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Viral RNA extracted from the cytoplasmic polyribosomes of human epidermoid carcinoma no. 2 cells infected with herpes simplex virus (mRNA) had a sedimentation coefficient between 10 S and 20 S while that from nuclei of infected cells varied in size from 10 S to >80 S . Estimates of the maximum molecular weight of viral RNA from its sedimentation coefficients suggest that at least 10 per cent of the viral genome is transcribed as a single molecule. The ratio of RNA of different sizes found in the nuclei of cells pulse labeled for 12 minutes was approximately the same as those found in cells labeled for longer intervals implying that either some classes of viral mRNA were made as small molecules or that the large viral RNA molecules were cleaved soon after synthesis. Cytoplasmic mRNA competed to a level of at least 80 per cent in viral DNA-RNA hybridization tests with >50 S RNA extracted from nuclei of infected cells. This is consistent with the hypothesis that viral mRNA is produced by cleavage of a large precursor RNA molecule.

The Vinca alkaloids vinblastine sulfate and vincrystine sulfate, which are mitotic poisons, inhibit RNA synthesis in human (HEp-2) cells cultured in vitro. Analyses of RNA synthesis by cells treated with these drugs by acrylamide gel electrophoresis show that 28s rRNA and to a lesser extent 18s rRNA are preferentially inhibited. The synthesis of tRNA is affected much less than that of rRNA. The present experiments suggest that the drugs inhibit both the synthesis and processing of the nucleolar RNA precursors of rRNA. An explanation is also given for previous reports that these alkaloids preferentially inhibit the synthesis of tRNA in animal cells in vitro.

HEp-2 cells were pulse-labeled at different times after infection with herpes simplex virus, and nuclear ribonucleic acid (RNA) and cytoplasmic RNA were examined. The data showed the following: (i) Analysis by acrylamide gel electrophoresis of cytoplasmic RNA of cells infected at high multiplicities [80 to 200 plaque-forming units (PFU)/cell] revealed that ribosomal RNA (rRNA) synthesis falls to less than 10% of control (uninfected cell) values by 5 hr after infection. The synthesis of 4 S RNA also declined but not as rapidly, and at its lowest level it was still 20% of control values. At lower multiplicities (20 PFU), the rate of inhibition was slower than at high multiplicities. However, at all multiplicities the rates of inhibition of 18 S and 28 S rRNA remained identical and higher than that of 4 S RNA. (ii) Analysis of nuclear RNA of cells infected at high multiplicities by sucrose density gradient centrifugation showed that the synthesis and methylation of 45 S rRNA precursor continued at a reduced but significant rate (ca. 30% of control values) at times after infection when no radioactive uridine was incorporated or could be chased into 28 S and 18 S rRNA. This indicates that the inhibition of rRNA synthesis after herpesvirus infection is a result of two processes: a decrease in the rate of synthesis of 45 S RNA and a decrease in the rate of processing of that 45 S RNA that is synthesized. (iii) Hybridization of nuclear and cytoplasmic RNA of infected cells with herpesvirus DNA revealed that a significant proportion of the total viral RNA in the nucleus has a sedimentation coefficient of 50 S or greater. The sedimentation coefficient of virus-specific RNA associated with cytoplasmic polyribosomes is smaller with a maximum at 16 S to 20 S , but there is some rapidly sedimenting RNA (> 28 S ) here too. (iv) Finally, there was leakage of low-molecular weight (4 S ) RNA from infected cells, the leakage being approximately three-fold that of uninfected cells by approximately 5 hr after infection.

A REPORT from this laboratory1 has shown that in HEp-2 cells infected with herpes simplex virus the incorporation of nucleosides into cellular DNA progressively decreases with time after infection. The purpose of this communication is to report on two other aspects of macromolecular synthesis, namely RNA and protein, in infected HEp-2 cells.

