Herpesviridae

Herpes Simplex-1 Herpes Simplex-2 Cytomegalovirus

 HERPESVIRIDAE: Subfamilies: i- Alpha: HSV-1 (Herpes simplex 1), HSV-2 (Herpes simplex 2), Varicella-Zoster (VZV) ii- Beta: Cytomegalovirus (CMV), Human Herpes Virus-6 & 7 (HHV-6 & HHV-7 from genus Roseolovirus) iii- Gamma: Epstein-Barr Virus (EBV from genus Lymphocryptovirus), and HHV-8 (Kaposis sarcoma)

Herpes Simplex Virus (Herpesviridae) from Greek to creep 1. History Since the Sme of the ancient Greeks, "herpes" has been recognized as a disease of spreading, ulceraSve skin lesions. In 1929 it was observed that the causaSve eSologic agent was not a bacterium; moreover, this agent was recognized to be a "nonlterable" virus that travels along the axonal route of infected animals to eventually reside in a latent state in the nerve cells of the ganglia. By the 1960s, it was demonstrated that disSnct viruses cause facial and genital lesions. Over the past two decades, criScal advances have been made in the development of anSviral chemotherapies against herpes viruses.

Cold sore

h#p://pathmicro.med.sc.edu/lecture/herpes.gif h#p://www.microbiologybytes.com/virology/3035pics/coldsore.jpg

There are over 100 members of the Herpesvirus family

2. ClassicaSon and Serologic RelaSonships The two herpes simplex virus serotypes (HSV1 and HSV2) are members of the family Herpesviridae. Within this family, both serotypes belong to the subfamily Alphaherpesvirinae and genus Simplexvirus. HSV1 and HSV2 share 47-50% sequence homology and their geneSc maps are collinear. They also dier in their restricSon endonuclease cleavage sites and the sizes of their viral proteins.

Three subfamilies are organized with respect to cell tropism, genomic organizaSon, etc.

ncbi.nlm.nih.gov

Three subfamilies are organized with respect to cell tropism, genomic organizaSon, etc.
Herpesvirus subfamilies: : short growth cycle, spreads rapidly, latent in sensory neurons. : long growth cycle, slow spread, restricted host range, latent in secretory glands and lymphoreScular cells. : growth primarily in epithelial cells and T or B cells. Latent in lymphocytes. Two main characterisScs of HSV infecSons: o Lesion formaSon o Latency: ager iniSal infecSon, viral DNA is harboured in a latent state within cell for months/years/lifeSme. The latent virus can be reacSvated and lead to recurrence of outbreak of the disease.lesions for HSV-1 and 2, or shingles for those who were infected with chickenpox.

Herpes Simplex Virus InfecSons HSV-1 and HSV-2 are closely related but anSgenically disSnguishable members of the alpha subfamily of Herpes viruses. Both types can cause oral and genital lesions. However, HSV-1 predominantly causes oral lesions while HSV-2 mainly causes genital lesions. Virus transmission through exposure to lesion uid, saliva, and vaginal secreSons.

3. Host Range and Epidemiology HSVs cause natural infecSons only in humans. Such infecSons occur world-wide and at all Smes of the year. No animal vectors are known. However, under experimental condiSons the virus will also infect hamsters, mice, rats, guinea pigs, rabbits, and embryonated chicken eggs. About 1/3 of people in developing countries are seroposiSve, with the highest prevalence in the lower socio-economic classes. HSV is also widespread in developed countries; for instance, prevalence data from the U.S., U.K. and Sweden indicate that 300,000- 500,000 new cases of HSV-2 appear each year in these countries. Factors found to inuence acquisiSon of HSV-2 include: gender (women more than men); race (some more than others); marital status (divorced more than single or married); place of residence (urban more than suburban); and number of sexual partners (higher number poses increased risk).

4. Transmission and Tropism Transmission of HSV1 and HSV2 occurs during close personal contact. Mucosal and skin surfaces are the primary sites of iniSal infecSon. While HSV1 is most commonly associated with facial lesions and HSV2 with genital lesions, HSV1 infecSon of the genitals can occur, resulSng from self inoculaSon or from oral sexual pracSces. Sexual contact is the primary route of HSV2 transmission. HSV2 can also be transmiped from mother to infants during passage through the birth canal. The CNS (centralnervous system is an addiSonal target of HSV infecSon, as are the liver, lungs, adrenal glands, pancreas, small and large intesSne and bone marrow.

