1. An electron dense core harboring the dsDNA viral genome
2. A protein capsid surrounding the virus core, the capsid is comprised of 162 capsomeres.
3. An amorphous layer surrounding the capsid termed the tegument.
4. An envelope (lipid bilayer) containing spikes that probably represent viral glycoproteins.

The herpesviruses are distinguished by their biological properties: a) they encode many enzymes involved in nucleic acid metabolism, b) their replication and assembly occur in the nucleus, c) the cell is killed (lysed) as an outcome of virus infection, d) they have the capacity to enter a latent state in which only a small subset of the viral gene complement is expressed.

The herpesviruses have been classified into three subfamilies.

Alphaherpesvirinae

Are characterized by a broad host range and are highly lytic in culture.

Betaherpesvirinae

Have a restricted host range, grow more slowly in culture and cells infected with this subclass often show enlargement.

Gammaherpesvirinae

Infect lymphoblastoid cells.

Human Herpesviruses

At least six human herpesviruses have been described. These include: Herpes simplex virus type 1 (HSV-1), Herpes simplex virus type two (HSV-2), Varicella zoster virus (VZV), Cytomegalovirus (CMV), Epstein-Barr virus (EBV), and Human herpesvirus six (HHV - 6). This handout will focus on HSV-1 and HSV-2. These two viruses share extensive nucleic acid sequence homology (50%) and for the purposes of this discussion can be considered together.

Herpes simplex virus diseases

Primary infection

Infection of individuals who have not been previously exposed to HSV.

Recurrent infection

Reactivation of latent virus that results in a second infection

Initial infection

Infection of an individual with HSV-1 who was previously exposed to HSV-2 (or the reverse)

Exogenous reinfection

Infection of an individual with the a second strain of the same HSV type.

Herpes labialis

(orolabial herpes). Usually occurs in children under five years of age, the primary infection usually presents as a gingivostomatitis. Pharyngitis may also be present. Predominantly associated with HSV-1.

Genital herpes

Sexually transmitted disease associated mainly with HSV-2.

Neonatal infection

Life threatening infection by HSV usually acquired by child during birth. Systemic infection.

Keratoconjunctivitis

HSV infection of conjunctiva and eye proper which can lead to blindness.

Skin infections

Usually acquired by health care workers or researchers by direct inoculation of virus from contaminated materials to a cut on the hand (whitlow).

CNS infections

Herpes encephalitis is a rare but devastating infection of the brain with high mortality and morbidity.

The virion

HSV virions contain over 30 proteins (virion polypeptides, VPs) including eight glycoproteins (gB, gC, gD, gE, gG, gH and gI) some of which are components of the envelope spikes. The tegument contains at least two proteins of known function: àTIF (alpha trans-inducing factor, also known as VP16 and vmw65) and VHS (virion host shut off).

Viral DNA

The viral genome is 150 kbp in size and contains single stranded nicks and gaps. It consists of two components, a long and short region flanked by inverted repeats. The structure can be written like this ab-UL-b'a'c'-Us-ca. The "a" sequence is highly conserved and consists of variable numbers of repeat elements. The long and short components can invert relative to each other yielding four linear isomers of the viral genome. The importance of inversion of viral genomes is uncertain in that mutants which do not invert grow normally in culture.

Virus Growth

Attachment and penetration

Five of eight viral glycoproteins are dispensable for virus growth in culture (gC, gE, gG, gI, gJ). Three glycoproteins (gB, gD, and gH) are essential and represent the minimal set of surface proteins necessary to sustain and carry out the dominant flow of events. Heparin sulfate proteoglycans appear to be the receptor molecules which are recognized by either gB or gC and which permit initial attachment of the virus. gB and gD are essential for virus penetration. Penetration occurs by direct fusion of the viral envelope with the cell membrane. Virions which attach to the plasma membrane which cannot fuse are internalized and degraded in endocytotic vesicles. Capsids are transported by the cellular cytoskeleton to nuclear pores and viral DNA is released into the nucleus where it accumulates. Virion components mediate the shut off of host macromolecular synthesis and àTIF acts to induce initial gene expression. HSV has the potential to encode at least 70 polypeptides but detectable HSV polypeptides do not exceed 50.

Gene expression

HSV transcription and protein synthesis is highly ordered. Although the absolute levels of viral protein synthesis may vary, different genes can be grouped on the basis of their requirements for synthesis. Hence, HSV genes have been subdivided into 3 broad groups based on their time and requirements for expression (alpha, beta and gamma).

Alpha genes

There are five alpha genes which have been identified and described as ICPs (infected cell proteins), these include ICP0, ICP4, ICP22, ICP27 and ICP47. The à genes are by definition expressed in the absence of viral protein synthesis and contain the sequence GyATGnTAATGArATTCyTTGnGGG upstream of their coding regions. Their peak synthesis occurs 2-4 hours post infection, but they continue to accumulate until late in infection. All alpha genes appear to function as regulatory proteins with the possible exception of ICP47.

Beta genes

These genes are not expressed in the absence of alpha proteins and their expression is enhanced in the presence of drugs which block DNA synthesis. They reach peak rates of synthesis 5-7 hr post infection. The genes have been subdivided into the beta 1 and beta 2 subclasses. beta 1 genes appear early after infection, but require the presence of à 4 protein for their synthesis. Examples of beta 1 genes include the large component of ribonucleotide reductase and the major DNA binding protein (ICP8). beta 2 genes include viral thymidine kinase (TK) and the viral DNA polymerase. beta gene synthesis immediately precedes the onset of viral DNA synthesis and most viral genes involved in viral nucleic acid metabolism appear to be beta genes.