Analysis of the kinetics of hybridization in liquid of labeled herpes simplex virus (HSV) 1 and 2 DNAs with excess unlabeled RNA extracted at 2 (early) and 8 (late) h postinfection revealed the following. (i) The RNA transcripts present in the HSV-1-infected cells at 2 and 8 h postinfection are complementary to 44 and 48% of HSV-1 DNA. The RNA transcripts present in the HSV-2-infected cells at 2 and 8 h postinfection are complementary to 21 and 50% of HSV-2 DNA. (ii) The transcripts present in 2-h HSV-1- or HSV-2-infected cells treated with cycloheximide are complementary to 44 and 45% of the respective DNAs. (iii) The RNA transcripts present in the HSV-1-infected cells at 2 h postinfection and in HSV-2-infected cells at 8 h postinfection form 2 classes, abundant and scarce, differing in molar concentrations. The RNA transcripts present in the HSV-2-infected cells at 2 h postinfection form only one abundance class. (iv) The transcripts present in the HSV-1-infected cells at 8 h postinfection are complementary to 24% of HSV-2 DNA and therefore 50% of the transcribed HSV-1 sequences are shared by the two viruses. Of the RNA sequences complementary to HSV-2 DNA, 13% arise from HSV-1 templates specifying abundant RNA and 11% arise from HSV-1 templates specifying scarce RNA. Thus, the DNA sequences shared in common by HSV-1 and HSV-2 DNAs constitute 71% of the HSV-1 templates specifying abundant RNA and 39% of sequences specifying scarce RNA.

A herpes simplex virus strain (MP) that causes the formation of polykaryocytes and a strain (mP) that causes rounding and clumping of cells were studied in a variety of tests designed to reveal differences in surface structure and composition of the virions. It was found that the patterns of elution from calcium phosphate gel are distinctive for each variant. Neutralization studies (multiplicity analysis) with anti-mP and anti-MP hyperimmune rabbit antisera revealed that the variants are antigenically closely related but not identical. However, no differences could be detected in kinetic or multiplicity analyses of inactivation by ethyl ether, chloroform, or urea. It is concluded that the mP and MP virions differ in surface structure and composition of the capsid or of the envelope. This variation in the envelope or the capsid may account for the difference in the apparent buoyant density between mP and MP virions reported in a previous communication. The possible relationship between the composition of the viral proteins or lipids and effects of the respective viruses on cells is discussed.

Herpes simplex virus strain MP dk − causes disaggregation of host polyribosomes in both permissive (HEp-2) and nonpermissive (DK) cells during the first 3 hours after infection. The following findings are discussed in this paper: (1) UV light-irradiated virus does not cause disaggregation of host polyribosome. (2) To obtain equivalent disaggregation of host polyribosomes, 1 hour after infection the nonpermissive DK cells must be infected with MP dk − virus at a multiplicity twenty- to thirty-fold higher than the permissive HEp-2 cells. (3) Actinomycin D (0–20 μg/ml of medium), 6-azauridine (0–10 −4 M ), or p -fluorophenylalanine (0–10 −3 M ) do not prevent the disaggregation of host polyribosomes in HEp-2 cells infected with 60 plaque-forming units (PFU)/cell of MP dk − virus. However, the drugs do prevent the disaggregation of host polyribosomes in DK cells infected with 1000 PFU/cell of MP dk − virus. (4) The differences between HEp-2 and DK cells with respect to multiplicity requirements to induce disaggregation and the ability of the drugs to prevent it are due to some property of the product specified by MP dk − in these cells. This conclusion is based on the finding that in DK cells productively infected with MP dk + sp , a multistep mutant of MP dk − virus, equivalent disaggregation of host polyribosomes is achieved with 40 PFU/cell and cannot be prevented by actinomycin D. The interpretation of these findings is based on data reported previously that the products specified by MP dk − virus in nonpermissive DK cells malfunction. We conclude that (1) in permissive cells the disaggregation of host polyribosomes is catalyzed by a highly efficient protein synthesized after infection; (2) in nonpermissive cells the protein malfunctions; to obtain comparable reduction in host functions the concentration of this protein must be higher in nonpermissive cells. This condition is satisfied by infecting nonpermissive cells at high multiplicity. (3) The reduction in synthesis or in function of this protein by the various drugs is sufficient to prevent the breakdown of host polyribosomes in nonpermissive cells but not in permissive cells.