HSV-2

HSV-1

5. Virion Structure HSV virions are characterized by large (150-200 nm) enveloped, icosahedral parScles surrounding a linear dsDNA genome that is densely packaged in a liquid- crystalline fashion. The capsid is made of 162 capsomeres consisSng of 150 hexons and 12 pentons (T=16). Each hexon contains six copies of the 155 kD major capsid protein (VP5), while each penton contains VP5 and 80-100 copies of the vertex protein VP26. An addiSonal capsid protein, VP24, projects into the interior and is thought to interact with the genome. The capsid oats in a matrix, called the tegument, which contains 20 disEnct structural proteins of various funcSons, including a virion- associated host "shut-o" funcSon. The viral envelope, acquired during budding of capsids through the cellular membranes of infected cells, is a trilaminar lipid membrane that has 8 nm spikes protruding from the surface. More than 10 viral-encoded glycoproteins reside in the envelope, at least three of which are involved in penetraSon into host cells. In total, more than 30 HSV proteins are associated with mature virions.

Tegument: large space between Capsid and envelope, contains ~14 dierent proteins VP26 (VP5)

Enveloped, spherical to pleomorphic, 150-200 nm in diameter, T=16 icosahedral symmetry. Capsid consists of 162 capsomers and is surrounded by an amorphous tegument. Glycoproteins complexes are embedded in the lipid envelope.

Of the 70 proteins or so that the PRV genome encodes, at least 33 are found in the final virion: 7 in the capsid 14 in the tegument 12 in the envelope

(PRV= pseudorabisvirus a porcine herpes virus)

h#p://www.molbio1.princeton.edu/labs/enquist/research/PRVstructure.html

6. Genomic OrganisaSon The virion-associated HSV genome consists of a linear, 120-230 kb (about 100x106 D) double-stranded DNA. Upon infecSon, viral DNA circularizes prior to any gene expression or replicaSon. The HSV genome is characterized by an unusually high G+C content and a complex arrangement of components (indicated by anbULbancUS'ca.). It consists of two covalently linked segments, one long (126 kb; UL) and one short (26 kb; US). The long (UL) segment contains several sequence moSfs: A terminal sequence (TRL), made up of the "a" sequence (200-500 copies of highly repeSSve G+C DNA) followed by the 8.8 kb "b" element; A 108 kb unique sequence element (UL); Another inverted copy of the "b" terminal repeat element (IRL), followed by a number of "a" elements. The short (US) segment contains the following moSfs: A 6.6 kb "c" sequence element (IRS); A 23 kb unique sequence element (US); A terminal sequence (TRS), made up of a second inverted copy of the "c" element followed by an addiSonal "a" element.

HSV Genomic OrganizaSon

anbULbancUS'ca

an b b an c c a

UL US

Four isomers produced during DNA replicaSon, via recombiaSon events between the repeated sequences. The "a" elements at the ends of HSV DNA are involved in packaging the genome within viral capsids. The arrangement of the inverted repeat (IR) elements allows the genomic segments to invert (or "isomerize") relaSve to each other into four sequence arrangements.

n= one or more copies of a = complementary sequence

Herpes Simplex Virus

Genome is large (approx 150 kb), linear dsDNA with repeated sequence elements
a b b a c c a

L S HSV encodes much of its own replicaSon machinery. a: no ORFs b: 4 ORFs c: 1 ORF UL: 65 ORFs Us: 14 ORFs HSV gene total: at least 84 genes

Genetic Map of HSV

(Map units 0-100 Circular format) Latency gene LAT

Viral genes are sequenSally expressed during replicaSon cycle. Immediate Early ( genes): transcribed a few hours ager infecSon Early ( genes): 4-8 hrs p. i., involved in viral DNA replicaSon

Late ( genes): many, expressed ager viral DNA synthesis (structural proteins).

hpp://www.genetherapyreview.com/images/stories/hsv_genome.jpg

Over 90% of HSV proteins are encoded by typical translaSonal ORFs, iniSated by ATG and terminated by TAA, TAG, or TGA. Generally, these ORFs are expressed as unique, unspliced mRNAs that are controlled by 40- 200 bp temporal class-specic promoters (i.e., recognised by eukaryoEc RNA polymerase II and having TATA box homologies) and terminated by the polyadenylaSon signal AATAAA. In fact, the HSV thymidine kinase promoter is widely used as a general prototype for eukaryoSc pol- II promoters. Most HSV ORFs also have a 150-250 base leader sequence between the transcript cap site and the transcripIon iniIator site. A few relaSvely abundant spliced transcripts are also expressed, namely those belonging to the latency associated transcript family (LATs).

