1. Varicella Zoster Virus Transcriptional Regulation and the Roles of VZV IE Proteins

William T. Ruyechan

Varicella zoster virus (VZV) is the causative agent of varicella (chickenpox) upon primary infection. The virus is spread from its initial sites of replication through T cell viremias to the internal organs and skin.VZV establishes latency in dorsal root ganglia and upon reactivation causes zoster (shingles). During productive infection the entire coding capacity of the VZV genome (some 69 unique open reading frames) is believed to be expressed. In contrast, during latent infection, a small number of lytic phase genes are expressed while the remainder of the genome is silent. Thus the biology of VZV infection indicates that control of expression of the viral genome is essential to the ability of the virus to replicate productively in a variety of cell types with minimal complications for the host, and to the establishment and maintenance of latency in neurons. In recent years, our understanding of some of the mechanisms of VZV gene expression and of the proteins which regulate those processes has significantly increased. This includes information on the roles of individual viral proteins and the involvement of ubiquitous cellular transcription factors.

2. The Modification of Cellular RNA Polymerase II During HSV-1 Infection

Stephen A. Rice and Kathryn A. Fraser

Like other nuclear-replicating DNA viruses, herpes simplex virus type 1 (HSV-1) utilizes the host cell RNA polymerase II (Pol II) to transcribe its genome during its productive infection. Moreover, HSV-1 has evolved mechanisms that allow it to effectively compete with the host cell for the use of Pol II. This review will focus on the ability of HSV-1 to alter the Pol II enzyme itself. Specifically, HSV-1 alters the localization of Pol II, recruiting it into virus-induced replication compartments, where viral DNA replication and transcription take place. In addition, infection alters the normal phosphorylation of the Pol II carboxyl-terminal domain (CTD), creating a novel form of the enzyme known as Pol III. Current evidence suggests that the HSV-1-induced changes to the CTD involve two separate pathways, both of which require viral immediate-early proteins. The biological ramifications of these viral changes to Pol II are discussed.

3. The Roles of ICP0 During HSV-1 Infection

Roger D. Everett

ICP0, one of the five immediate-early proteins encoded by herpes simplex virus type 1 (HSV-1), plays a central role in regulating whether the virus progresses to lytic or latent infection. ICP0 is a member of the family of E3 ubiquitin ligase enzymes that have a so-called RING finger zinc-binding domain. This domain confers on ICP0 the ability to induce the proteasome-dependent degradation of a number of cellular proteins. This process results in multiple consequences, including the disruption of cellular nuclear sub-structures known as ND10 or PML nuclear bodies. Interestingly, prior to the disruption of ND10, parental HSV-1 genomes become closely associated with these structures during the early stages of HSV-1 infection. The E3 ubiquitin ligase activity of ICP0 correlates very well with its role in the stimulation lytic virus infection and induction of reactivation of latent or quiescent viral genomes. However, despite intensive investigation, the mechanism of the connection between the E3 ubiquitin ligase activities of ICP0 and its roles during HSV-1 infection remain poorly understood. This chapter summarises recent work on the biochemical and biological activities of ICP0, and discusses the possible mechanisms that might explain how ICP0 regulates HSV-1 infection.

4. The Functions and Activities of Herpes Simplex Virus Protein ICP27, a Multifunctional Regulator of Gene Expression

Rozanne M. Sandri-Goldin

ICP27 is a multifunctional regulator of gene expression that assumes different roles throughout the course of infection. At early times, ICP27 is an inhibitor of cellular splicing, whereas, at later times it is involved in recruiting RNA polymerase II to viral sites of replication and in facilitating viral RNA export. Evidence also suggests that at late times, it may be involved in the stimulation of translation of viral transcripts. ICP27 performs its activities by interacting with RNA and with a myriad of proteins. ICP27 binds viral RNAs to facilitate their export. Further, ICP27 interacts with itself to form multimers, and with HSV-1 proteins ICP4 and ICP8, as well as with an increasing number of cellular proteins. The regions of ICP27 involved in these interactions have been broadly mapped to both the N-terminal and C-terminal halves of the molecule. Several protein motifs have been identified based upon sequence comparisons; however, no structural information has yet been generated. Although much has been learned about the mechanisms by which ICP27 performs its roles, little is known about how its varied activities are regulated. The roles and activities of ICP27 are detailed in this review.

5. HSV-1 DNA Replication

Sandra K. Weller

Herpes Simplex Virus (HSV) DNA replication exhibits a complex relationship between DNA replication and recombination. In this respect HSV is similar to the large DNA bacteriophages, T4 and lambda which replicate via concatemers of tandemly repeated monomeric units using rolling circle replication as well as multiple pathways to carry out homologous recombination. The seven replication proteins of HSV may work in combination with both viral and cellular recombination proteins during this process, the details of which remain unclear. It appears that HSV DNA replication occurs in two stages. The first stage involves an origin-UL9 dependent step and in the second stage DNA replication may proceed in an origin-independent manner via a recombination-driven and/or rolling circle mechanism.