SummaryHamsters injected intracere–brally or by peripheral routes with NDV developed encephalomyelitis and pneumonitis. The amount of virus required to produce infection varied for each route, being least by the intracerebral route and highest by the subcutaneous route. Virus was recovered from the brain but not from the lungs or blood in the prodromal stages. The manifestations and pathology of the resultant encephalomyelitis were similar irrespective of route of inoculation.

Deoxyribonucleic acid (DNA) extracted from purified nucleocapsids of Marek's disease herpesvirus (MDV) was cosedimented with T4 and with herpes simplex virus (HSV) DNA in neutral sucrose density gradients and with T4 DNA in alkaline sucrose density gradients. These experiments indicated that the intact MDV DNA had a sedimentation constant of 56 S corresponding to a molecular weight of 1.2 × 10 8 daltons. In the alkaline gradients, the largest and most prominent band contains a DNA sedimenting at 70 S corresponding to 6.0 × 10 7 daltons in molecular weight. The DNA is therefore double-stranded and not cross-linked. Isopycnic sedimentation of the MDV DNA molecules with SPO1, Micrococcus lysodeikticus , and HSV DNA gave a density of 1.705 g/cm 3 corresponding to 46 guanine plus cytosine moles per cent. Lastly, in hybridization tests the DNA hybridized with RNA of infected cells but not with that of uninfected cells supporting the conclusion that it is viral.

Herpesviruses specify new glycoproteins that bind to cell membranes and also appear in the envelope of the virion. Incubation of purified smooth membranes from infected cells with antiviral antibody results in an increase in the density of the membranes as determined by flotation in sucrose density gradients. The magnitude of this increase depends on the amount of antibody used; densities as high as 1.16 grams per cubic centimeter have been obtained (the density of the untreated membranes is 1.08 grams per cubic centimeter). Antiviral antibody does not increase the density of uninfected cell membranes nor do saline or normal rabbit serum change the densities of infected or uninfected cell mnembranes. Viral antigens—presumably the glycoproteins specified by the virus—are probably on the surface of the infected cell membranes and bind to them strongly enough to withstand the hydrodynamic forces applied to them in the sucrose gradient.

Evidence is presented by use of radiolabeling and pancreatic and T1 ribonuclease digestion that some of the ribonucleic acid specified by herpes simplex virus contains polyadenylic acid sequences. The polyadenylic sequences are not transcribed from viral DNA.

This chapter deals with the structure and replication of herpesviruses in susceptible cells. Having stated its scope, we shall take license to say what what we think of it.

Herpes simplex virus strain MP dk − multiplies in HEp-2 cells, but not in dog kidney (DK) cells. Strain MP dk + sp , a multistep mutant of MP dk − , multiplies in both HEp-2 and DK cells. Stabilized lysates of productively infected cells yield three macromolecular aggregates of viral deoxyribonucleic acid and protein banding in CsCl gradients at densities of 1.285 g/cm 3 (α), 1.325 g/cm 3 (β), and 1.37 to 1.45 g/cm 3 (γ). Similar lysates from abortively infected cells yield only the β and γ bands. Electron microscopic examination revealed that (i) the α band contained enveloped nucleocapsids, whereas the β band contained naked nucleocapsids and particles tentatively identified as internal components of the nucleocapsids, and that (ii) the enveloped virions and reduplication of cellular membranes observed in thin sections of productively infected cells were absent from abortively infected cells. Studies of the surface antigens of infected cells in a cytolytic system described previously revealed that abortively infected cells contained approximately 10-fold less virus-induced surface antigen than did productively infected cells. From these and other data published previously, we concluded that infectious MP dk − virions are not made in DK cells because (i) functional viral products necessary for the envelopment of the nucleocapsid are not made, and (ii) capsid proteins and some nonstructural products specified by the virus malfunction.

Lysates of HEp-2 cells productively infected with herpes simplex virus yielded two bands on isopycnic centrifugation in CsCl gradients, ranging from 1.2 to 1.6 g/cm 3 . One band, designated α, had a mean buoyant density of 1.27 g/cm 3 and contained herpes virions. Band β had a mean density of 1.305 g/cm 3 and contained primarily complement-fixing viral antigens and little or no viral deoxyribonucleic acid (DNA). The products banding in the α and β bands were unstable; fivefold or higher amounts were recovered by treating the cell extract with formaldehyde prior to centrifugation. Formaldehyde treatment increased the buoyant density of viral products in both the α and β bands by about 0.015 g/cm 3 . In addition, it stabilized hitherto inapparent products, forming a broad band γ with a density range of 1.37 to 1.45 g/cm 3 . The material in the γ band was heterogeneous; it contained viral DNA, cellular DNA, and viral antigen. Formalinized lysates of DK cells abortively infected with herpes simplex virus yielded a β band undifferentiated from that formed by extracts of productively infected cells. The γ band was less dense and narrower. The α band was entirely missing.