GENE EXPRESSION Each viral transcript usually encodes a single protein and has a promoter/regulatory sequence, a TATA box, a transcripSon iniSaSon site, a 5 leader sequence of 30-300 bp (not translated), a 3 nontranslated sequence of 10-30 bp, and a poly A signal. There are many gene overlaps. There are only few spliced genes. Some of the expressed ORFs are anSsense to each other. Some ORFs can be accessed from more than one promoter. There are some non-coding genes.

7. TranscripSon Sequence analysis indicates that the HSV genome expresses more than 70 proteins during the lySc infecSon cycle. About 25 of these are absolutely essenSal to virus replicaSon (as seen in cultured cells), while the rest are involved in determining host range, Sssue tropism and suppressing the host response to infecSon. The overall papern of HSV lySc-phase transcripSon is similar to that of other animal viruses, with viral DNA replicaSon marking a clear switch from early (a and b) to late (bg- g1 and g-g2) gene expression. All HSV transcripSons are mediated by host cell RNA polymerase II (pol-II) and cellular transcripSon factors. The basic temporal class-specic promoter for all early viral transcripts is a typical pol-II promoter. TranscripSon begins when a cellular transcripSon factor, Oct-1, interacts with the tegument-associated TIF late protein, which in turn interacts with HSV sequence elements to produce a rapid increase in (immediate early) class viral transcripts, the rst produced during lySc infecSons. Prior to DNA replicaSon, (delayed early) class transcripts are then expressed at maximal levels. Following DNA replicaSon, the (intermediate or leaky late) class transcripts are expressed. Among the transcripts are a number of capsid and structural proteins. The promoters controlling transcripts also include pol-II elements but have addiSonal regulatory elements near and downstream of the cap site; these sites appear to have a role in blocking transcripSonal shut-o. Moreover, the funcSonal porSon of the promoters extends 5' of the cap site only 25-30 bases to a clear TATA box, while elements at and 3' of the cap site are also present; it is thought that the lack of early transcripSon of sequences may thus be due to the truncated nature of their promoters 5' of the cap site. Finally, latency associated transcripts (LATs) are mediated by a single promoter.

Temporal Regulation of HSV Gene Expression

Immediate Early Gene Expression
(promoters have identical cis-element)

-cellular transcripSon factors Oct-1, HCF-1 -tegument-associated TIF (carried in TIF: trans-inducing factor infecSng virion)

Many Immediate early proteins act as transcription factors for early virus gene expression (expression of some cellular

Early Gene Expression

genes as well) and late gene expression.

Early genes encode proteins involved in viral DNA synthesis

HSV DNA Synthesis

Late Gene Expression

Late proteins are involved in virus assembly release

8. HSV replicaSon cycle:

Entry

gC binds to heparan sulfate gD binds to addiSonal receptors These two receptors are dierenSally expressed in dierent cell types. gB, gH and gL are fusion proteins. Fusion can take place either at the plasma membrane or within endosomes. Nucleocapsid is transported via a microtubular network to the nuclear pore, where the viral DNA is released, leaving the empty capsid on the outside of the nuclear membrane.
h#p://www.dbc.uci.edu/~faculty/wagner/entry.jpg

Plasma membrane

endocytosis

HSV enters the cell, nucleocapsid reaches nucleus, viral DNA enters nucleus and is circularized, either by direct ligaSon or by base pairing between the ends.

Nuclear membrane

New virions are released

Late gene expression assembly

Cellular ligase?

