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Microbilogy Year 2 > Molecular Virology > Flashcards

Molecular Virology Flashcards

0
Q

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

A

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

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1
Q

What does the 3D gene in picornaviruses encode for?

A

RNA dependant RNA polymerase (RdRp)

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2
Q

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

A

Uncoating then endosome disruption HRV14

Endosome disruption then Uncoating once capsid has left the endosome

Pore formation then genome is ejected (HRV2)

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3
Q

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

A

Receptor binding then:

Membrane disruption and Uncoating or pore formation then genome ejected

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4
Q

How does HIV enter the cell then enter the nucleus

A

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

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5
Q

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

A

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

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6
Q

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

A

Enveloped

Leaner, single stranded positive sense RNA genome

7-12kb

Virions = 80-100nm in diameter

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7
Q

Examples of a simple and a complex retrovirus?

A

Simple = murine leukaemia virus

Complex = HIV-1

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8
Q

Retrovirus lifecycle: basic outline of steps

A 
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
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9
Q

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

A

Virus particles contain 50-100 tRNA’s per virion

Recruited by RT during virus assembly

Different retrovirus utilise different tRNA’s

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10
Q

HIV-1 reverse transcriptase

A

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

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11
Q

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

A

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

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12
Q

Endogenous retroviruses

A

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

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13
Q

What does omina cellulae e cellulae man?

A

All cells from cells

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14
Q

Poxviruses: genome and site of assembly

A

Large DNA viruses: vaccinia, smallpox

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

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15
Q

Vaccinia: different types of virions and number of membranes

A

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

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16
Q

The poliovirus polyprotein is myristoylated

A

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

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17
Q

How are pentamers stabilised in polio virus?

A

Via interactions between VP3 and myristate residues on VP0

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18
Q

Weight of polio virus protomer?

A

5S

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19
Q

Weight of pentamers (five protomers) polio

A

14S

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20
Q

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

A

73S

155S

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21
Q

How is poliovirus RNA genome encapsidated?

A

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

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22
Q

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

A

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

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23
Q

Role of 2C and 2BC proteins

A

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

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24
Q

What is the role of the 2B protein

A

Is an ion channel protein that alters membrane permeability.

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25
Q

Picornavirus inhibition of cellular cap-dependant translation

A

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.

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26
Q

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

A

25,000-100,000 per cell

Ratio can be as low as 1:1000

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27
Q

Genome of HIV

A

2 strands of ssRNA postive sense

9.5kb

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28
Q

Where does HIV assembly take place

A

At the plasma membrane and Intracellular vesicles

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29
Q

What does Gag encode for?

A

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

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30
Q

How is gag-pol fusion peptide translated

A

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

31
Q

How much gag is produced compared to gag-pol

A

95% gag 5% Gag-Pol

19 gags for every 1 Gag-Pol

32
Q

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

A

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

33
Q

Macropinocytosis

A

Membrane ruffling

Large vesicles

Involves fluid uptake

Used by large viruses such as: herpes pox also Ebola

34
Q

Non Clathrin dependant endocytosis

A

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

35
Q

Clathrin mediated Endocytosis

A 
Clathrin cage causes membrane curvature 
Pinched off to vesicles 
Target to endosomes 
Acdification 
E.g influenza virus
36
Q

Poliovirus receptor

A

CD155

Normal function: Intracellular adhesion in epithelial cells

37
Q

HRV receptors

A

Major group 14/16: use Intracellular adhesion molecule-1 (ICAM-1)

Minor group 2: LDLR: normal function Is uptake of lipids

38
Q

Why are HRV 14/16 resistant to neutralising antibodies?

A

Receptor binding site is at the base of a canyon

ICAM-1 can reach this but antibodies can not.

39
Q

Name the virus attachment factors

A

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

40
Q

Describe the concept of cell tropism

A

Only cells with the appropriate receptors can become infected

Cell tropism can determine disease or pathogenesis

41
Q

Viral helicase’s

A

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

42
Q

Structure of a CD4 receptor

A

Four Extracellular (IgG) like domains, short cytoplasmic tail

Normal function= bind MHC class II, bind IL16 also

43
Q

Papillomaviruses

A

Circular DNA genome 8kb

Early and late genes expressed

Overlapping reading frames

HPV 16 + 18 = can produce carcinomas
HPV 2 = warts

44
Q

Name the picornaviruses

A 
FMDV
Polio
Enterovirus 71
Cocksackie virus A16
human rhinovirus 
Hepatitis A
45
Q

Paramyxovirus

A 
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
46
Q

Rabies

A

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

47
Q

Bornaviridae

A

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

48
Q

Negative strand RNA viruses

A

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

49
Q

RNP

A

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

50
Q

VSV genes

A

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

51
Q

VSV transcription

A

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

52
Q

VSV 2

A

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)

53
Q

VSV3

A

mRNA’s are capped at their 5’ ends

mRNA’s have poly (A) tails at their 3’ ends of the genome

54
Q
  • sense RNA a replication
A

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

55
Q

Classic example of forward genetics

A

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

56
Q

Reverse genetics principles

A

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

57
Q

Rift Valley fever virus

A 
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

58
Q

MP12 RVFV

A

Non pathogenic strain of the virus

59
Q

Comparison WT and MP12

A

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

60
Q

HRSV in knock out experiments

A

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

61
Q

VSV motif KO

A

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)

62
Q

First engineered virus

A

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

63
Q

RNP assembly

A

Requires PB1, PB2, PA and NP

64
Q

Flu season peak

A

February

65
Q

Influenza surveillance

A

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

66
Q

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

A

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

67
Q

Draw backs of old method of generating influenza vaccine

A

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

68
Q

Proteins required to form RNP in influenza

A

PB1
PB2
PA
N

69
Q

Reverse genetics vaccine

A

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

70
Q

IFN

A

Cytokines that have wide scale antiviral activity

71
Q

RIG-I

A

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

72
Q

RIG-I detecs

A 
Paramyxovirus 
Influenza + other -sense RNA viruses
Hep C
Japanese encephalitis virus
In vitro transcribed dsRNA

Recognises 5’PPP

73
Q

Influenza

A

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

74
Q

What part of RIG-I detects ds RNA

A 
CTD domain 
(In the ATPase/helicase domain)
75
Q

Mda-5

A

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

Microbilogy Year 2 (3 decks)

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