We examined the electrophoretic properties of the proteins and glycoproteins in the smooth membranes and virions purified from cells infected with herpes simplex virus strains differing with respect to their effects on the interaction of cells among themselves. The data show the following: (1) The glycoproteins in virions and binding to membranes share common features but vary quantitatively and qualitatively depending on the virus strain. (2) The binding to smooth membranes is ordered and not random. (3) Differences in the glycosylation of membrane proteins in African green monkey (VERO) and human (HEp-2) cells indicate that glycosylation is at least in part determined by the host.

Publisher Summary This chapter deals with the effects of cytolytic viruses on macromolecular biosynthesis of animal cells. One of the earliest manifestations of a productive infection of animal cells with many viruses is a rapid decline and cessation of synthesis of host macromolecules. The inhibition of host macromolecular synthesis probably results from the specific effect of one or more viral gene products on the synthesis of products of host genes. As a model system for the study of gene interactions in animal cells, virus infection has several advantages: (1) prior to infection, the cell lacks viral genes or their products and (2) the introduction of viral genes or their products into the cell is amenable to considerable experimentation. The inhibition of host macromolecular synthesis is a frequent sequella of infection. Not all viruses have been reported to affect host metabolism, but both abortive and productive infection nearly always cause some phenotypic modification of the cell. Thus, viruses that are unable to inhibit a particular host may induce the production of interferon.

A human cervical tumor, free of detectable infectious herpes simplex 2 virus, contained a fragment comprising 39% of herpes viral DNA. Renaturation kinetics indicate that an average of 1 to 3.5 DNA fragments of herpes simplex virus are present per cell, depending on the ploidy of the cells in this particular tumor. Virus-specific sequences were found linked to highly repetitive sequences of host DNA, which reassociated under conditions designed to preclude reassociation of viral sequences. The tumor also contained RNA transcripts complementary to 5% of the viral DNA. The fraction of viral DNA template transcribed in the cervical tumor is considerably less than that transcribed in productively infected cells (50%).

Herpes simplex virus particles of a strain which causes syncytium formation (macroplaques) and a strain which causes rounding and clumping of cells (microplaques) were ultracentrifuged in a solution of cesium chloride. Assays of infectious macroplaque and microplaque virus particles contained in fractions collected from the bottom of the tube after the ultracentrifugation revealed that the two strains differ in their apparent buoyant density by 0.8%. The macroplaque virus which localized in less dense zones may be less homogeneous with respect to density than the microplaque variant of herpes simplex virus. It is concluded that the physical difference between the two strains may be due either to a difference in nucleic acid, protein, or lipid content of the particles or to preferential interaction of particles of one strain with cesium chloride or impurities

Previous papers in the series have shown that the surface membranes of herpesvirus-infected cells acquire new immunological specificities and that purified infected cell membrane preparations, characterized by their physical properties rather than topology in the cell, contain new glycoproteins genetically determined by the virus. In this study, we prepared purified plasma membrane identified by its 5′ nucleotidase, fucose, and reduced nicotinamide adenine dinucleotide-diaphorase content. Analysis of the membrane proteins and glycoproteins by electrophoresis in acrylamide gels indicated the following. (i) Purified plasma membranes from infected cells contained two sets of proteins, i.e., host proteins were present both before and after infection and viral proteins were present only after infection. (ii) After infection, no appreciable selective or nonselective loss of host proteins from membranes was demonstrable. However, no new host proteins were made. (iii) Electropherograms of plasma membrane proteins from infected cells indicated the presence of at least 12 virus-specific proteins ranging in molecular weight from 25 × 10 3 to 126 × 10 3 daltons. Of these, at least nine were glycosylated. Proteins and glycoproteins with similar electrophoretic mobilities but in somewhat different ratios were also present in preparations of highly purified virions.

