The replication of the prototypical alphaherpesvirus HSV The HSV virion

All herpesviruses possess similar enveloped icosahedrons. The envelope of HSV contains 10 or more glycoproteins. The matrix (called the tegument for obscure reasons) lies between the envelope and the capsid and contains at least 15—20 proteins. The capsid itself is made up of six proteins; the major one, VP5, is the 150,000-dalton major capsid protein. VP5 is also called Ul19 for the position of its gene on the viral genetic map. A computer-enhanced model of the HSV capsid structure is shown in Fig. 9.3. A more conventional electron microscopic view is shown in Fig. 17.1. The molar ratio of HSV capsid proteins is tabulated in Table 11.2 - various capsid proteins are present in widely differing amounts.

The viral genome

While each herpesvirus is different, a number of general features can be illustrated with the HSV-1 genome. The HSV-1 genome is linear, and is 152,000 base pairs long. With HSV, the left end of the genome is set as 0 map unit and the right is 1.00 map unit; therefore, each 0.1 map unit is 15,200 base pairs. Although the virion DNA is linear, the genome becomes circular upon entering latency. An electron micrograph of this DNA, which is about 50 microns long, is shown in Fig. 11.9.

A high-resolution genetic and transcription map of the HSV genome is shown in Fig. 17.2. Because the HSV genome spends the majority of its life in the latent state, the genome becomes circular, the map is shown as a circle, but note that the genome's ends are indicated at the top of the circle. Since the virus encodes nearly 100 transcripts and more than 70 open translational reading frames (ORFs), the map is complex. Still, the basic methods of interpreting it are the

  1. 17.1 Electron micrograph of an enveloped HSV-1 virion revealing specific features, especially glycoprotein spikes projecting from the envelope. The capsid has a diameter of about 100 nm and encapsidates the 152,000-base pair viral genome. The interior of the capsid does not contain any cellular histones, in contrast to smaller DNA viruses. Rather, it contains relatively high levels of polyamines such as spermidine and putrescine, which serve as counterions to allow compact folding of the viral DNA needed in the packaging. (Photograph courtesy of Jay Brown.)
  2. 17.1 Electron micrograph of an enveloped HSV-1 virion revealing specific features, especially glycoprotein spikes projecting from the envelope. The capsid has a diameter of about 100 nm and encapsidates the 152,000-base pair viral genome. The interior of the capsid does not contain any cellular histones, in contrast to smaller DNA viruses. Rather, it contains relatively high levels of polyamines such as spermidine and putrescine, which serve as counterions to allow compact folding of the viral DNA needed in the packaging. (Photograph courtesy of Jay Brown.)
Trig Pie Chart
  1. 17.2 The HSV-1 genetic and transcription map. Specific features of the genome are discussed in the text, and tabulated in Table 17.1. Individual transcripts are controlled by their own specific promoters, and splicing is uncommon. Each transcript is headed by its own promoter, and most are terminated with individual cleavage/polyadenylation signals. The time of expression of the various transcripts is roughly divided into immediate-early (a), early (p), late (Py), and strict late (y). This is, in turn, based on whether the transcripts are expressed in the absence of viral protein synthesis (a), before viral DNA replication and shutoff following this (p), before viral DNA replication but reaching maximum levels following this (Py), or only following viral DNA replication (y). The genome is about 152,000 base pairs and contains extensive regions of duplicated sequences.
  2. 17.2 The HSV-1 genetic and transcription map. Specific features of the genome are discussed in the text, and tabulated in Table 17.1. Individual transcripts are controlled by their own specific promoters, and splicing is uncommon. Each transcript is headed by its own promoter, and most are terminated with individual cleavage/polyadenylation signals. The time of expression of the various transcripts is roughly divided into immediate-early (a), early (p), late (Py), and strict late (y). This is, in turn, based on whether the transcripts are expressed in the absence of viral protein synthesis (a), before viral DNA replication and shutoff following this (p), before viral DNA replication but reaching maximum levels following this (Py), or only following viral DNA replication (y). The genome is about 152,000 base pairs and contains extensive regions of duplicated sequences.

same as with the simpler SV40 map. Interpretation of the HSV genetic and transcription map is aided by the fact that few viral transcripts are spliced and most ORFs are expressed by a single transcript, each with a contiguous promoter.

The genetic map of HSV-1 is summarized in Table 17.1, where viral proteins and other genetic elements are listed. The number of viral proteins that are not required for replication of the virus in cultured cells is large. Many of these dispensable proteins have a role in aspects

Table 17.1 Some genetic functions encoded by herpes simplex virus type 1.

Required

Required

Table 17.1 Some genetic functions encoded by herpes simplex virus type 1.

