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Virology > Herpesviruses > Flashcards

Herpesviruses Flashcards

1
Q

What are some examples of alphaherpesvirinae?

A

HSV-½
VZV (chicken pox)
Latent in neurones

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2
Q

What are some examples of betaherpesvirinae?

A

HCMV
HHV-6 A/B
HHV-7
Latent in monocytes

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3
Q

What are some examples of gammaherpesvirinae?

A

EBV
KSHV
Latent in B lymphocytes

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4
Q

Describe HSV-1 infection

A

Infects a 40-80% of people worldwide
Lytic infection at mucosal surfaces (oro-facial/genital)
Latent in sensory ganglia
Mostly asymptomatic/mild symptoms such as cold sores
Can get more severe disease in immunocompromised patients
Rapid and efficient replication in cell cultre

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5
Q

What is the structure of a herpesvirus?

A 
Large, complex, enveloped DNA viruses
dsDNA genome (150-200kbp)
Icosahedral capsid
Tegument
Lipid envelope
Envelope proteins
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6
Q

Give an overview of herpesvirus assembly and egress

A

Capsid and DNA encapsidation happens in the nucleus, capsid then moves into the cytoplasm by budding at the inner nuclear membrane - get perinuclear virions that then fuse with the outer nuclear membrane
In the cytoplasm, the capsid recruits the tegument proteins
The virus acquires envelope proteins by budding into the lumen of the TGN/endosomes containing envelope proteins
The vesicles fuse with the plasma membrane, releasing the virus

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7
Q

How do herpesviruses leave the nucleus?

A

Requires a conserved nuclear egress complex of pUL34 and pUL31 (HSV)
pUL34 is a type II membrane protein that complexes with pUL31 in the nuclear membrane
The complex recruits PKC and/or viral kinases that phosphorylate the lamins
Interaction of the pUL34/31 complex with the capsid drives budding and scission

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8
Q

What is the tegument of herpesviruses?

A

Essential structural role - links the capsid to the envelope
Contains factors to modify the host cell (immune evasion, host shut off, nucleotide synthesis etc)
Has several enzyme activities
Has 2 layers - capsid bound (inner) and envelope associated (outer)

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9
Q

How is tegument recruited into the herpesvirus?

A

Can either be by recruitment to capsids or by recruitment to the cytoplasmic domain of viral envelope protein.
An interaction between capsid tegument and envelope tegument is likely to drive envelopment

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10
Q

What is the composition of herpesvirus B capsids?

A

Has pentons made of VP5 (portal complex in place of one made of UL6)
Hexons of VP5
Triplexes of VP19c and VP23

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11
Q

What is the composition of herpesvirus C capsids?

A

Found in the nucleus
As B capsids with:
CVSC (capsid vertex specific component) made up of CL17 and UL25 - may contribute to signal for nuclear egress of DNA filled capsids or be a binding site for inner tegument

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12
Q

Describe the capsid - inner tegument interaction of herpesviruses?

A

Most inner tegument is thought to be VP1/2 (UL36) that can interact with UL37, UL25 and VP16. Cryo-EM has shown tegument associated with capsid pentons and CVSC - thought to be the C terminus of VP1/2 that binds UL25 (part of CVSC)

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13
Q

What are the components of the inner and outer tegument of herpesviruses?

A

Inner: pUL36 (VP1/2), pUL37, pUS3
Outer: pUL46 (VP11/12), pUL47 (VP13/14, pUL48 (VP16), pUL49 (VP22)

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14
Q

Describe tegument protein VP1/2 (UL36) in herpesviruses

A

Conserved in every herpes virus, is essential for assembly. One of the first tegument proteins to be recruited to the capsid. Required for recruitment of pUL37 and VP16 to the capsid. Very large - could be a scaffold for tegument assembly

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15
Q

Describe tegument protein VP16 (pUL48)

A

Transactivator of IE gene expression, major component of the tegument. Essential for HSV-1 assembly. Interacts with some outer tegument proteins (vhs, pUL46,4749). Could be a key connection between inner and outer tegument

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16
Q

What is the importance of the VP16-VP1/2 interaction in HSV-1?

A

Mutating K343 of VP16 removes the interaction of VP1/2 with VP16. This delays tegument recruitment, but doesn’t significantly effect viral replication

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17
Q

How are HSV-1 envelope proteins localised to TGN and endosome vesicles?

A

Many have a motif to mediate internalisation and targeting (the way the cell does it - active retrieval). Internalisation is controlled by gM and/or gK/pUL20.
Some proteins don’t, including essential pD and gH/gL

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18
Q

How are HSV-1 proteins gD and gH/gL internalised?