6. Hailing the Arrival of the Messenger: Strategies for Translational Control in Herpes Simplex Virus-infected Cells

Ian Mohr

Like all viruses, the translation of a-herpesvirus mRNAs relies completely upon components in their host cells. Thus, ensuring that viral mRNAs are able to effectively engage cellular translation factors and compete with cellular mRNAs for access to this equipment is of paramount importance. Notwithstanding the onslaught of a potent cellular antiviral response focused on incapacitating the translation machinery, the significance of viral protein production is further accentuated by viral functions devoted to neutralizing this host retort and preserving the activity of the host translational apparatus. Here, interactions between a-herpesviruses, exemplified by herpes simplex virus-1, with host translation initiation factors are discussed and recent developments concerning how viral functions control translation in infected cells are presented.

7. The Development and Use of Microarrays for Analyzing HSVGene Expression

Edward K. Wagner

The design and construction of long (75mer) oligonucleotide-based DNA microarray for quantitative analysis of Herpes simplex virus transcripts expressed in infected cells and tissues is described. The HSV-1 probes used in the current array are able to specifically cross-hybridize with HSV-2 sequences, and thus, can be used to study both HSV-1 and HSV-2 transcripts. Data presentation and the sensitivity of several methods of detection hybrids on such microarrays are presented. The quantitative measure of HSV transcripts expressed in cells infected with mutations in specific regulatory genes is utilized to illustrate the application of the information gained towards the general understanding of the virus-host interaction.

8. HSV Glycoproteins and Their Roles in Virus Entry and Egress

Gabriella Campadelli-Fiume and Tatiana Gianni

In the past several years there has been remarkable progress in our understanding of the events leading to herpes simplex virus (HSV) entry into the cells and its egress. Foremost was the realization that HSV entry requires a multigene system; this property differentiates HSV, and herpesviruses in general, from the majority of viruses. The glycoproteins absolutely required for the fusion that leads to HSV entry are gB gD, gH, gL. The receptor binding glycoprotein gD interacts with multiple alternative receptors that belong to unrelated protein families. They are nectin1 and 2, and the herpesvirus entry mediator. In addition, gD encodes a pro-fusion domain, thought to trigger fusion. Fusion is executed by gB, gH, gL. gH is likely to be the real fusion glycoprotein, as its ectodomain carries a a-helix with attributes of an internal fusion peptide, and two coiled coil heptad repeats. As far as HSV egress is concerned, controversy still exists about the possible routes of egress (luminal versus de-envelopment re-envelopment). A growing consensus in favor of the latter rests on studies of tegument assembly and the possible role of non essential glycoproteins in the cytoplasmic re-envelopment. is discussed. The lines of evidence in favor of, or against each route, as well as a novel possible route of egress are discussed.

9. Endocytosis of Varicella-Zoster Virus Glycoproteins: Virion Envelopment and Egress

Charles Grose, Lucie Maresova, Guruprasad Medigeshi, Gregory K. Scott, and Gary Thomas

Endocytosis from the infected cell surface is a property of the four major glycoproteins of varicella-zoster virus (VZV), designated gE, gI, gB and gH. Each of these glycoproteins has a functional endocytosis motif in its cytoplasmic tail. Three have tyrosine based motifs, while gI has a dileucine motif. The YAGL motif of gE is located adjacent to an acidic cluster, which also contains serine and threonine residues variably phosphorylated by both a viral kinase (ORF 47 kinase) and casein kinase II. The acidic cluster interacts with a connector protein called prosphofurin acidic cluster sorting protein 1 (PACS-1) after clathrin mediated internalization. Although the function of endocytosis has been elusive, recent research suggests that the internalized VZV glycoproteins traffic to the site of virion assembly in the trans-Golgi network and are subsequently incorporated into the envelopes of nascent virions. In turn, the enveloped virions reside within vacuoles, which travel toward and fuse with the plasma membrane. A recombinant VZV genome containing a gE gene lacking only its endocytosis motif cannot replicate. Taken together, the above results indicate the importance of VZV glycoprotein endocytosis in the life cycle of this alpha herpesvirus.