Location (map

for replication

Name of element

unit) (Fig. 17.2)

in culture?

or protein

Function

0.0

Yes

"a"

Cis genome cleavage, packaging signal

0.00-0.06

Yes

RL

See below

0.05

No

ICP34.5

Reactivation (?)

0.01 (RL)

Yes

a0

Immediate-early transcription regulator (mRNAspliced)/interferon

inhibitor

0.02 (RL)

No

LAT

Approximately 600 bases in 5' region facilitate reactivation; no

protein involved

0.04 (RL)

No

LAT-intron

Stable accumulation in nucleus of latently infected neurons,

unknown function

0.06

Yes

gL

Viral entry, associates with gH

0.07

No

UL2

Uracil DNA, glycosylase DNA repair

0.08

No

UL3

Nonvirion membrane-associated protein

0.09

No

UL4

Tegument protein, unknown function

0.1

Yes

Helicase-primase

DNA replication

0.1

Yes

UL6

Capsid protein, capsid maturation, DNA packaging

0.11

No

Ul7

Unknown

0.12

Yes

Helicase-primase

DNA replication

0.13

Yes

ori-binding protein

DNA replication

0.14

No

gM

Glycoprotein of unknown function

0.14

Yes

UL11

Tegument protein, capsid egress and envelopment

0.16

Yes

Alkaline exonuclease

DNA packaging (?), capsid egress

0.15

No

UL12.5

C-terminal two-thirds of UL12, expressed by separate mRNA;

specific function unknown

0.17

No

Protein kinase

Tegument associated

0.18

No

UL14

Unknown

0.16/0.18

Yes

UL15

DNA packaging, cleavage of replicating DNA(?), (spliced mRNA)

0.17

No

UL1 6

Unknown

0.2

Yes

Ul17

Cleavage and packaging of DNA

0.23

Yes

Capsid

Triplex

0.25

Yes

Capsid

Major capsid protein, hexon

0.27

Yes

UL20

Membrane associated, virion egress

0.28

No

UL21

Tegument

0.3

Yes

gH

Viral entry, functions with gL

0.32

No

UL23

Thymidine kinase

0.33

No

UL24

Unknown

0.33

Yes

UL25

Tegument protein, capsid maturation, DNA packaging

0.34

Yes

UL26

Maturational protease

0.34

Yes

UL26.5

Scaffolding protein

0.36

Yes

gB

Glycoprotein required for virus entry

0.37

Yes

UL28

Capsid maturation, DNA packaging

0.4

Yes

UL29

ssDNA-binding protein, DNA replication

0.41

No

OriL

Origin of replication

0.42

Yes

DNA po1

DNA replication

0.45

No

UL31

Nuclear phosphoprotein, nuclear budding

0.45

Yes

UL32

Capsid maturation, DNA packaging

0.46

Yes

UL33

Capsid maturation, DNA packaging

0.47

No

UL34

Membrane phosphoprotein, nuclear budding

0.47

Yes

UL35

Capsid protein, capsomer tips

0.50

No

Ul36

ICP1/2, tegument protein

Table 17.1 continued

Requ

ired

Location (map

for replication

Name of element

unit) (Fig. 17.2)

in cu

lture?

or protein

Function

0.55

No

Ul37

Tegument phosphoprotein

0.57

Yes

UL38

Capsid protein, triplex

0.58

Yes

UL39

Large-subunit ribonucleotide reductase

0.59

Yes

UL40

Small-subunit ribonucleotide reductase

0.6

No

UL41

VHS, virion-associated host shutoff protein, destabilizes mRNA, envelopment

0.61

Yes

UL42

Polymerase accessory protein, DNA replication

0.62

No

UL43

Unknown

0.62

No

UL43.5

Antisense to UL43

0.63

No

gC

Initial stages of virus-cell association

0.64

No

UL45

Membrane associated

0.65

No

UL46

Tegument associated, modulates a-TIF

0.66

No

Ul47

Tegument associated, modulates a-TIF

0.67

Yes

a-TIF

Virion-associated transcriptional activator, enhances immediate-early, envelopment transcription through cellular Oct-1 and CTF binding at TATGARAT sites

0.68

No

UL49

Tegument protein

0.68

No

UL49.5

Unknown

0.69

No

dUTPase

Nucleotide pool metabolism

0.7

No

UL51

Unknown

0.71

Yes

Helicase/primase

DNA replication

0.73

No

gK

Virion egress

0.74

Yes

a27

Immediate-early regulatory protein, inhibits splicing

0.75

No

UL55

Unknown

0.76

No

UL56

Tegument protein, affects pathogenesis

0.76 to 0.82

Yes

Rl

See RL above

0.82

Yes

Rl/Rs junction

Joint region, contains "a" sequences

0.82-0.86

Yes

Rs

See below

0.82-0.86 (RS)

