membranes
Allows movement of virus to cell surface on microtubules

Cell-associated enveloped virus (CEV) = 2membranes
Induces formation of actin tails to drive CEV away from cell

Extracellular enveloped virus (EEV) = 2 membranes
Mediates long-range spread of virus

Question 16

The poliovirus polyprotein is myristoylated

Answer 16

Addition of a 14-carbon saturation fatty acid to an N-terminal glycine residue
Amine linkage = irreversible
Catalysed by cellular enzyme N-myristoyltransferase (NMT)

Question 17

How are pentamers stabilised in polio virus?

Answer 17

Via interactions between VP3 and myristate residues on VP0

Question 18

Weight of polio virus protomer?

Answer 18

5S

Question 19

Weight of pentamers (five protomers) polio

Answer 19

14S

Question 20

Weight of polio pro capsid (12 pentamers), and capsid with genome?

Answer 20

73S

155S

Question 21

How is poliovirus RNA genome encapsidated?

Answer 21

Only VP-g RNA is encapsidated
However no evidence for specificity, can swap different VP-g from different picornaviruses
Majority of particles in infected cells are empty, large amount of genome RNA without VPg
Thought that capsid forms around genome

Question 22

During encapsidation of RNA there is a conformational change in the capsid proteins. (Polio)

Answer 22

Cleavage of VP0 to VP4 and VP2
Auto cleavage via a histidine residue
Stabilises the mature virus so is more resistant to heat, pH and detergents.
Virion is only composed of VPg RNA and VP1-4- no other viral or cellular proteins

Question 23

Role of 2C and 2BC proteins

Answer 23

Involved in reorganising cellular membranes into vesicles where virus assembly takes place.
These membranes are derive from the ER
2C also binds to 3’end of genome of RNA

Question 24

What is the role of the 2B protein

Answer 24

Is an ion channel protein that alters membrane permeability.

Question 25

Picornavirus inhibition of cellular cap-dependant translation

Answer 25

Polio and rhinovirus 2A protease cleaves a component of the eIF4F complex p220 So inhibits cap dependant initiation-host cell shut off. However virus translation continues due to IRES As a result this induces predefined sequence of host cell death and lysis.

Question 26

How many virus are released when an polio infect cells lysis? And what is the infectivity ratio?

Answer 26

25,000-100,000 per cell | Ratio can be as low as 1:1000

Question 27

Genome of HIV

Answer 27

2 strands of ssRNA postive sense | 9.5kb

Question 28

Where does HIV assembly take place

Answer 28

At the plasma membrane and Intracellular vesicles

Question 29

What does Gag encode for?

Answer 29

The capsid proteins (p55) = MA,CA,NCp6

Question 30

How is gag-pol fusion peptide translated

Answer 30

Normally ribosome falls off once it reaches stop signal after stem loop However about 1in 20 times the stem loop (shift sequence) causes the ribosome to bounce back and therefore reads through a different reading frame so no longer reads the stop codon. Therefore translation continues

Question 31

How much gag is produced compared to gag-pol

Answer 31

95% gag 5% Gag-Pol 19 gags for every 1 Gag-Pol

Question 32

How are gag and gag-pol polyprotein targeted to Membranes?

Answer 32

They are myristoylated. But myristate does not interact with amino acid sequence. Gag myristate inserts into lipid bilayer Interaction with membrane is stabilised by electrostatic interactions between basic amino acids and acidic phospholipids

Question 33

Macropinocytosis

Answer 33

Membrane ruffling Large vesicles Involves fluid uptake Used by large viruses such as: herpes pox also Ebola

Question 34

Non Clathrin dependant endocytosis

Answer 34

Caveolae formed from cholesterol rich lipid rafts that are then target to the ER Used by viruses such as polyomavirus, bunyaviruses