The core of the herpes simplex virion consists of an electron-dense toroidal structure 50 nm high, with an inside diameter of 18 nm and an outside diameter of 70 nm, penetrated by a less dense cylindrical mass. The toroid contains deoxyribonucleic acid (DNA), as evident from the observation that uranyl ion staining is removed by ethylenediaminetetraacetic acid under conditions which result in the extraction of uranyl ions bound to nuclear DNA. Studies on negatively stained preparations of purified capsids suggest that the toroid consists of DNA arranged as if it were spooled around the cylindrical mass.

Publisher Summary The herpesviruses are formally defined as large enveloped virions with an icosahedral capsid consisting of 162 capsomeres and arranged around a deoxyribonucleic acid DNA core. The family Herpesviridae does not have many known members. There are several reasons for this: Herpesviruses are complex viruses, cantankerous, and at times difficult to grow. This chapter discusses the structure and morphogenesis of the herpesvirus virion. It describes the base compositions of the DNA's of the herpesviruses. The molecular weights of the DNA of several herpesviruses (equine abortion, herpes simplex, and Marek's disease) are of the order of 10 8 daltons. One objective of the studies of the multiplication of herpesviruses is a complete description of the structure, functions, amount, and time of synthesis of all the products specified by the virus in the infected cells. At present, the number of products and their functions are uncertain. According to current accounting practices, herpesvirus DNA carries information sufficient to specify the sequence of 140,000 amino acids.

The DNA of herpes viruses associated with Marek's disease of fowl contains 56-57 moles of guanine and cytosine per 100. The composition of its DNA and lack of infectiousness of cell-free preparations suggest that the herpes virus associated with Marek's disease belongs to the herpes virus group B which contains predominantly cytomegaloviruses.

EPIZOOTICS caused by polyoma virus have been reported1,2 in mouse colonies maintained in proximity to mice infected experimentally with this virus. Establishments without facilities to house infected and uninfected mice in separate buildings run the risk of contaminating their polyoma-free stock. It was of interest to determine, therefore, whether similar problems would be encountered when the Syrian hamster was used as the experimental animal to study this disease. Our experience indicates that infected hamsters rarely transmit polyoma virus to other hamsters or mice. The evidence that polyoma infection is a much less contagious disease of hamsters than of mice is shown by the following experiments.

In this paper we report that viral polypeptides from herpes simplex virus 1 (HSV-1) and 2 (HSV-2)-infected cells electrophoretically separated in sodium dodecyl sulfate-polyacrylamide-agarose gels and transferred to diazobenzyloxymethyl paper can react with rabbit hyperimmune sera, both polyvalent and prepared against specific antigens. The polyvalent hyperimmune sera against HSV-1 reacted with 17 HSV-1 polypeptide bands and 8 HSV-2 polypeptide bands. Concordantly, polyvalent sera against HSV-2 reacted with at least 16 HSV-2 polypeptide bands and 8 HSV-1 polypeptide bands. The antisera prepared against the specific antigens reacted with a smaller number of polypeptide bands. Preimmune sera and immune sera did not react with electrophoretically separated polypeptides from infected and uninfected cells, respectively. The immune localization of separated antigens test provides a powerful technique for identification of immunogenic viral polypeptides, especially those which are normally insoluble and therefore unavailable for immunological reactivity in immune precipitation tests.

