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

Jeffrey I. Cohen

Varicella-zoster virus (VZV) encodes three immediate-early proteins, IE4, IE62, and IE63; however, only IE62 has TAATGARAT-like sequences in its promoter which are present in the promoters of each of the herpes simplex virus immediate-early proteins. The TAATGARAT-like elements on the IE62 promoter bind to VZV ORF10 protein, Oct, and HCF-1. In addition, histone methyltransferases are recruited to the IE62 promoter to modify chromatin to a transcriptionally active form. VZV IE62, the major VZV transactivator binds to VZV IE4 and IE63, and Med25, part of the mediator complex which upregulates gene expression. VZV IE62, IE4, and IE63 are present in the viral tegument where they may help to regulate transcription early in infection. IE63 binds to several cellular proteins including ASF1 and RNA polymerase II. Two hypotheses have been proposed for regulation of VZV gene expression during latency. First, relocalization of HCF-1 from the cytoplasm to the nucleus of sensory ganglia in response to stimuli associated with reactivation may help to augment transcription of IE62 to reactivate VZV from latency. Second, promoters of latent genes are maintained in a euchromatic state allowing their transcription, while promoters of genes not associated with latency are in a heterochromatic state resulting in repression of transcription.

2. Functions and Mechanism of Action of the Herpes Simplex Virus Type Regulatory Protein, ICP4

Neal A. DeLuca

ICP4 is expressed from the HSV genome very early in infection. It is a large structurally complex nuclear phosphoprotein that is essential for viral growth largely due to its requirement for the transcriptional activation of most HSV early and late genes. It also acts a repressor of transcription under certain circumstances. The HSV genome is transcribed by RNA polII, and ICP4 interacts with components of the RNA polII transcription machinery to carry out is functions in transcription. The interactions that are important for its functions can be genetically defined implicating a modular composition of the ICP4 protein. ICP4 also plays a specific role in virus growth in sympathetic neurons implicating a specific function in pathogenesis. This is a review about what is known about ICP4 from many genetic, biological and biochemical studies, from many laboratories.

3. The Functions and Activities Of HSV-1 ICP27, a Multifunctional Regulator of Gene Expression

Rozanne M. Sandri-Goldin

Herpes simplex virus 1 (HSV-1) protein ICP27 is a multifunctional regulator that is essential for HSV-1 infection. ICP27 performs a number of different functions during infection that include inhibiting cellular pre-mRNA splicing, stimulating viral early and late gene transcription by recruiting cellular RNA polymerase II to viral replication sites, binding and exporting viral RNA to the cytoplasm and stimulating translation of some HSV-1 transcripts by binding translation initiation factors. ICP27 also recruits Hsc70 to nuclear foci (VICE domains) that are enriched in chaperones and components of the proteasome, and which are believed to be involved in nuclear protein quality control. ICP27 interacts with a number of proteins and it binds RNA. Post-translational modifications have been demonstrated to regulate ICP27's interactions with several proteins. NMR analysis of the N-terminus showed that it is highly flexible, which may be necessary for switching between different protein interactions. Further, ICP27 undergoes a head-to-tail intramolecular association that may also regulate its interactions, especially with proteins that require that both the N- and C-termini of ICP27 be intact for interaction. This review will cover the different activities of ICP27 and what we know about how these activities are regulated.

4. The Role of ICP0 in Counteracting Intrinsic Cellular Resistance to Virus Infection

Roger D. Everett

In recent years it has become apparent that, in addition to the acquired and innate defences against virus infection, there is also a third aspect to antiviral defences that operates at the intracellular level. This concept is known as intrinsic resistance, intrinsic antiviral defence or intrinsic immunity. Its key features include constitutively expressed cellular proteins that restrict viral gene expression, and viral regulatory proteins that counteract the actions of the cellular inhibitors. This chapter reviews the cellular proteins and pathways that are thought to be involved in intrinsic resistance to HSV-1 infection, and the mechanisms by which these are inactivated by ICP0, an important viral regulatory protein. The phenotype of ICP0 null mutant HSV-1 is described to give a background to the phenomenon, then the principal properties of ICP0 itself are summarised. The effects of ICP0 on components of cellular nuclear structures known as ND10 or PML nuclear bodies are reviewed, then the possible roles of these proteins in intrinsic resistance are discussed. The relationships between ICP0, intrinsic resistance and the regulation of viral chromatin structure are considered, and finally the parallels between ICP0 and related proteins expressed by other alphaherpesviruses are described. Intrinsic resistance and the manner in which viruses overcome it are important aspects of the biology of virus infection, but we have much to learn before we achieve a complete understanding of the viral and cellular proteins that are involved.