ReplicaSon, Late transcripSon

circularizaSon

+histones minichromosome

hpp://wpcontent.answcdn.com/wikipedia/en/thumb/3/35/HSV_replicaSon.png/350px-HSV_replicaSon.png

HSV Life Cycle Movies

HSV Entry
h#p://www.youtube.com/watch?v=moBtPyuPXrE TranscripSon h#p://www.youtube.com/watch?v=_R7ARxKh84&NR=1 DNA synthesis and encapsidaSon h#p://www.youtube.com/watch?v=6EAI4_NGNTM&NR=1 HSV envelopment and release h#p://www.youtube.com/watch?v=bgj1YpevA6A&NR=1

(note: -trans-inducing factor (-TIF), a viral tegument protein, binds to promoters of genes to trans-acSvate them

8. ReplicaSon DNA synthesis occurs in the nucleus. The HSV genome contains three cis-acEng origins of replicaEon (ORIs). These ORIs have a high degree of dyad symmetry and include palindromic sequences; intriguingly, removal of one of the two symmetrical regions does not necessarily disrupt their funcSon. Seven viral proteins are required for HSV replicaSon. The process is iniIated when UL9, an ORI binding protein, interacts with the criIcal sequence GTTCGCAC in the US ORI. Next, the viral helicase/primase (UL5, 8 and 52) associates with this region to generate an iniSaSon "bubble" and then with the polymerase (UL30), which funcSons as a complex with the ds DNA binding protein UL42. DNA synthesis is conSnuous along one strand and disconSnuous along the other (lagging) strand, with another major viral DNA binding protein, UL29, maintaining the growing forks. A number of other viral proteins are also indirectly involved in replicaSon by altering the pool of deoxyribonucleoSde precursors and by having a role in repair and proofreading funcSons. Newly synthesised HSV DNA is concatameric. During lySc infecSons in cell culture, as much as 50% of the host cell DNA content (by mass) is of a viral nature ager 20 h of replicaSon.

HSV-1 proteins involved in viral DNA synthesis

genes, 3 origins of replicaSon
UL9: ori binding protein UL29:single-stranded binding protein UL5/UL8/UL52: helicase-primase complex UL30: DNA polymerase UL42: Processivity factor

8. HSV ReplicaSon cont..d

Establishment of bidirecSonal DNA replicaSon
Three origins of replicaSon (2 Ori S (short), 1 Ori L (large)

ICP = Infected Cell Protein

Herpes Simplex Virus

h#p://bio.lundberg.gu.se/research/project2.html

Much of DNA replicaSon machinery is encoded by the virus.

Rolling circle replicaSon subsequently produces mulSmeric concatemers of viral DNA

Viral nucleocapsids are assembled on a scaold in the nucleus

HSV capsids assemble around viral scaolding proteins in the nucleus, and then other viral proteins interact with replicated viral DNA to allow DNA encapsidaSon. The encapsidated DNA is not associated with histones, but highly basic polyamines (perhaps synthesized with viral enzymes) appear to facilitate the encapsidaSon process and full capsids presumably associate with tegument (matrix) proteins near the nuclear membrane.

h#p://www.dbc.uci.edu/~faculty/wagner/encap.jpg

Summary of ReplicaSon
The replicaSon cycle of Herpes Simplex virus.
1.Specic proteins in the viral envelope apach to host cell receptors on the cell membrane. 2. PenetraSon is achieved when the viral envelope fuses with the cell membrane releasing the nucleocapsid directly into the cytoplasm. 3. The virion is uncoated and the viral DNA is transported into the nucleus. 4. In the nucleus, the viral DNA is transcribed into early mRNAs which are transported to the cytoplasm for the translaSon of early proteins. These early proteins are brought back into the nucleus and parScipate in the replicaSon of the virus DNA into many copies. The viral DNA is then transcribed into the late mRNAs which exit to the cytoplasm for translaSon into the late (nucleocapsid and envelope) proteins. 5. The capsid proteins encapsidate the newly replicated genomes. The envelope proteins are imbedded in the nuclear membrane. 6. The nucleocapsids are enveloped by budding through the nuclear membrane, and the mature viruses are released from the cell through cytoplasmic channels.

 9. Latency HSV has the ability to persist in a latent (non-replicaSng) state. This condiSon is compaSble with cell survival but is ogen followed by subsequent episodes of virus reacSvaSon and clinical symptoms. In the latent process, HSV virions (or possibly just capsids) are transported intracellularly to the sensory nerve cell bodies in the ganglia; in the ganglia, HSV DNA is believed to persist in an extra chromosomal state, likely as a circular episome. It is not understood why inducSon of the lySc cascade fails during latency; one possible explanaSon is that one or more important cellular transcripSon factors may be lacking in such cells. HSV1 establishes reacSvatable latency in the trigeminal ganglia,

while HSV2 latency occurs in the sciaSc nerve ganglia.

Latently infected neurons represent <1% of the total neurons in the ganglia, and infected cells typically contain no more than 5-10 copies of viral DNA. Axonal injury is thought to be involved in reacSvaSon of the latent virus, which normally results in the development of lesions at the site of primary infecSon or along an intact nerve route.