In cells infected with herpes simplex viruses the capsids acquire an envelope at the nuclear membrane and are usually found in the cytoplasm in structures bound by membranes. Infected cells also accumulate unenveloped capsids alone or juxtaposed to cytoplasmic membranes. The juxtaposed capsids have been variously interpreted as either undergoing terminal deenvelopment resulting from fusion of the envelope with the membrane of the cytoplasmic vesicles or undergoing sequential envelopment and deenvelopment as capsids transit the cytoplasm into the extracellular space. Recent reports have shown that (i) wild-type virus attaches to but does not penetrate cells expressing glycoprotein D (G. Campadelli-Fiume, M. Arsenakis, F. Farabegoli, and B. Roizman, J. Virol. 62:159-167, 1988) and that (ii) a mutation in glycoprotein D enables the mutant virus to productively infect cells expressing the wild-type glycoprotein (G. Campadelli-Fiume, S. Qi, E. Avitabile, L. Foa-Tomasi, R. Brandimarti, and B. Roizman, J. Virol. 64:6070-6079, 1990). If the unenveloped capsids in the cytoplasm result from fusion of the cytoplasmic membranes with the envelopes of viruses transiting the cytoplasm, cells infected with virus carrying the mutation in glycoprotein D should contain many more unenveloped capsids in the cytoplasm inasmuch as there would be little or no restriction in the fusion of the envelope with cytoplasmic membranes. Comparison of thin sections of baby hamster kidney cells infected with wild-type and mutant viruses indicated that this was the case. Moreover, in contrast to the wild-type parent, the mutant virus was not released efficiently from infected cells. The conclusion that the unenveloped capsids are arrested forms of deenveloped capsids is supported by the observation that the unenveloped capsids were unstable in that they exhibited partially extruded DNA.

Earlier studies have shown that herpes simplex viruses adsorb to but do not penetrate permissive baby hamster kidney clonal cell lines designated the BJ series and constitutively expressing the herpes simplex virus 1 (HSV-1) glycoprotein D (gD). To investigate the mechanism of the restriction, the following steps were done. First, wild-type HSV-1 strain F [HSV-1(F)] virus was passaged blindly serially on clonal line BJ-1 and mutant viruses [HSV-1(F)U] capable of penetration were selected. The DNA fragment capable of transferring the capacity to infect BJ cells by marker transfer contains the gD gene. The mutant gD, designated gDU, differed from wild-type gD only in the substitution of Leu-25 by proline. gDU reacted with monoclonal antibodies which neutralize virus and whose epitopes encompass known functional domains involved in virus entry into cells. It did not react with the monoclonal antibody AP7 previously shown to react with an epitope which includes Leu-25. Second, cell lines expressing gDU constitutively were constructed and cloned. Unlike the clonal cell lines constitutively expressing gD (e.g., the BJ cell line), those expressing gDU were infectable by both HSV-1(F) and HSV-1(F)U. Lastly, exposure of BJ cells to monoclonal antibody AP7 rendered the cells capable of being infected with HSV-1(F). The results indicate that (i) gD expresses a specific function, determined by sequences at or around Leu-25, which blocks entry of virus into cells synthesizing gD, (ii) the gD which blocks penetration by superinfecting virus is located in the plasma membrane, (iii) the target of the restriction to penetration is the identical domain of the gD molecule contained in the envelope of the superinfecting virus, and (iv) the molecular basis of the restriction does not involve competition for a host protein involved in entry, as was previously thought.

SummaryRate of formation and release of poliovirus Type 1 were studied at 32° and 37°C in FL cell cultures. Virus accumulated almost as rapidly and reached practically the same intracellular levels at 32°C as at 37°C. However, release of virus was much slower at 32° than at 37°C and as a result, at 32°C the intracellular concentration of virus remained at or near peak levels for 4 to 6 hours. From these observations, highly potent virus for use in immunological studies was prepared by mechanical disruption of infected cells maintained at room temperature (26–32°C) for 18–20 hours.

HERPES simplex is an infectious disease of man caused by a virus once known by the same name but which is now euphemistically known as herpesvirus hominis1. A characteristic of this disease is localization to particular parts of the body, frequently on the skin and near the lip, cornea, thigh or genitals. Immunological analysis2 of herpes viruses isolated from a large number of patients has led to the suggestion that there are two types of herpes viruses. Viruses isolated from infections on all parts of the body except the genitals frequently fall into type 1. Conversely, viruses transmitted venereally and isolated from lesions on the genitals fall into type 2. Recently there has been considerable interest in the properties, classification and evolution of herpes viruses, particularly in view of the reports linking carcinoma of the cervix with type 2 herpes viruses3,4 and cancer of the lip with type 1 viruses5,6. With respect to the extent of the relatedness of type 1 and type 2 viruses and the role of the virus in determining the site of infection, we suggest that (1) herpes simplex viruses in nature and in the laboratory form a spectrum of variants differing with respect to structural components of the virus and, in certain cases, biological properties and mode of transmission; (2) the viruses isolated from genital infections (type 2) are most probably closely related to viruses causing infection of the face (type 1); and (3) herpesvirus hominis is not a suitable name for herpes simplex virus.