10 . Nucleocapsid Assembly and Envelopment of Herpes Simplex Virus

Joel D. Baines and Carol Duffy

Herpes simplex virus capsids assemble in the nuclei of infected cells in a number of ordered steps. The spherical procapsid is the first capsid formed and consists of an inner and outer shell. Proteolytic cleavage of the inner capsid shell, followed by subsequent expulsion or degradation of the inner shell and insertion of genomic DNA, results in a dramatic conformational change in which the outer shell of the procapsid angularizes and matures into a stable icosahedron. The completed, DNA-filled nucleocapsid is then actively transported to the nuclear membrane where it attaches and buds into the perinuclear space to become a virion. These events are detailed in the following review.

11. The Development and Use of Alpha Herpesviruses Expressing Fluorescent Proteins

Gregory A. Smith and Bruce W. Banfield

The development of fluorescent proteins as tools to monitor gene expression and protein trafficking within living cells and intact organisms has had a tremendous impact on virtually every discipline of the biological sciences including virology. In this chapter we review the development and application of recombinant alpha herpesviruses that express a variety of fluorescent proteins. First, we cover some general considerations, concerns and caveats associated with the use of these viruses. Next, we describe the use of viruses that express fluorescent protein fusions to further understand the trafficking and assembly of subvirion components within living cells. Finally, we discuss the use of recombinant alpha herpesviruses expressing fluorescent proteins to monitor virus infections in experimental animal model systems.

12. The Induction of Apoptosis by HSV-1

Christine M. Sanfilippo and John A. Blaho

Herpes simplex virus type 1 (HSV-1) is a cytolytic alphaherpesvirus which profoundly impacts its host cells. It is now widely recognized that consequences of HSV-1 infection include the induction of programmed cell death, also known as apoptosis, and the concomitant synthesis of proteins which act to block this process. While our current understanding of the precise mechanism by which apoptosis induction occurs remains elusive, several breakthrough studies have revealed much about how HSV-1 modulates the apoptotic process. In this chapter, we will review recent data focusing on the triggering of apoptosis by HSV-1, as well as evidence showing how this important human pathogen interferes with the fundamental cell death process.

13. Mechanisms of Subversion of Type I Interferon Responses by Alpha Herpesviruses

Karen L. Mossman

Interferons are pleiotropic cytokines that were first discovered based on their ability to interfere with virus replication. In vivo, interferons have been shown to play a central role in limiting the spread of alpha herpesviruses in that strains of mice that are resistant to virus infection rapidly produce high levels of this cytokine whereas strains that are susceptible to infection do not. Given the evolutionary pressure for alpha herpesviruses to delay or subvert the antiviral activities of interferons, it is not surprising that multiple viral proteins have been identified that collectively function to limit interferon production, signaling and effector function. This chapter will summarize the current knowledge surrounding mechanisms utilized by alpha herpesviruses to ensure efficient replication and spread in the face of an active interferon-mediated host antiviral response.

14. HSV-1 and the Host Cell: A Story of Global Conspiracies, Plots and Counterplots

Bernard Roizman and Brunella Taddeo

Studies on viral gene function revealed that infected cells mount an unsuccessful multi-layered defense that includes attempts to silence the viral genome at early stages of infection, activation of interferon pathways, turnover of cellular proteins required by the virus, and finally attempts to commit suicide through programmed cell death to salvage the infected host. The surprising finding is that multiple viral functions expressed by different viral proteins or domains thereof target the same cellular pathways to render them dysfunctional. The grand design of viral conquest of the host cell is to sequester and redirect cellular proteins to (i) preclude the infected cells from signaling it status to the environment or be the recipient of signals from the environment, (ii) disrupt the interferon defense mechanisms, and (iii) perform novel functions required by the virus. Considering that current knowledge is based on analyses of the functions of a few viral proteins, a wealth of data awaits to be discovered through analyses of the many multifunctional proteins expressed by virus.

15. Investigations of the Molecular Mechanisms of Varicella Zoster Virus Pathogenesis

Ann M. Arvin, Anne C. Schaap, Chia Chi Ku, Jeremy O. Jones, Marvin Sommer and Leigh Zerboni

Two major advances have provided new opportunities for understanding the molecular mechanisms of VZV pathogenesis. First, VZV cosmids permit construction of VZV recombinant viruses with targeted genetic mutations. Second, the development of the SCIDhu mouse model, in which skin, T cell and dorsal root ganglia xenografts are infected in vivo makes it possible to investigate VZV replication in differentiated human cells within their unique tissue microenvironments. These two approaches have yielded new insights about the molecular mechanisms of VZV pathogenesis that are involved in viral tropism for skin, T cells and neurons in vivo and about the host cell response to VZV infection.

