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Whether herpes simplex virus (HSV) is viewed as a pathogen or as a model eukaryotic system, it is virtually certain that any experimental work will require the virus to be grown and assayed. The following chapter is therefore seen as the fundamental first step before embarking on more intellectually and technically challenging technology. Its importance should not however be underestimated. It never fails to surprize us that people who describe themselves as virologists have little understanding of the basic requirements needed to attain a contamination-free, high-titer, low particle:plaque-forming units (PFU) ratio, genetically pure virus stock [ABSTRACT FROM AUTHOR]

The study of genome structure and gene function is pivotal in understanding the mechanisms of replication, pathogenesis, and virulence of herpesviruses. In this respect, mutagenesis and sequence analysis of genes encoded by the virus are of great importance. However, the herpesvirus genomes are large, with sizes ranging between 120 and over 200 kbp and encoding between 70 and 200 genes (see ref. 1 for a review). This large size hampers handling and systematic mutagenesis of the virus genome using standard modern molecular biology techniques. Most current methods of mutagenesis therefore do not rely on direct modification of the viral genome in vitro but depend on exchange in vivo, by homologous recombination, of a viral gene by a copy of the latter gene that is truncated in vitro by insertion of a marker gene. Mutant virus progeny can be screened or selected for, depending on the marker gene that is used. Commonly used marker genes are thymidine kinase and lacZ. This procedure is generally used, reliable, and has yielded a wealth of information on the function of herpers simplex virus type 1 (HSV-1) encoded genes. However, it requires prior mapping and cloning of every gene to be mutagenized and is therefore less feasible if the virus is a novel or less-well-known herpesvirus. [ABSTRACT FROM AUTHOR]

Herpes simplex virus (HSV) is a human pathogen that causes diseases ranging in medical importance from herpes labialis, through genital herpes and herpes keratitis, to herpes encephalitis-a life-threatening disease. HSV types 1 and 2 have the ability to enter a latent phase during in vivo infection, during which time the virus is able to evade immune surveillance, and from which state it is able to escape from time to time to cause disease, especially in immunocompromised individuals. This characteristic makes antiviral chemotherapy an indispensable weapon in the management of recurrent herpesvirus infection. [ABSTRACT FROM AUTHOR]

A number of aspects of the natural biology of herpes simplex virus (HSV) make it an attractive candidate for a vector to express foreign genes within the nervous system. Some of the advantages of an HSV vector are 1Establishment of a life-long latent infection within peripheral and central nervous system neurons (for a review, see ref,1);2Latent HSV genomes exist as multiple episomal copies/neuron and integration is not known to occur (2), and3.Nonreplicating HSV recombinants can establish a latent infection efficiently (3) [ABSTRACT FROM AUTHOR]

The polymerase chain reaction (PCR) technique is a sensitive method for detection of nucleic acids that can be used to detect herpes simplex virus (HSV)-DNA and RNA in tissue samples with greater sensitivity than hybridization with specific probes (1,2). In its most basic form, PCR involves multiple cycles of denaturation of DNA, annealing with specific primers and replication of specific DNA using a thermostable DNA polymerase like Taq polymerase, resulting in amplification of a specific DNA sequence. Reverse transcriptase polymerase chain reaction (RT-PCR) employs a preliminary reverse transcription step of RNA, using either a specific 3′ or an oligo (dT) primer, to produce complementary DNA (cDNA), followed by PCR using primers specific for the transcript of interest. In its standard application, PCR offers qualitative information regarding the presence or absence of target sequences, and has been used to analyze latently infected ganglia and brain for HSV-DNA (6,8-11) and RNA (6-10,12). As described in the following, with the inclusion of mutated templates as internal standards, PCR can be used to determine a quantitative estimate of the number of HSV genomes and transcripts in tissue extracts. Histologically, in situ hybridization (ISH) can be used to detect HSV-DNA and RNA in specific cells in the nervous system (3-5), although it has not been successfully applied to detect HSV genomes during latency (6,7). PCR methods can be applied to tissue sections (in situ PCR), making it possible to identify individual cells harboring HSV genomes, even during latency (9,13). [ABSTRACT FROM AUTHOR]