5. Multiple Roles of Immediate-Early Protein ICP22 in HSV-1 Replication

Stephen A. Rice

ICP22 is the least characterized of the five herpes simplex virus type 1 (HSV-1) immediate-early (IE) proteins. However, accumulating evidence indicates that it carries out a number of interesting regulatory activities inside the infected cell. These include the enhancement of viral gene expression, the modification of RNA polymerase II (RNAP II), and the reorganization of host cell molecular chaperones into nuclear inclusion bodies. Recent studies of engineered HSV-1 mutants indicate that certain of ICP22's activities are genetically separable from each other. Thus, similar to several other of the IE proteins, ICP22 appears to be a multifunctional, multi-domain polypeptide. This chapter summarizes the current state of knowledge concerning ICP22 and its varied regulatory roles during the productive HSV-1 infection.

6. HSV-1 DNA Replication

Stacey A. Leisenfelder and Sandra K. Weller

The cis- and trans-acting elements required for DNA synthesis of Herpes Simplex Virus (HSV) have been identified, and genetic and biochemical analyses have provided important insights into how they work together to replicate the large double-stranded viral genome. Furthermore, viral enzymes involved in DNA replication have provided a rich store of useful targets for antiviral therapy against herpesviruses. Despite these advances, many questions remain unresolved concerning the overall mechanism of genome replication. For instance, it has long been recognized that the products of viral DNA replication are head-to-tail concatemers; however, it is not clear how these concatemers are generated. In this chapter we will summarize the known functions of viral replication proteins and explore the possibility that these viral proteins may function in combination with cellular proteins to produce concatemers suitable for packaging into preformed viral capsids.

7. Translational Control in Herpes Simplex Virus-infected Cells

Ian Mohr

Like all viruses, α-herpesviruses are completely reliant upon the protein synthesis machinery resident in their host cells. In particular, viral mRNAs must effectively compete with cellular mRNAs to engage ribosomes. To ensure high-level production of the polypeptides required for their lytic replication, multiple independent gene products expressed by the model α-herpesvirus HSV-1 effectively seize control of critical host cell translational control pathways. Surprisingly, while host protein synthesis is profoundly suppressed by global changes in mRNA metabolism, the assembly of a multi-subunit, cap-binding translation initiation factor complex required to recruit 40S subunits to mRNA is directly stimulated. This involves both inactivation of a cellular translational repressor by viral functions, and direct interaction between specific viral proteins and select cellular translation initiation factors. In addition to their dependence on cellular components required for mRNA translation, virus-encoded functions must preserve its activity by neutralizing potent host responses capable of incapacitating the translation machinery, one of which senses stress within the endoplasmic reticulum lumen and another of which functions as a host innate defense component by sensing double-stranded RNA, a molecular signature of viral infection. This chapter discusses in detail the many virus-host interactions that are presently known to control translation in cells productively infected with HSV-1 and highlights recent developments in this area.

8 . Recent Progress in Understanding Herpes Simplex Virus Entry: Relationship of Structure to Function Entry

Roselyn J. Eisenberg, Ekaterina E. Heldwein, Gary H. Cohen and Claude Krummenacher