Question 35

Clathrin mediated Endocytosis

Answer 35

``` Clathrin cage causes membrane curvature Pinched off to vesicles Target to endosomes Acdification E.g influenza virus ```

Question 36

Poliovirus receptor

Answer 36

CD155 Normal function: Intracellular adhesion in epithelial cells

Question 37

HRV receptors

Answer 37

Major group 14/16: use Intracellular adhesion molecule-1 (ICAM-1) Minor group 2: LDLR: normal function Is uptake of lipids

Question 38

Why are HRV 14/16 resistant to neutralising antibodies?

Answer 38

Receptor binding site is at the base of a canyon ICAM-1 can reach this but antibodies can not.

Question 39

Name the virus attachment factors

Answer 39

Glycosaminoglycans (GAG's) Linear polysaccharide linked to cell surface protein Negatively charges and often sulphated Bind viruses by electrostatic interactions

Question 40

Describe the concept of cell tropism

Answer 40

Only cells with the appropriate receptors can become infected Cell tropism can determine disease or pathogenesis

Question 41

Viral helicase's

Answer 41

Are accessory proteins Unwind duplex DNA/RNA/ DNARNA Also can be used to unwind single stranded RNA as has complex secondary structure Requires energy from a NTPase enzyme Requires

Question 42

Structure of a CD4 receptor

Answer 42

Four Extracellular (IgG) like domains, short cytoplasmic tail Normal function= bind MHC class II, bind IL16 also

Question 43

Papillomaviruses

Answer 43

Circular DNA genome 8kb Early and late genes expressed Overlapping reading frames HPV 16 + 18 = can produce carcinomas HPV 2 = warts

Question 44

Name the picornaviruses

Answer 44

``` FMDV Polio Enterovirus 71 Cocksackie virus A16 human rhinovirus Hepatitis A ```

Question 45

Paramyxovirus

Answer 45

``` Example: human respiratory syncytial virus Infect super and lower respiratory tract Infects all human by age of 2 200,000 deaths a year Linked to life long asthma No vaccine ```

Question 46

Rabies

Answer 46

99.99999% fatal-less than 20 survivors 60,000 deaths a year Pre and post vaccines

Question 47

Bornaviridae

Answer 47

Example:Borna disease virus BDV infects several mammals: horse sheep rabbit humans Spread is suspected via small mammals such a rodents Causes a persistent infection of neuronal tissue, dysfunction, death

Question 48

Negative strand RNA viruses

Answer 48

The RNA genome is covered entirely covered from end-end RNA becomes highly ordered into a helical structure In many cases, the RNA a in the RNP is resistant to RNase

Question 49

RNP

Answer 49

10 N molecules per ring each rings binds 90nt long RNASE in cleft which is to small for RNase to access the RNA to degrade it Viral RNA a binds via its phosphate backbone K and R amino acids on N bind electrostaticly 37.5 N per turn

Question 50

VSV genes

Answer 50

Nucleocapsid protein N = wraps RNA into RNP a helix Phosphate protein P = RdRp cofactors Matrix protein M = links N with glycoproteins to form virus particle Glycoproteins G = forms spikes on virus exterior-used in cell attachment Large protein L = RdRp

Question 51

VSV transcription

Answer 51

Requires 2 virus encoded proteins P and L RdRp binds to the -genome at the 3' end RdRp locates the gene start (nt 44) and then copies the template strand into +strand RNA RdRp responds to signals in the RNA a genome to stop and start copying, and then begin again

Question 52

VSV 2

Answer 52

Each of the 5 genes has a start signal and stop signal The RdRp respond to these signals-generates one mRNA per gene Transcription products are not equally abundant mRNA gradient is caused by 3 factors 1) single 3' RdRp entry site 2) The RdRp can only move in one direction The RdRp can fall off the template at the gene junctions (30% chance)

Question 53

VSV3

Answer 53

mRNA's are capped at their 5' ends | mRNA's have poly (A) tails at their 3' ends of the genome

Question 54

- sense RNA a replication

Answer 54

During replication the - sense genome is copied to make a perfectly complementary RNA a called the anti-genome, which is + sense This is then used as a template to make more -sense RNA RdRp binds at 3' end and is copied from position 1 RdRp copies genome ignoring the gene junctions As the new anti-genome is made it is wrapped in N protein Replication can only occur after transcription as N protein needed

Question 55

Classic example of forward genetics

Answer 55

Maize: mutation in the C1 gene responsible for formation of the anthocyanin pigment The Belgian blue: 11nt deletion in the gene for myostatin

Question 56

Reverse genetics principles

Answer 56

Change a specific gene in a virus Cause change of phenotype Link the gene to the observed phenotype

Question 57

Rift Valley fever virus

Answer 57