A

Requires gM and (gH/gL)/or (gD). Measured with an antibody feeding assay - apply antibodies to cell surface which are specific to the extracellular domain. Incubate for a bit, then fix and observe - see if antibodies have been internalised.

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19
Q

How do we know that HSV-1 glycoproteins travel to assembly sites via endocytosis from the plasma membrane?

A

Virus neutralisation by antibody feeding assay. Add neutralising antibodies to the medium for a period of replication. Destroy extracellular viruses with a protease treatment. Titrate remaining infectivity. Found that with application of neutralising antibodies to gD and gH virus infectivity was reduced from the control.
Experiments with a dynamin inhibitor with fluorescent capsid and antibody feeding against gD shows that dynamic is essential for internalisation of gD (see virus particles with no gD inside cell). Implies clathrin mediated endocytosis is a route for internalisation

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20
Q

What do the ESCRT complexes do?

A

ESCRT 0,1 and 2 mediate cargo recruitment and initiate bud formation
ESCRT 3 polymerisation into filaments around the bud neck mediates membrane scission. It is recycled by VPS4 ATPase.

21
Q

How do we know that HSV-1 requires the ESCRT pathway?

A

In dominant negative cells (overexpress VP34 ATPase dead), virus titre is significantly reduced

22
Q

How do we know which parts of the ESCRT machinery are required for HSV-1 budding?

A

Use RNAi to silence and then measure virus titre
Know not ESCRT 0,1,2 or ALIX as they are relatively dispensable.
HSV-1 has multiple redundant pathways for recruiting and regulating ESCRTs.

23
Q

What is the lifecycle of HSV-1?

A

Acute primary infection including a multiplication lesion
Virus becomes latent in ganglia
Reactivation gives an acute recurrent infection

24
Q

What are the similarities and differences of HSV-1 and HSV-2?

A

Primary infections of the pro-facial area (mild or asymptomatic) - HSV-1. Primary infection of genital tract may be severe - HSV-2
Latency in trigeminal ganglia (HSV-1) or sacral ganglia (HSV-2)
HSV-1 is acquired early in life, HSV-2 is acquired sexually
Similar genomic organisation and DNA sequence homology

25
Q

What is the operational definition of latency?

A

Acute disease: take sensory ganglia, disrupt tissue and lyse cells - see infectious virus
Latent infection: take sensory ganglia, disrupt tissue and lyse cells - no infectious virus. If cells are cultivated instead, get infectious virus

26
Q

How is HSV IE gene expression activated?

A

VP16 in the virus particle interacts with Oct1 and HCF (host cell factor - a transcriptional coregulator). This complex binds IE promoters and stimulates IE gene production. HCF recruits lysine specific demethylases (LSD) to prevent H3K9 methylation at IE promoters. These then self regulate and initiate early and late gene expression for a productive infection

27
Q

Why may IE genes fail to activate in HSV-1 infection?

A

Insufficient VP16 reaching the neuronal nucleus
Limiting amounts of Oct1/presence of Oct2 isoforms that can’t associate
Cytoplasmic localisation of HCF

28
Q

What is the structure of latent HSV DNA?

A

No free ends - has be circularised
Variable copy number in infected cells
Nucleosomal organisation
LAT promoter is associated with acetylated H3 - transcriptionally active? Whilst the DNA Pol gene is hypoacetylated - repressed

29
Q

What are HSV LATs?

A

Latency associated transcripts. Aid establishment of latency - suppress viral IE gene expression and prevent neuronal apoptosis.
Make some miRNAs to repress IE genes
Have been suggested to have a role in maintaining repressive chromatin - LAT negative mutants can be latent but latent genomes are enriched in active histone marks
Is important for maintenance of infected cells

30
Q

How are host miRNAs implicated in herpesvirus infection?

A

Have been shown to down regulate lytic genes, encouraging latency

31
Q

How can herpesvirus infected cells be observed and quantified in mice?

A

Cassette of a promoter (Rosa26) followed by an inhibitory gene surrounded by lox P sites followed by lacZ. Herpesviruses expressing Cre infect the cells, cut out the inhibitory gene and lacZ is the reporter gene.

32
Q

How is HSV-1 reactivated using ICP0?

A

Somehow, ICP0 is transcribed (specific activation of promoter/VP16 independent)
ICP0 is an E3 ubiquitin ligase, mediating degradation of cellular proteins (HDAC function disrupted to alleviate repression, inhibits IRF-3 and7, PML, SP100 etc)
Get global de-repression and histone acetylation
Leads to reactivation

33
Q

How is HSV reactivated using VP16?

A

Likely to also require VP16 - if lack transactivation function then virus doesn’t exit latency. The promoter of VP16 has a unique regulatory activity in neutrons and can be reactivated in response to to stress. Get transcription of IE genes

34
Q

What is EBV?