Membrane fusion allows exchange of materials between cellular compartments enclosed by lipid membranes. Similarly, entry of enveloped viruses into cells allows the viral contents to be delivered by fusion of the envelope with a target cell membrane. Fusion requires disruption of both layers of the two membranes. For most enveloped viruses, a single surface glycoprotein undergoes conformational changes that bring the bilayer of the virus in proximity with that of the host cell and fusion ensues. In contrast, herpesvirus entry requires three virion glycoproteins, gB and a gH/gL heterodimer, that function as the core fusion machinery. Some herpesviruses require additional proteins, e.g. alphaherpesviruses (with a few exceptions) initiate fusion by binding of glycoprotein gD to a cell receptor. A conformational change then exposes the normally hidden receptor binding residues of gD. This change and/or the exposed residues trigger gB and gH/gL to effect virus-cell and cell-cell fusion. Because of the multiplicity of proteins involved in HSV entry as opposed to entry of enveloped RNA viruses, it has been difficult to unravel the mechanism of how the four entry glycoproteins function. Some favor formation of a multiprotein fusion complex while others suggest it may be more of a stepwise process. Solution of the structures of all four entry proteins, coupled with existing and new information has solved much of this mystery. We now have a much better idea of the outline of the process, but the challenge for the future will be to fill in important details. It is clear that entry of HSV occurs in an exquisitely regulated stepwise process that begins with binding of gD to a receptor, activation of the regulatory protein gH/gL which in turn up-regulates the fusogenic activity of gB. Thus, in some ways, HSV entry is remarkably similar overall to entry by simpler RNA viruses, such as influenza. A single fusion protein gB carries out fusion. What distinguishes HSV entry is the double regulation of this process. This chapter will focus primarily on what we know about the structures of gD, gH/gL and gB along with supporting data for how those proteins function.

9. Structure-function Profiles of Nine Varicella-Zoster Virus Glycoproteins: Endocytosis, Entry And Egress

Charles Grose, Susan Vleck, Odd Andre Karlsen and Eduardo A. Montalvo

Varicella zoster virus has a smaller genome than herpes simplex virus and therefore encodes fewer glycoproteins. In this chapter, nine VZV glycoproteins are profiled, including gE, gI, gC, gH, gL, gB, gK, gM, and gN. Although all VZV glycoproteins have HSV homologs, functions occasionally have greatly shifted. For example, VZV gE is the predominant VZV glycoprotein and exists as a monomer, dimer and trimer, as well as a gE/gI complex. VZV gE is essential, unlike HSV gE. Even though essential, mutations in gE had been detected in wild type VZV strains that exhibit an accelerated cell-spread phenotype. The VZV gC glycoprotein differs from HSV gC in that both transcription and translation of VZV gC are remarkably delayed in cultured cells; often VZV gC protein is difficult to detect altogether. The VZV gH/gL complex resembles the HSV gH/gL complex is that both are critical for virus induced fusion. Fusion is a prominent feature of VZV infected cells. Neutralization antibody to VZV gH dramatically reduces the spread of virus and limits pathogenesis in the skin. The VZV gB glycoprotein is also involved in virus-induced fusion. Of interest, four VZV glycoproteins (gE, gI, gH and gB) have functional endocytosis motifs in their cytoplasmic tail. Thus, all four are internalized from the cell surface in clathrin coated vesicles. This pathway appears critical for the process of virion envelopment in the assembly compartment. Even though abundant amounts of most glycoproteins are produced in cell culture, assembly of fully enveloped and infectious VZV particles rarely occurs. The particle:plaque forming unit ratio remains an extremely high 40,000:1. Likewise, the aberrant assembly process severely limits any assessment of egress mechanisms.

10. Nucleocapsid Structure, Assembly and DNA Packaging of Herpes Simplex Virus

James F. Conway and Fred L. Homa

The herpes simplex virion consists of an external membrane envelope, a proteinaceous layer called the tegument, and an icosahedral capsid containing the double-stranded linear DNA genome. The capsid shell is 125 nm in diameter and consists of 162 capsomers (150 hexons, 11 pentons and a portal) which lie on a T=16 icosahedral lattice. The capsid shell consists of four major structural proteins VP5, VP19C, VP23 and VP26 which are the products of the HSV UL19, UL38, UL18 and UL35 genes. In addition to the four major structural proteins the HSV-1 capsid contains a number of minor capsid proteins. These include the UL6, UL15, UL17, UL25, UL28 and UL33 proteins, all of which (along with the HSV-1 UL32 protein) are required for the processing and packaging of replicated viral DNA into preformed capsid shells. The UL6, UL17, UL25 and UL33 proteins remain associated with DNA containing capsids while UL15 and UL28 do not. This review is a summary of our present knowledge with respect to how the capsid is assembled, how DNA is packaged and what is known about the role of the seven packaging proteins in this process. In addition, recent advances in our understanding the structure of the four distinct types of capsids that are present in HSV infected cells as determined by three dimensional image reconstructions from cryo-electron microscopy (cryoEM) will be presented and discussed.