PREVIOUS reports have shown that the buoyant density of herpes simplex virus in a solution of caesium chloride is genetically determined by the virus1–3. Thus plaque purified mutants obtained from a single parent and grown in the same host differ with respect to buoyant density in caesium chloride1,2. Mutants differing in buoyant density also differ, in a predictable fashion, with respect to patterns of elution from calcium phosphate gel, surface antigenic structure, stability on heating at 40° C, and plaque size2,3. These findings led to the conclusion that all these properties of the herpes virus virion are covariant and may reflect the structure of the capsid or of the envelope of the virus2,3.

HSV at a glance Family name: Herpesviridae (herpesviruses derive their name from the Greek herpes, meaning “creep”) Subfamilies: Nine human herpesviruses subdivided in the three subfamilies: alpha- beta- gamma-herpesvirinae Herpes simplex viruses 1 and 2 (human herpes virus-1 and -2) Genome: Linear double stranded DNA (about 150 kbp) consists of two covalently linked unique regions UL and US, each bracketed by inverted repeats. One ORIl at midlle of UL sequence, and two Oris at each terminus of Us. The repeats of the L component are designated ab and a’b’. The repeats of the S component are a’c’ and ca. The number of a sequence repeats at the L-S junction and at the L terminus are variable. The genome can be represented as alanb-Ul-b’a’mc’-Us-cas (Fig. 1). Gene products: At least 84 unique transcriptional units (six in two copies) encode 84 proteins (Fig. 1). The genes are classified into kinetic classes as alpha or immediate early, beta or early, and gamma- or late. Six alpha proteins encode transactivators (Table 2) The proteins can be clustered into functional groups (Table 1) Virion morphology: Capsid has icosahedral symmetry, is overlayed by tegument and envelope 6 proteins in capsid – ICP5 is the major capsidic protein 14 protein in tegumant 11 glycoproteins in envelope.

Herpes simplex virus (HSV) represents the most comprehensive example of virus-receptor interaction in the Herpesviridae family, and the prototype virus encoding multipartite entry genes. Whereas small enveloped viruses package the functions required for entry and fusion into one or two fusion glycoproteins, in HSV the same functions are distributed over several distinct glycoproteins, each with a specialized activity. In addition, HSV encodes a highly sophisticated system for promoting and blocking fusion between the viral envelope and cell membrane. Because the most obvious models of virus entry into the cell do not fit with the HSV complexity, and despite our detailed knowledge of the HSV receptors and of the crystal structure of glycoprotein D (gD), the receptor-binding glycoprotein, and of gB, HSV entry is still, in part, a puzzle (WuDunn and Spear, ; Cocchi et al., ; Geraghty et al., ; Carfi et al., 2001).

A commonly accepted concept in herpesvirology holds that herpesvirions are formed by budding of nucleocapsids at the inner nuclear membrane and the enveloped virions are released into the perinuclear space (see Chapter 13). This is a closed compartment that virions need to exit, in order to reach the extracellular space and start a new infection cycle. How alphaherpesviruses accomplish this goal is a controversial issue. Of the two pathways of virus exit proposed, the single envelopment and the double envelopment, also referred to as de-envelopment–re-envelopment, each has evidence and supporters in the literature (the topic has been covered in excellent reviews and papers (Enquist et al., ; Skepper et al., ; Johnson and Huber, ; Mettenleiter, 2002). Part of the uncertainties that still dominate this topic comes from the difficulties in interpreting static electron microscopy images. Thus cytoplasmic virions juxtaposed to curved vesicles were interpreted in some studies as budding virions, i.e., as evidence for secondary envelopment and for the deenvelopment-reenvelopment pathway. In other studies they were interpreted as virions undergoing fusion with encasing vesicles, i.e., as evidence of de-envelopment (Campadelli-Fiume et al, : Roizman and Knipe, 2001). To solve these ambiguities, several approaches have been undertaken in recent years, including the generation of genetically modified mutants and cytochemistry.