A

Epstein Barr Virus. Discovered in Burkitt’s lymphoma. A B-lymphotropic virus, efficient at transformation. In immunocompetent hosts, cancer is rare

35
Q

Describe EBV life cycle in humans

A

Infects at the nasopharynx, goes to B lymphocytes. Infection is asymptomatic in children or infectious mononucleosis (glandular fever) in young adults. End up with latency and occasional shedding from the oropharynx

36
Q

What is the evidence for EBV epithelial cell infection?

A

Can be detected in a benign lesion of the oral epithelium (common in AIDS patients)
Can transform epithelial cells (nasopharyngeal carcinoma)
Can infect in vitro even if cells lack B cell virus receptor CR2 and class II - an alternative receptor on epithelial cells?

37
Q

How does EBV enter B cells?

A

Uses CR2 and MHC class II on B cell surface. Virus binds using gp350 and gH/gL+gp42. Binding of CR2 activates B cells from resting state. Virus genome goes into the nucleus

38
Q

How does EBV establish latency?

A

First gene expressed is EBNA-2, a master regulator of latency. It activates the B cell and up regulates latency genes in the virus. Latency III program is initiated, consisting of 6 nuclear antigens, 2 membrane antigens and 2 small RNAs (not miRNAs)

39
Q

What are the stages of EBV latency?

A

The proliferative stage from EBNA-2 activation
The maintenance stage - EBNA-1 is expressed (required for plasmid maintenance in rapidly dividing populations). Seen in Burkitts lymphoma.
As latency progresses, more and more genes are switched off

40
Q

How is EBV DNA replicated during latency?

A

Ori P interacts with EBNA-1 to facilitate replication by the host DNA polymerase. (Different to lytic phase in which origins are recognised by virally encoded DNA pol). Ori P has a family of repeats which binds EBNA-1 dimers. The DNA is looped to help unwind DNA at the dyad symmetry element (to facilitate DNA replication). EBNA-1 tethers the plasmid to histones to ensure equal segregation

41
Q

Why isn’t EBNA-1 recognised by CTLs?

A

Has an extended glycine-alanine repeat that restricts MHC class I antigen presentation by interfering with proteosomal processing

42
Q

What is the state of the EBV latent genome?

A

Multiple copies of episomal DNA in B cells
Extensively methylated and histone associated
Efficiently segregated into daughter cells

43
Q

What do EBV encoded RNAs do?

A

Known as EBERs. 2 non-polyadenlyated RNAs transcribed by RNA pol III. Expressed to high levels in all forms of latency and function to inhibit IFNalpha induced apoptosis. They bind PKR to prevent activation and also induce IL-10 production to suppress CTLs.

44
Q

How does EBV establish latency in memory B cells?

A

Development of memory B cells is normally antigen specific
EBV latency genes promotes B cell proliferation and differentiation
An EBV latent membrane protein LMP1 mimics the growth signal normally received from a T cell receptor and is constituently active. Signalling is via JAK/STAT and NFkB
The second EBV latent membrane protein, LMP2a mimics the B cell receptor and is constituently active.
Together, the 2 membrane proteins provides the signals for differentiation of B cells

45
Q

How is the growth program down regulated? Why is this important?

A 
First switch from promoter Wp to Cp occurs when there is sufficienct EBNA-2 - occurs during the latency III program
Switch from Cp to Qp to initiate the latency II program. No more EBNA-2 is made. As EBNA-2 levels drop, get less latent membrane proteins. Cp drives high expression of nuclear antigens
True latency (latency I) is then established. Only EBNA-1 is produced (and can be on or off) which is required for  plasmid maintenance.
46
Q

How is the first promoter switch in establishment of EBV latency controlled?

A

EBNA-2 and CBF-1 binds the Cp promoter and activates the growth program, including production of EBNA3A and EBNA3C (nuclear antigens)
EBNA3A/C then binds CBF1 and displaces EBNA-2 to repress the Cp promoter. A region across the Cp and Wp promoters is epigenetically silenced.

47
Q

What miRNAs does EBV encode?

A

25 miRNAs
Viral targets: DNA polymerase, LMP1
Cellular targets: PUMA (pro-apoptotic), CXCL11 (chemokine, T cell attractant)

48
Q

How can EBV reactivation be stimulated?

A

Treatment with phorbol esters or cross linking of surface immunoglobulin

49
Q

What is the first gene transcribed upon EBV reactivation?

A

BZLF1. An immediate early transactivator that initiates the lytic cascade. It binds CpG methylated DNA and transactivates repressed promoters - novel way to overcome genetic silencing.

Virology (13 decks)

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