11. Nuclear Egress and Envelopment of HSV

Joel D. Baines

In a process unique in biology, all herpesviruses obtain their initial virion envelope by budding through the inner nuclear membrane. In the most prominent model of virion egress, the envelope of the perinuclear virion then fuses with the luminal surface of the outer nuclear membrane, releasing the de-enveloped capsid into the cytosol for subsequent budding events. The pUL31/pUL34 protein complex is a major player in the initial budding event, and mediates several relevant functions including disruption of the nuclear lamina, recruitment of other viral proteins to the inner nuclear membrane and perinuclear virion, and budding of the nucleocapsid through the inner nuclear membrane. This review focuses on pUL31, pUL34, and other proteins known or believed to be involved in the initial budding event and egress from the perinuclear space.

12. Apoptosis Modulation During Herpes Simplex Virus Replication

Christopher R. Cotter and John A. Blaho

Consequences of human herpes simplex virus (HSV) infection include the induction of apoptosis and the concomitant synthesis of proteins which act to prevent this process from killing the infected cell. Recent data has clarified our current understanding of the mechanisms of induction and prevention of apoptosis by HSV; which ultimately establishes a delicate balance of pro- and antiapoptotic modulating factors in infected cells. These findings emphasize the fact that modulation of apoptosis by HSV during infection is a multicomponent phenomenon involving a combination of viral and cellular factors. We review recent evidence showing how this important human pathogen modulates the fundamental cell death process.

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

Paul T. Sobol and Karen L. Mossman

Key to the innate immune response to alpha herpesvirus infection is the expression and secretion of type I interferons (IFNs). This family of cytokines bolsters a host offensive to invading pathogens by inducing IFN stimulated genes (ISGs). Not surprisingly, the evolutionary pressure faced by alpha herpesviruses to adapt to the type I IFN response has shaped alpha herpesvirus evolution at the very interface of the virus-host interaction. The cumulative effects of type I IFN expression on alpha herpesvirus replication in vitro and dissemination in vivo are discussed in this chapter, along with mechanisms employed by these viruses to subvert the type I IFN response. Specifically, this chapter will summarize how alpha herpesviruses block type I IFN production, inhibit the effects of type I IFN signal transduction and suppress downstream IFN-dependent effector pathways with the aims of augmenting viral replication and dissemination.

14. Molecular Chaperones and Alphaherpesvirus Infection

Christine M. Livingston, Christos Kyratsous, Saul Silverstein and Sandra K. Weller

Molecular chaperone proteins have long been recognized to play diverse and important roles in the life cycles of viruses from bacteriophage to SV40 to herpesviruses. The alphaherpesviruses HSV-1 and VZV not only interact with and reorganize cellular chaperones and co-chaperones but alphaherpesviruses also encode their own molecular chaperones. Cellular chaperones such as Hsp70, Hsc70 and Hsp90 are required for efficient production of infectious virus in that they play essential roles in nuclear transport of viral proteins, protein quality control and maintenance of cellular homeostasis during viral infection. These findings raise the possibility that molecular chaperones could be utilized as effective targets for antiviral therapy. Here we review the evidence that replication of the human alphaherpesviruses herpes simplex virus type 1 and 2 (HSV-1 and 2) and varicella zoster virus (VZV) requires the activities of cellular and viral molecular chaperones.

15. Interactions Between HSV-1 and the DNA Damage Response

Matthew D. Weitzman and Sandra K. Weller

The cellular DNA damage machinery responds to virus infection and the foreign genomes that accumulate in the nuclei of infected cells. Many DNA viruses have been shown to manipulate the cellular DNA damage response pathways in order to create environments conducive to their own replication. Some cellular factors are activated during infection while others are inactivated. In this chapter we will focus on the complex cellular responses triggered by infection with HSV-1 and the consequences of its manipulation by viral factors.