Assays in use for the analysis of herpes simplex virus (HSV) gene expression during the establishment and maintenance phases of infection in the nervous system include: 1The use of reporter genes, for example, the lacZ gene from Escherichia coli, which is inserted by homologous recombination into the viral genome, and which may be driven either by viral promoters or by an exogenous promoter, such as the major immediate early (IE) promoter of cytomegalovirus. In our hands, the detection of lacZ activity in neuronal tissue infected with recombinant HSV constructs has proven to be a simple and effective means of monitoring viral activity in the peripheral nervous system.2.Analysis of virally encoded RNA transcripts, either by in situ hybridization (ISH) using radioactive or nonradioactive indicator molecules, or by Northern analysis (this technique is described in Chapters 13 and 24).3.Immunohistochemistry to demonstrate the presence of viral proteins, which technique can also be used in combination with ISH (dual labeling) [ABSTRACT FROM AUTHOR]

Protective immunity against a eajority of viral infections is mediated by a combination of both humoral and cell-mediated immune responses. However, in the case of herpesvirus infections, where viral spread is largely cell-to-cell, cell-mediated immune mechanisms (which facilitate the clearance of virally infected cells) are particularly important (1-4). Moreover, cell-mediated immunity (CMI) has also been implicated in the establishment and/or reactivation of latent herpes simplex virus (HSV) infection (5,6). Thus, a major focus of herpesvirus immunology continues to be the identification of those herpesvirus antigens that serve as targets for CMI and the means by which protective responses can be optimally induced. Clearly this information is critical for the rational development of effective vaccine strategies. [ABSTRACT FROM AUTHOR]

We have developed an in vitro model of herpes simplex virus (HSV) latency in primary neurons that mimics many aspects of HSV latency in animal models and the human disease (1-3). Using this model, we demonstrated that HSV-1 and HSV-2 establish latent infections in vitro in the same neuronal cell types that are shown to harbor latent HSV in humans (3). Latent HSV infections can be produced in neuronal cultures from ganglia of rodents and primates with similar results (3). In all cases examined, the neurotrophin, nerve growth factor (NGF), is required to maintain the latent infections. Depletion of NGF results in the reactivation of latent virus (1-3). Depending upon the conditions and the use of a high multiplicity of infection, latent HSV-1 infections are established in the majority of primary sensory or sympathetic neurons in tissue culture (2,4). To achieve high efficiency of establishment of latency with little or no evidence of lytic infection, an antiviral agent (e.g., acyclovir) is added to the neuronal cultures during the first week after inoculation with virus. However, latency can be established in the absence of antiviral treatment provided that the multiplicity of infection (MOI) is very low (1,2). At least one of the actions of the antiviral treatment is to prevent amplification of the input virus in the nonneuronal cells that are present in the culture at the outset of the infection. These nonneuronal cells are destroyed in the presence of acyclovir and virus (4). Latency is maintained in neurons in culture for as long as 10 wk in the presence of NGF. Viral transcripts and antigens associated with the productive infection are not detected during the latent infection (2,3,5). Viral transcription is restricted to the latency-associated transcripts (LAT) during the latent infection and is present in the nuclei of 80-90% of the neurons by 3 wk postinfection (4,5) Upon removal of NGF from the culture medium, for as brief as 1 h, reactivation of latent virus is induced (3), and viral antigens associated with the productive infection and infectious virus are detected between 48-72 h after NGF deprivation. [ABSTRACT FROM AUTHOR]

Research on herpesvirus infections has commanded the attention of a diverse group of scientists for over a century. Until the advent of the human immunodeficiency virus, the herpes simplex viruses (HSV) were the most intensively studied of all viruses. During the early part of the nineteenth century, long before the infectious agent responsible for cold sores (fever blisters) was identified, studies suggested that damage to the trigeminal nerve could induce peripheral herpetic vesicles (1). Gruter demonstrated that a particle of material from a herpetic blister inoculated into a rabbit eye could cause herpes and that, in this way, the disease could be transmitted in series from one rabbit to another (2). Following Gruter's basic discovery, research conducted in many parts of the world showed a variety of clinical forms of herpes to be similarly transmissible by inoculation (3). At roughly the same time, Goodpasture proposed that "the virus remains in the ganglia in a latent state after the local lesion has healed" and discussed in detail the pathology of herpetic infection in humans and animals (4,5). [ABSTRACT FROM AUTHOR]