Viral DNA enters nucleus of the neuron, circularizes and remains dormant.

Latency-associated transcripts (LAT) map to the b region of the genome, expressed as RNAs which accumulate in the nucleus, prevent apoptosis SSmuli such as stress, UV light, etc can induce reacSvaSon of the virus. Virus is transported back to mucosal sites, where it can replicate and form lesions.

Another movie hpp://www.youtube.com/watch?v=zMLu1kRLP4Y&feature=related

h#p://darwin.bio.uci.edu/~faculty/wagner/latency.jpg

The Big Picture

h#p://www.dbc.uci.edu/~faculty/wagner/cycle03.jpg

10. Pathogenicity The clinical severity of HSV infecSon varies among individuals, ranging from an asymptomaSc to a severe (and possibly fatal) systemic illness. HSV-induced skin lesions involve a combinaSon of virus mediated cell death and an associated inammatory response. MulSnucleated giant cells are commonly formed at these lesions, as is a clear (vesicular) uid that contains large quanSSes of virus and cell debris. Upon healing, this uid becomes pustular and then scabs.

The infected cell has mulSple nuclei that have moulded together and become lled with viral parScles.

HSV infecSons in the Neonate: This is a generalized and severe infecSon caused by HSV-2. It is usually fatal due to the uncontrolled viral replicaSon and disseminaSon to mulSple body organs. It is usually acquired from a primary genital infecSon in the mother either in utero or, more frequently, perinatally during passage through the genital canal at delivery when mother is acSvely shedding virus. This can be avoided by having a C-secSon.

TransformaSon/oncogenecity:

Many epidemiological studies have shown a clear associaSon between HSV2 infecSon and an increased risk of squamous cervical cancer.

Experimental studies have demonstrated that mice inoculated intracervically with HSV2 can undergo cervical neoplasSc transformaSon. While the exact role of HSV2 in carcinogenesis is sSll unknown, the HSV2-encoded N-terminal domain of a ribonucleoSde reductase (large subunit) is believed to be an oncoprotein that acts as a growth factor receptor protein kinase. This oncogene is presumed to have been acquired from human cells by an ancestral HSV.

Cytomegalovirus HHR-5 (CMV) (Herpesviridae): cytomegalo means

large cell CMV is a common human pathogen. Most CMV infecSons are acquired subclinically
during childhood. CMV is the most common cause of congenital

defects. CMV becomes parScularly important as a pathogen in immunocompromised

paSents (e.g. AIDS paSents). 1. Structure, classicaSon and replicaSon: CMV is a betaherpesvirus and infects only humans. Genome is the largest amongst herpes viruses. It normally infects epithelial cells, broblasts and macrophages. Latent infecSon occurs in lymphocytes and bone marrow. Cell-mediated immunity is essenSal for controlling CMV infecIon. Immunosuppression (e.g. organ transplant, HIV-1 infecSon etc.) results in reacSvaSon of CMV with serious consequences. 2. Clinical symptoms and epidemiology: . Transmission is through contact with body uids (oral, sexual, congenital routes, blood transfusion, and organ transplantaSon). ReacSvaSon of latent CMV is one of the commonest opportunisSc infecSons leading to death in AIDS paSents.

11. Treatment

HSV infecSons readily respond to treatment by ACYCLOVIR. The drug requires acSvaSon by virus-encoded thymidine kinase enzyme, which is the basis for Acyclovir specicity. Acyclovir specically inhibits HSV DNA polymerase. The drug also incorporates into viral DNA causing chain terminaSon. It has minimal eect on cellular DNA polymerases. There are no severe toxic side eects associated with Acyclovir, even ager prolonged treatment. Acyclovir may be used topically, orally, or systemically to treat genital herpes. The drug has also been used prophylacScally to protect against potenSal exposure.

L44 Study Guide

What is the dierence between HSV-1 and HSV-2? Describe in general the structure of HSV and its genomic organizaSon. What is meant by a, b, c elements and UL, US? Approximately how many genes does the virus have? What is meant by temporal classes of gene expression? Describe in general the replicaSon strategy of HSV. What is meant by latency? How is this achieved? What is the host range of HSV? Who are most at risk of infecSon by cytomegalovirus? Describe the mechanism of acSon of the anSviral acyclovir.