16. Varicella-zoster Virus Pathogenesis and Latency

Leigh Zerboni and Ann M. Arvin

Varicella-zoster virus (VZV) is a human alphaherpesvirus and the etiological agent of varicella during primary infection and herpes zoster upon reactivation from latency. In this chapter we describe clinical observations and experience with VZV disease in the natural human host. We present a refined paradigm for VZV pathogenesis that emphasizes the importance of VZV lymphotropism. We also present key findings in our investigations of VZV pathogenesis and latency using SCID mouse-human tissue xenografts. Experimental infection with over 100 recombinant VZV viruses containing mutations in open reading frames encoding viral glycoproteins, viral kinases, regulatory proteins and as well as promoter elements has revealed tissue-specific requirements for VZV gene motifs and promoter elements for replication in skin, T-lymphocytes and neurons. We have repeatedly observed that replication phenotypes of VZV mutants in cultured cells are not predictive of behavior within differentiated intact tissues under physiological (in vivo) conditions. These observations underscore the importance of in vivo investigations in identifying molecular mechanisms of VZV pathogenesis. These experiments provide insight into VZV interactions with human host cells and have potential relevance in the design of 'second generation' recombinant VZV vaccines.

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

David C. Bloom and Dacia L. Kwiatkowski

Herpes simplex virus type 1 (HSV-1) latency is characterized by the persistence of viral genomes 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. In addition, a number of microRNA (miRNA) and non-miRNA small RNAs have recently been mapped to the LAT region of the genome. HSV-1 recombinant viruses with deletions of the LAT promoter exhibit reactivation deficits in a number of animal models, and there is evidence that other LAT deletion mutants also possess altered establishment and virulence properties. The phenotypic complexity associated with this region, as well as evidence that the LATs may play a role in suppressing latent gene expression, suggests that the LAT locus may function as a regulator to modulate the transcription of key lytic and latent genes. 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. Vaccines and New Antiviral Strategies Against Herpes Simplex Virus

Timothy E. Dudek and David M. Knipe

Vaccines have been among the most effective public health approaches for protecting individuals against viral disease, with two of the world's most successful vaccines being against smallpox virus and poliovirus. Herpes simplex virus 1 (HSV-1) is a nearly ubiquitous pathogen, and the worldwide prevalence of herpes simplex virus 2 (HSV-2) continues to increase. These two pathogens cause significant morbidity and mortality among the general population, but in particular in neonates and immunocompromised individuals. Perhaps most significantly, there is a 3-4 fold increased risk of HIV acquisition in HSV-2 infected individuals. To date, attempts at producing a vaccine against HSV have not been successful, but each attempt has brought insights into what may be required for an effective vaccine. Furthermore, intense studies into the immunology of HSV infection and the resources that have been put into vaccine design and development have recently yielded knowledge that will be necessary to achieve the goal of a highly effective vaccine against HSV. Here we will discuss several vaccine constructs that have contributed to the advancement of the HSV vaccine field and some of the new antiviral strategies to counter HSV.

19. Immunity to Herpes Simplex Virus

Keith R. Jerome

HSV presents unique challenges to the human immune system. Most of these result from the ability of the virus to establish latency in neurons of the dorsal root ganglia. The first line of defense against the initial establishment of latent infection is the innate immune response. The innate response relies on a variety of cell types recognizing HSV infection via pattern recognition receptors, including toll-like receptors. After exposure, the adaptive immune response is triggered. However, the adaptive response must deal with reactivation of HSV from the latently infected neuron, which in turn seeds mucosal sites with virus. T cells are especially important in this, and likely control both the extent of reactivation from latently infected neurons as well as the extent of viral replication at mucosal sites. Not surprising, HSV has evolved a wide variety of immune evasion mechanisms to tip this balance in its favor and facilitate transmission to new hosts. The study of HSV and its interaction with the host immune system has provided insights into the function of both, and may ultimately facilitate the development of an effective HSV vaccine.