Animal models remain essential for studies of many aspects of the biology of herpes simplex virus (HSV). Such studies include basic experiments on pathogenesis (including characterization of viral mutants), tests of antiviral drugs, and methods of immunization. With reference to models of recurrent infection, high levels of recurrence and clinical disease have been achieved with guinea pigs (particularly with genital infection) and rabbits (particularly with ocular infection; reviewed in ref. 1). However, in contrast to these animals, with the laboratory mouse there are many inbred and congenic lines; a major advantage for immunological studies. To this can now be added the growing technology of transgenic and "knockout" animals. For these reasons we have expended considerable effort in developing various mouse models of infection, particularly with HSV type 1 (HSV-1). [ABSTRACT FROM AUTHOR]

The herpes simplex virus (HSV) lytic cycle is dependent on a precise temporal pattern of viral gene expression with the initial expression of the immediate-early (IE) genes, followed by the early genes, and finally late gene expression (1). Although such a temporal cascade of viral gene expression involves the action of virally encoded regulatory proteins, such factors act, at least in part, by interacting with cellular transcription factors that are present in the uninfected cell. Thus, although the HSV virion protein Vmw65 is essential for transactivation of the viral IE genes in lytic infection by binding to the TAATGARAT sequences in the promoters (2), it can only achieve this by forming a complex with the cellular transcription factor Oct-1 (3,4) and other cellular factors (5). Similarly, the IE promoters contain binding sites for other cellular transcription factors such as Spl (6) and this is also observed in the promoters for viral genes of other kinetic classes, such as the early gene encoding thymidine kinase (7). [ABSTRACT FROM AUTHOR]

In order to identify the nature of posttranslational modifications and enzyme functions of herpes simplex virus 1 and 2 (HSV-1 and HSV-2) proteins, it is necessary to apply both biochemical and genetic analyses. The experimental methods described in this chapter have been applied to cells cultured in vitro and infected with HSV-1 or to isolated nuclei of infected cells, to nuclear or cytoplasmic fractions, and, in some instances, to purified extracts of infected eukaryotic cells or of prokaryotic cells expressing a viral gene. [ABSTRACT FROM AUTHOR]

Herpes simplex virus (HSV) amplicons are defective virus vectors capable of introducing amplified foreign genes into variable types of eukaryotic cells, such as fibroblasts, macrophages, glia, and neurons in different organisms including rodents, monkeys, and human (refs. 1-3; reviewed in ref. 4). The defective viruses follow their nondefective counterparts in the ability to infect mitotic, as well as postmitotic cells. This makes them potentially useful vectors for use in nondividing cells, such as in nerve cells. Available retrovirus vectors employed to date for gene therapy require cell division and therefore cannot be used to target neurons. [ABSTRACT FROM AUTHOR]

Investigations of genome replication in DNA viruses involve several facets. These include characterizing the sites at which synthesis is initiated (origins of DNA replication), identifying the viral and host proteins that participate, understanding the enzymatic activities of these proteins, and elucidating the mechanisms of DNA synthesis and maturation. For several viruses cell-free systems capable of carrying out faithful viral origin-dependent DNA synthesis have been described that have provided important insights into these areas. Unfortunately, such an assay is not yet available for HSV and other approaches therefore have been required. One of the most useful and widely employed has involved transient assays for viral origin-dependent DNA synthesis in transfected tissue culture cells. Such assays played important roles in the initial identification of the viral replication origins and the virus-coded proteins essential for DNA synthesis and more recently have helped provide detailed information on the structure and function of these elements. Similar approaches also have been exploited to study genome replication in other herpesviruses. [ABSTRACT FROM AUTHOR]

Since the herpes simplex virus type 1 (HSV-1) genome has been sequenced and most HSV-1 RNAs are not spliced (1), detailed information about the structure of many HSV-1 RNAs can be obtained without the considerable time and effort that is required to construct and analyze cDNA libraries. Once the 5′ and 3′ ends of an RNA have been mapped precisely, the RNA nucleotide sequence can be deduced simply from the genomic DNA sequence. However, there are certain situations, such as the analysis of spliced RNAs or of chimeric RNAs expressed from foreign genes inserted into HSV-1 vectors, where cDNA cloning of HSV-1 transcripts may be informative. There are families of transcripts that arise by alternate splicing in several human herpesviruses: HSV-1, cytomegalovirus (CMV), and Epstein-Barr virus (EBV). For example, HSV-1 encodes several overlapping latency-associated transcripts or LATs (2-4). The splice junctions of the intron within the HSV-1 2.0-kb LAT have been determined by RNA-PCR with primers located on either side of the intron, followed by direct DNA sequence determination of the PCR product (5). The construction of partial cDNAs by PCR saves much time-consuming effort and expense compared with the analysis of cDNA libraries. In addition, by sequencing PCR products directly, the need to analyze several cDNA clones in order to be assured of obtaining the consensus sequence is eliminated. [ABSTRACT FROM AUTHOR]