Yes

a4

Immediate-early transcriptional activator

0.86 (RS)

Yes

OriS (cis-acting)

Origin of replication

0.86

No

a22

Immediate-early, protein affects virus's ability to replicate in certain cells

0.87

No

Us2

Unknown

0.89

No

US3

Tegument-associated protein kinase, phosphorylates UL34 and US9

0.9

No

gG 4

Glycoprotein of unknown function

0.9

No

gJ

Glycoprotein of unknown function

0.91

Yes

gD

Virus entry, binds HVEM

0.92

No

gI

Glycoprotein that acts with gE, binds IgG Fc, and influences cell-to-cell spread of virus

0.93

No

gE

Glycoprotein that acts gI, binds IgG-Fc, and influences cell-to-cell spread of infection

0.94

No

Us9

Tegument-associated phosphoprotein

0.95

No

US10

Tegument-associated protein

0.95

No

US11

Tegument-associated protein phosphoprotein, RNA binding, post-transcriptional regulation

0.96

No

a47

Immediate-early protein that inhibits MHC class I antigen presentation in human and primate cells

0.96-1.00

Yes

Rs

See RS above

1

Yes

"a"

Cis genome cleavage, packaging signal

of the pathogenesis of the virus. The exact function of such proteins, in theory, can be established by studying the effect of the deletion of the genes encoding them on the way the virus replicates in its natural host. Because the natural host of HSV is humans, this analysis must be carried out in animal models instead. This study can be a difficult task, and the actual biological functions of many virus-encoded proteins and enzymes are still unknown.

The genome can be divided into six regions, each encoding a specific function as follows:

1 The ends of the linear molecules. The ends of the genome contain repetitive DNA sequences made up of various numbers of repeats of three basic patterns or groupings termed "a," "b," and "c." The "a" sequences also are found at the junction between the long and short segments of the genome (see a later section). They also contain the signals used in the assembly of mature virions for packaging of the viral DNA.

2 The long repeat (RL) region. The 9000-base pair repeat (RL) encodes both an important immediate-early regulatory protein (a0) and the promoter of most of the "gene" for the latency-associated transcript (LAT). This transcript functions in reactivation from latency by an as yet unknown mechanism.

3 The long unique (UL) region. This region (UL), which is 108,000 base pairs long, encodes at least 56 distinct proteins (actually more because some ORFs are spliced and expressed in redundant ways). It contains genes for the DNA replication enzymes and the capsid proteins, as well as many other proteins.

4 The short repeat (RS) regions. The 6600-base pair short repeats (RS) encode the very important a4 immediate-early protein. This is a very powerful transcriptional activator. It acts along with a0 and a27 (in the UL region) to stimulate the infected cell for all viral gene expression that leads to viral DNA replication.

5 The origins of replication (ori's). HSV contains three short regions of DNA that serve as ori's. In the laboratory, any two can be deleted and virus replication will occur, but the three ori's are always found in clinical isolates. oriL is in the middle of the UL region; oriS is in the RS, and thus is present in two copies. All sets of ori's operate during infection to give a very complicated network of concatemeric DNA and free ends in the replication complex.

6 The unique short (US) region. The 13,000-base pair unique short region (US) encodes 12 ORFs, a number of which are glycoproteins important in viral host range and response to host defense. This region also encodes two other proteins, a22 and a47, which are expressed immediately upon infection. The latter serves to block the infected cell's ability to present viral antigens at its surface.

HSV productive infection

HSV has a very complex genome, and the herpesviruses are the first ones described that have diploid copies of some of their genes. Still, the pattern of productive infection is roughly similar to that seen for smaller DNA viruses. In an HSV infection, the virus supplies most of the components it needs to replicate, and each HSV gene is encoded by an mRNA that has its own promoter and polyadenylation signal. Most (but not all) HSV transcripts are unspliced and the relationship between gene structure and encoded polypeptide is relatively simple.

During the productive replication (vegetative) cycle, HSV gene expression is characterized by a progressive cascade of increasing complexity where the earliest genes expressed are important in "priming" the cell for further viral gene expression, in mobilizing cellular transcriptional machinery, and in blocking immune defenses at the cellular level. This phase is followed by the expression of a number of genes that are either directly or indirectly involved in viral genome replication. And, finally, upon genome replication, viral structural proteins are expressed in high abundance. The time of maximum expression of each viral gene is shown in Fig. 17.2, and the cascade of increasingly complex transcription is shown schematically in Fig. 17.3. The time required for completion of a replicative cycle of HSV and other alpha-herpesviruses is fast compared with beta- and gamma-herpesviruses as well as smaller nuclear-replicating DNA viruses such as adenoviruses and papovaviruses. HSV is able to replicate in a wide selection of animals, tissues, and cultured cells.

IRL IRS us trs

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