20. Immunity and Immune Evasion Strategies Induced by Varicella Zoster Virus

Paul R. Kinchington and Allison Abendroth

Of three human alphaherpesviruses, only Varicella Zoster Virus (VZV) induces a lifelong immunity that protects against clinical signs of exogenous re-infection and, for most of the population, from any sign of reactivation from the latent state. The importance of VZV specific immunity is exemplified by its absence: severity and morbidity of the primary infection (varicella) and incidence of reactivated disease (zoster) are greatly increased in those with immune compromise, particularly those impaired in the cell mediated immune responses. The protection afforded by VZV specific immunity underlies successful live attenuated vaccines that have greatly impacted the incidence of varicella, and reduce the incidence, severity and complications of zoster. Consequently, the important components of VZV induced immunity and their contribution to the protective state has been well studied and is outlined in this chapter. Less is known of the strategies exploited by VZV to evade the innate and adaptive arms, but their activities are presumed to be critical to extend the life of the infected cell and to enhance viral production and dissemination. Evasion appears to include distinct strategies from those used by Herpes simplex viruses and includes expression of novel immune evasion proteins. Our current understanding of these strategies and the underlying mechanisms is outlined, along with areas in which there is need for further study.

21. Herpesviruses and the Control of Autophagy

Philipe A.M. Gobeil and David A. Leib

Autophagy is a rapidly growing area of biomedical research with broad relevance to fields including microbiology, cell biology, immunology, cancer biology, and neurodegeneration. In infection and immunity, it is emerging as a pivotal pathway mediating direct pathogen degradation as well as for the development of robust innate and adaptive immune responses. Successful pathogens have evolved to either evade or harness the autophagy pathway to further their replication and pathogenesis. In this chapter, the basic aspects of autophagy will be described, along with its role in cellular homeostasis, and the development of immunity. The primary focus is a survey of past and recent research defining the interplay of autophagy and the herpesviruses, with particular reference to immune evasion and pathogenesis.

22. Human Alpha-herpesvirus MicroRNAs: Expression and Functions

Jennifer L. Umbach and Bryan R. Cullen

MicroRNAs (miRNAs) are an extensive class of ~22 nucleotide long regulatory RNAs expressed by all mammalian cells and also by several DNA viruses, including many members of the herpesvirus family. Using deep sequencing technology, it has now been demonstrated that Herpes Simplex Virus 1 (HSV-1) encodes at least eight viral miRNAs, seven of which are expressed in latently infected human neurons. Similarly, HSV-2 has also been shown to encode at least six miRNAs, four of which are evolutionarily conserved between HSV-2 and HSV-1. Perhaps surprisingly, varicella zoster virus does not appear to express any viral miRNAs in latently infected cells. This review discusses the potential functions of the currently known HSV-1 and HSV-2 miRNAs, focusing on a possible role in stabilizing viral latency in infected neurons.

23. Oncolytic HSV Vectors for Cancer Therapy

Samuel Rabkin

Oncolytic HSV (oHSV) virotherapy is a promising new strategy for cancer therapy, converting a human pathogen into a therapeutic agent. This takes advantage of the biology of HSV, by introducing genetic alterations that limit virus replication and cytotoxicity to transformed cancer cells while making the virus non-permissive in normal cells. HSV encodes a large number of genes that are non-essential for growth in tissue culture cells, but are nevertheless important for growth in post-mitotic cells and for interfering with intrinsic antiviral and innate immune responses. Many of the cellular pathways regulating growth and antiviral responses are disrupted in cancer cells, which means that viral gene products allowing replication in normal cells are not necessary in cancer cells. In considering the development of an infectious agent for human use, safety is a critical consideration. Therefore mutations targeting cancer cells must be combined with mutations in genes that play important roles in vivo; causing pathogenicity, spread through the nervous system and other organs, latency and reactivation, and adaptive immune responses. This review will focus more on the virological aspects of oHSV vectors and less on the cancer cell target, and describe the multiple strategies and genes involved in generating oHSV vectors. However, it is important to bear in mind that the effect of different HSV mutations will be highly dependent upon the physiology of the particular type of cancer cell and tumor, and that each oHSV vector will be more effective in some tumor types, so that it is unlikely that any one oHSV will be optimal for all types of cancer.