During lytic virus replication, herpes simplex virus (HSV) exhibits a closely regulated pattern of viral gene expression and of DNA replication, resulting in virion production (1). Broadly, HSV genes can be divided into immediate early, early, and late categories based on the kinetics of their expression. The five immediate early genes are expressed in the absence of prior viral protein synthesis although their expression is stimulated by a viral tegument protein. Two immediate early proteins are essential for virus replication in vitro and act at the transcriptional (IE 175) and posttranscriptional (IE63) levels to regulate early and late gene expression. Throughout infection, mRNA is synthesized using cellular RNA poly-merase II, which is modified by the action of an immediate early protein (2). [ABSTRACT FROM AUTHOR]

Expression of herpes simplex virus (HSV) polypeptides in bacterial expression systems has provided a useful tool for the generation of large quantities of specific viral proteins for use in both biochemical and functional analysis, and as immunogens for antisera production. Proteins can be expressed either in the full-length native form or as fusion proteins with affinity tails. [ABSTRACT FROM AUTHOR]

Gold particles in colloidal suspension are particularly well suited as markers for immune electron microscopy. Their extreme electron opacity ensures that they are detected with accuracy even at particle sizes of less than 3 nm. Gold spheres can be made easily and inexpensively by reduction of gold chloride with mild acid and heat (1), and particles can be prepared in a variety of sizes by varying the nature of the reducing agents (2). [ABSTRACT FROM AUTHOR]

The last decade has seen a remarkable flourishing of the biological structure field. This blossoming has brought an explosion of stereochemical information, and has been made possible by the combined improvement in techniques of X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and the bulk preparation of biological materials. Most crucial, however, has been the desire of the experimental biologists to follow research problems to the level of stereochemistry, which is the ultimate reductionist limit of molecular biology. This aim has been driven by the anticipation that such knowledge may permit better understanding and even engineering of biological function. [ABSTRACT FROM AUTHOR]

Herpes simplex virus (HSV) remains a major human pathogen worldwide (25 causing cold sores, eye and genital infections, blindness, encephalitis, and neonatal infections. Most adults have antibodies against the oral form of the virus HSV-1 (9), and a significant number are infected with the genital form, HSV-2. Both serotypes establish lifelong latent infections and reactivate periodically to produce recurrent disease (25). After infection, virus-encoded glycoproteins are expressed on all cellular membranes and are major targets of the host's immune response. The virion envelope contains 10 glycoproteins that are important for infection and pathogenesis of HSV-1 and HSV-2. Because HSV contains so many glycoproteins, sorting out their functions in virus entry remains a difficult task. Our approach has focused on establishing structure-function relationships of the individual glycoproteins with particular emphasis on gC and gD. After many years of studying the properties of these proteins in HSV-infected and plasmid-transfected mammalian cells, we have now begun to overexpress the proteins using a baculovirus expression system. [ABSTRACT FROM AUTHOR]

The isolation of an individual polypeptide from a heterogeneous mix is an essential process in characterizing a protein of interest. In purified form a protein can be used to generate specific polyclonal and monoclonal antibodies for in vivo studies, in vitro the enzymic properties or interactions with nucleic acids or other proteins can be studied in detail and related to in viva function and, ultimately, the purified proteins can be used in structural determinations that define how polypeptide chains fold and amino acids interact to create a protein with a specific function. Protein purification exploits the properties a polypeptide derives from its unique amino acid composition and separation techniques rely on variations in solubility, size, charge, hydrophobicity and specific affinities to achieve fractionation. A combination of these methods is sufficient to isolate an individual protein from a complex mix. A prerequisite for any purification is the ability to unambiguously distinguish the protein of interest at all stages. This can be achieved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting with specific antisera, or by use of an assay specific for an activity of the protein. [ABSTRACT FROM AUTHOR]