Gamma genes

This class of genes is for convenience also separated into two groups. gamma 1 genes are expressed early in infection and are only minimally affected by inhibitors of DNA synthesis (example, major capsid protein). In contrast, gamma 2 genes are expressed late in infection and are not expressed in the presence of inhibitors of viral DNA synthesis.

The location of the gene classes within the HSV genome is of interest. alpha genes map at the termini of the long and short components and tend to cluster together. In particular, alpha genes surround the HSV origin of replication in the short region (oris). Each alpha gene has its own promoter-regulatory region and transcription initiation and termination sites. beta and gamma genes are scattered in both the long and short components. Interestingly, the beta genes specifying the DNA polymerase and the DNA binding protein flank the origin of replication in the long region (oriL). There is little gene overlap and few instances of gene splicing for any of the HSV gene classes.

Essential and nonessential genes

Large numbers of viral mutants have been generated and have led to identification of genes that are essential or nonessential for HSV growth in tissue culture. essential: gB, gD, major DNA binding protein (ICP8), alpha 27 and alpha 4. nonessential: all genes in the unique short region (except for gD), dUTPase, gC, alkaline DNAse, thymidine kinase, ribonucleotide reductase, uracil DNA glycosylase.

Synthesis of viral DNA

HSV specifies a large number of enzymes involved in viral DNA synthesis. Viral DNA synthesis begins 3 hrs postinfection and continues for 9-12 hrs. DNA replication occurs in the nucleus and evidence suggests that late in infection HSV DNA replicates by the rolling circle mechanism.

Origins of replication

Three origins of replication have been identified within the HSV genome. One origin is present in each "c" component of the short region and one origin is present in the unique long region between the major DNA binding protein (ICP8) and the DNA polymerase. oriL has A/T rich sequences with a near perfect palindrome. oriL and one copy of oris can be deleted without affecting the ability of the virus to multiply. Both origins are situated between transcriptional initiation sites.

Functional requirements for viral replication.

1. Proteins essential for viral origin dependent amplification
2. Enzymes involved in nucleic acid metabolism (thymidine kinase, ribonucleotide reductase, dUTPase, uracil DNA glycosylase, alkaline exonuclease.

Using the Challberg assay seven genes have been identified which are necessary for origin dependent replication: viral DNA polymerase, ICP8 (single stranded DNA binding protein), origin binding protein, dsDNA binding protein ,and three other proteins which may be involved in primase and helicase activities.

The importance of the virally encoded enzymes in HSV replication is detailed below.

• Alkaline DNase: essential for viral growth and DNA replication
• Thymidine kinase (TK): broad substrate specificity
• Ribonucleotide reductase: reduces ribonucleotides to deoxyribonucleotides creating a pool of substrates for DNA synthesis. It is comprised of two subunits 140kd and 38kd . Both subunits are required for activity.
• Uracil DNA glycosylase: involved in DNA repair and proof reading, corrects insertion of dUTP into DNA.
• dUTPase: converts dUTP to dUMP preventing dUTP incorporation into DNA and providing a pool of dUMP for conversion to dTMP.

Capsid assembly occurs in the nucleus. Cleavage and packaging of HSV DNA are probably linked processes. The packaged DNA can be defined by the distance between two directly repeated "a" sequences. Evidence suggests that a head full mechanism may also operate once a minimal amount of DNA has been packaged. Virus matures at nuclear membranes at "patches" where cellular proteins have been displaced by viral glycoproteins. How glycoproteins are targeted to and enter the nuclear membranes is unclear.

Regulation of viral gene expression

RNA polymerase II is responsible for viral mRNA synthesis. In general viral mRNAs are capped and polyadenylated just like cellular mRNAs. Only a small portion are spliced. DNA sequences upstream of HSV genes determine their capacity to be expressed as alpha or beta genes. alpha gene expression is directed by alpha-TIF (VP16, vmw65) which complexes with cellular proteins to bind a TAATGArATT motif. Despite controversy over the ability of ICP4 to bind DNA their appears to be support for specific ICP4 binding sites upstream of the TK gene (beta gene) and of ICP4 itself. In general expression of HSV genes appear to be controlled by three means:(a) cis-acting sites for both viral transacting factors and cellular factors. (b) trans-acting signal proteins specified by the virus and (c) viral and cellular factors involved in viral DNA synthesis and post-translational modification of viral proteins.

Thus far there are three "top guns" for viral gene regulation.

Alpha-TIF: alpha trans-inducing factor (also known as VP16 and vmw65) is a structural component of the HSV virion (located in the tegument). In conjunction with cellular proteins alpha-TIF binds the TAATGArATT motif upstream of alpha genes and induces their transcription. alpha-TIF does not bind DNA directly and there are about 500-1000 copies of alpha-TIF per virus particle.

ICP0: also known as IE110, it is an alpha gene which can promiscuously transactivate transfected genes. Its function in infected cells is not known. ICP0 deletions mutants are still viable in cell culture.

ICP4: (also known as IE175) This is the big guy. It is the major transactivator of HSV genes and is an essential gene product. ICP4 is autoregulatory and probably turns off its own synthesis and ICP0 synthesis as well. ICP4 functions as both a transactivator and a repressor and may be regulated by post-translational modifications, position of the DNA binding site or strength of binding to DNA.