16. Varicella Zoster Virus Neuropathogenesis and Latency

Donald H. Gilden, Ravi Mahalingam, Steven Deitch, and Randall J. Cohrs

Varicella zoster virus (VZV) is a highly neurotropic alphaherpesvirus. VZV causes chickenpox in 4 million children in the United States annually, after which virus becomes latent in cranial nerve, dorsal root and autonomic nervous system ganglia along the entire neuraxis. Decades later, a declining VZV host immunity allows virus to reactivate spontaneously, resulting in zoster, the primary neurologic complication of VZV reactivation, characterized by pain and rash restricted to 1-3 dermatomes. This chapter reviews the clinical features of zoster, including the two most serious neurologic complications: postherpetic neuralgia (PHN) and VZV vasculopathy. Evidence is presented to support the notion that VZV vasculopathy and PHN as well, may be caused by persistent virus infection. The chapter also provides an update on the physical state of VZV during latency in human ganglia, including the distribution and prevalence of virus in ganglia, virus abundance and configuration, cell type infected, and extent of virus transcription and translation. Finally, because VZV is an exclusively human virus, no animal model of latency and reactivation has been achieved. However, simian varicella virus (SVV), the primate counterpart of human VZV, does produce chickenpox and reactivate in monkeys. The chapter provides an update of studies, which have used SVV in primates as a model to study varicella latency and persistence.

17. HSV-1 Latency and the Roles of the LATs

David C. Bloom

Herpes simplex virus type 1 (HSV-1) latency is characterized by the persistence of viral genome as episomes in the nuclei of sensory neurons. During this period only one region of the genome is abundantly transcribed: the region encoding the latency-associated transcripts (LATs). The LAT domain is transcriptionally complex, and while the predominant species that accumulates during latency is a 2.0 kb stable intron, other RNA species are transcribed from this region of the genome, including a number of lytic or acute-phase transcripts. HSV-1 recombinant viruses with deletions within the LAT region exhibit reactivation deficits in a number of animal models, however there is evidence that some LAT deletion mutants also possess altered establishment and virulence properties. The phenotypic complexity associated with this region, including some evidence that the LATs may play a role in suppressing latent gene expression, suggests the that LAT locus may function as a regulator that modulates the transcription of key lytic and latent functions. The goal of this review is to provide an overview of our current understanding of the role(s) of the HSV-1 Latency Associated Transcripts (LATs) in the pathobiology of HSV-1 infections in vivo.

18. Immunity to Herpes Simplex Virus: Present but Not Perfect

Christopher D. Pack and Barry T. Rouse

Herpes simplex virus types 1 and 2 both represent significant human pathogens that infect a majority of the population. They should be controllable by vaccines, but despite notable effort, satisfactory vaccines remain unavailable. One hopes that fundamental studies of both the virus and how it interacts with the host will uncover clues useful for the design of effective prophylactic and therapeutic vaccines. This review focuses on host responses to the virus revealed in both animal models and human patient studies.

19. Anti-alphaherpesvirus Drugs

Donald M. Coen

The human alphaherpesviruses, herpes simplex virus 1 and 2 and varicella zoster virus, cause significant disease in patients. This has led to the development of drugs active against these viruses, in particular, acyclovir, which is one of the most successful antiviral agents. Acyclovir and its relatives act via a two step mechanism, entailing selective phosphorylation by viral thymidine kinase and selective inhibition of viral DNA polymerase. Molecular genetic, biochemical, and crystallographic studies have illuminated the details of the mechanisms of these drugs, as well as how the virus can mutate to evade the drug, yet retain pathogenicity. Similar studies of how alphaherpesviruses replicate in cells have identified interesting new drugs and drug targets. However, much more work will be needed to pinpoint molecular targets and specific strategies that could cure latent alphaherpesvirus infections.

20. Imaging HSV1 in Living Animals

Gary D. Luker

Studies of viral and host factors that influence pathogenesis largely have used experimental mouse models that rely upon sacrifice of infected mice to determine distribution and titers of virus. While this experimental paradigm has provided important data, it precludes real-time investigations of the same animal over the entire course of disease progression. Therefore, conventional assays may miss unexpected sites or patterns of viral dissemination because appropriate tissues were not collected or analyzed properly. Our laboratory and others have begun to exploit recent advances in technology for imaging small animals for studies of viral-host pathogenesis. In particular, we have shown that bioluminescence imaging is a sensitive, reproducible method for monitoring recombinant a recombinant strain KOS HSV-1 virus that expresses firefly luciferase. Infection with luciferase-expressing viruses can be detected at multiple anatomic sites, including brain, and quantitative differences in emitted light correlate with relative differences in viral titer measured by standard plaque assays. In this chapter we summarize current technologies for small animal imaging and their use in studies pathogenesis of HSV-1 and other microbial pathogens. We also highlight future directions in which new imaging technologies may provide innovative approaches to interrogate viral-host pathogenesis in living animals.