Complementary cell lines have become an accepted tool for the functional analysis of viral genes (1,2) and proteins, as well as an essential component in strategies for the construction of mutant viruses. More recent applications include the propagation of replication-defective virus products with potential as viral vaccines (3,4) or as vehicles for gene therapy (5,6). The perceived requirements for such systems are low recombination frequencies between complementing cell and recombinant virus, stable expression of the complementing protein within the cell, and efficient complementation. The standard techniques of eukaryotic cell transfection and clonal selection are routinely employed in the generation of complementary cell lines, and are described briefly in this chapter. Perhaps a more novel introduction to this field is the possibility of using transgenic technology. Transgenic animals have the potential to provide both an in vivo model of complementation (7) and a comprehensive library of novel complementing cell types, particularly cell types resistant to traditional transfection protocols. [ABSTRACT FROM AUTHOR]

Herpes genomes are large and complex, with many interactions among herpes encoded proteins, herpes DNA and RNA, and the host cell. These interactions begin as the virus enters the cell, and continue as the decision for latency or lytic replication is made. Correctly regulated gene expression then allows herpes genes to be expressed in a temporally regulated manner and to subvert the host cell metabolism in favor of virus production. Finally, infectious progeny virions are assembled and released. Exploring the function of the many herpes-encoded proteins and the mechanisms to control their expression during these processes thus requires a fine dissection of the herpes genome, so as to allow protein-coding regions to be linked with function and control DNA regions to be identified. [ABSTRACT FROM AUTHOR]

This chapter deals with (1) the preparation of herpes simplex virus (HSV) virion DNA of a quality and purity suitable to be used for the generation of infectious virus, and (2) its use in the preparation of infectious virus. An important development in the understanding of virus genetics and gene products has been the ability to carry out reverse genetics. This is dependent on the ability to manipulate the genome in vitro and reconstitute infectious virus. Our understanding of DNA viruses and positive stranded RNA viruses (where DNA and RNA/cDNA, respectively, are generally infectious) is considerably greater than for negative stranded RNA viruses, where until recently, it had been impossible to generate virus.from either RNA or cDNA. Within the herpesviridae, knowledge of the function of HSV gene products is one of the more advanced owing to the relatively straightforward techniques required to generate virus from HSV-DNA, and to introduce desired mutations by cloning small parts of the genome, manipulating them, and then reintroducing the mutations by a process of cotransfection and in vivo recombination with intact virus DNA. Other α-herpesviruses, such as EHV-1 and PRV, are equally amenable to such manipulation, and knowledge of their gene products is also well advanced. In contrast, this technology is only now, and with much less success, being applied to other members of the family, such as EBV, HCMV, and VZV, and knowledge of their genetics is much less advanced. The use of cosmids to reconstitute intact virus will aid in the advance of knowledge for these viruses. For examples of uses of recombinant DNA technology, the reader is referred to other chapters (especially those on cloning and mutagenesis). I will concentrate on the techniques currently in use in my laboratory, but will also mention other techniques in use elsewhere that may be more appropriate in certain cell types. [ABSTRACT FROM AUTHOR]

This chapter deals with assays commonly used to follow herpes simplex virus type 1 (HSV-1) entry into and spread between cells in tissue culture These are complex processes, known to involve several of the 20 or more HSV-encoded membrane proteins (see refs. 1 and 2 for recent reviews). HSV entry is mediated by a number of proteins on the surface of the virus particle. Recognition of and binding to target cells are known to involve at least three glycoproteins—gB, gC, and gD. gC mediates the initial interaction with cells, recognizing heparan sulfate proteoglycans on the cell surface. gB also interacts with heparan sulfate proteoglycans, and can substitute for gC in gC negative viruses. This initial, heparin-sensitive attachment to cells is relatively weak, and is followed by a more stable attachment to cells, apparently mediated by gD. Following attachment, the virus particle fuses with the cell membrane to mediate entry. Fusion is known to require gB and gH/gL, and possibly also gD, but their precise functions are uncertain. The roles of other virus-encoded membrane proteins in entry are unclear, but it is possible that different proteins may be required for entry into different cell types. [ABSTRACT FROM AUTHOR]