``` RVFV is a bunyavirus 3 segments, 4genes, 6proteins Infects insects and mammals 1997-1998 Kenya outbreak 90,000 human cases- 470 deaths S segment = N, NSs ``` M segment = GN + GC + NSm L segment = L

Question 58

MP12 RVFV

Answer 58

Non pathogenic strain of the virus

Question 59

Comparison WT and MP12

Answer 59

25 nt changes 11 coding changes One or more must confer to pathogenicity 11 virus made each with a different mutation Observe to see if pathogenicity is affected Reverse genetics showed residue 513 is responsible for the MP12 phenotype

Question 60

HRSV in knock out experiments

Answer 60

Ns2 KO Virus grows, but poorly NS2 blocks IFN production allowing rapid virus growth

Question 61

VSV motif KO

Answer 61

PPPY LATE motif in M protien deleted Lead to viruses not being able to leave cell PPPY like PPXY motif invloved in virus release It interacts with Nedd4 (cellular protein)

Question 62

First engineered virus

Answer 62

First achieved in 1976 generation of a hybrid SV40 + phage lambda

Question 63

RNP assembly

Answer 63

Requires PB1, PB2, PA and NP

Question 64

Flu season peak

Answer 64

February

Question 65

Influenza surveillance

Answer 65

Global influenza surveillance response system (GISRS) 105 countries 175,000 sequences sampled GISRS predict 3 likely strains in March

Question 66

How are the 3 chosen strains weakend to reduce their ability to cause disease

Answer 66

Weakend by allowing segment exchange with the PR8 strain This segment exchange is called re assortment PR8 and WT viruses co infect same cell Viruses replicate, segments can mix 256 possible new genotypes Reference strain = seed strain

Question 67

Draw backs of old method of generating influenza vaccine

Answer 67

Generation and selection of 3 reassortants is slow-3months Dependence on egg production 170 million eggs Some H5N1 grow poorly in eggs

Question 68

Proteins required to form RNP in influenza

Answer 68

PB1 PB2 PA N

Question 69

Reverse genetics vaccine

Answer 69

Allow rapid response to unpredictable pandemics Seed in 2 weeks Rag has been recently approved FDA approved for flucelvax Produced by Novartis Seeds are cDNA derived Seeds are grown in culture, no need for eggs

Question 70

IFN

Answer 70

Cytokines that have wide scale antiviral activity

Question 71

RIG-I

Answer 71

Discovered in 2004 Ubiquitously expressed in human tissues Composed of two CARD domains and a carboxyl terminal helicase domain

Question 72

RIG-I detecs

Answer 72

``` Paramyxovirus Influenza + other -sense RNA viruses Hep C Japanese encephalitis virus In vitro transcribed dsRNA ``` Recognises 5'PPP

Question 73

Influenza

Answer 73

100nm sphere 13kb Replication in the nucleus, assembly in the cytoplasm

Question 74

What part of RIG-I detects ds RNA

Answer 74

``` CTD domain (In the ATPase/helicase domain) ```

Question 75

Mda-5

Answer 75

Has same domain order and sequence similarity as RIG-I Can bind to synthetic dsRNA a Mice deficient in Mda-5 are more susceptible to infection with picornaviruses and caliciviruses Detects large > 4kb dsRNA sequences in viruses Detects VpG