Herpes Simplex Virus Type 1 Glycoprotein gC Mediates Immune Evasion In Vivo

Many microorganisms encode proteins that interact with molecules involved in host immunity; however, few of these molecules have been proven to promote immune evasion in vivo. Herpes simplex virus type 1 (HSV-1) glycoprotein C (gC) binds complement component C3 and inhibits complement-mediated virus neutralization and lysis of infected cells in vitro. To investigate the importance of the interaction between gC and C3 in vivo, we studied the virulence of a gC-null strain in complement-intact and C3-deficient animals. Using a vaginal infection model in complement-intact guinea pigs, we showed that gC-null virus grows to lower titers and produces less severe vaginitis than wild-type or gC rescued virus, indicating a role for gC in virulence. To determine the importance of complement, studies were performed with C3-deficient guinea pigs; the results demonstrated significant increases in vaginal titers of gC-null virus, while wild-type and gC rescued viruses showed nonsignificant changes in titers. Similar findings were observed for mice where gC null virus produced significantly less disease than gC rescued virus at the skin inoculation site. Proof that C3 is important was provided by studies of C3 knockout mice, where disease scores of gC-null virus were significantly higher than in complement-intact mice. The results indicate that gC-null virus is approximately 100-fold (2 log₁₀) less virulent that wild-type virus in animals and that gC-C3 interactions are involved in pathogenesis.     

Anterograde Spread of Herpes Simplex Virus Type 1 Requires Glycoprotein E and Glycoprotein I but Not Us9

Anterograde neuronal spread (i.e., spread from the neuron cell body toward the axon terminus) is a critical component of the alphaherpesvirus life cycle. Three viral proteins, gE, gI, and Us9, have been implicated in alphaherpesvirus anterograde spread in several animal models and neuron culture systems. We sought to better define the roles of gE, gI, and Us9 in herpes simplex virus type 1 (HSV-1) anterograde spread using a compartmentalized primary neuron culture system. We found that no anterograde spread occurred in the absence of gE or gI, indicating that these proteins are essential for HSV-1 anterograde spread. However, we did detect anterograde spread in the absence of Us9 using two independent Us9-deleted viruses. We confirmed the Us9 finding in different murine models of neuronal spread. We examined viral transport into the optic nerve and spread to the brain after retinal infection; the production of zosteriform disease after flank inoculation; and viral spread to the spinal cord after flank inoculation. In all models, anterograde spread occurred in the absence of Us9, although in some cases at reduced levels. This finding contrasts with gE- and gI-deleted viruses, which displayed no anterograde spread in any animal model. Thus, gE and gI are essential for HSV-1 anterograde spread, while Us9 is dispensable.Alphaherpesviruses are parasites of the peripheral nervous system. In their natural hosts, alphaherpesviruses establish lifelong persistent infections in sensory ganglia and periodically return by axonal transport to the periphery, where they cause recurrent disease. This life cycle requires viral transport along axons in two directions. Axonal transport in the retrograde direction (toward the neuron cell body) occurs during neuroinvasion and is required for the establishment of latency, while transport in the anterograde direction (away from the neuron cell body) occurs after reactivation and is required for viral spread to the periphery to cause recurrent disease. In addition to anterograde and retrograde axonal transport within neurons, alphaherpesviruses spread between synaptically connected neurons and between neurons and epithelial cells at the periphery (19, 22).The alphaherpesvirus subfamily includes the human pathogens herpes simplex virus type 1 (HSV-1), HSV-2, and varicella-zoster virus (VZV), as well as numerous veterinary pathogens such as pseudorabies virus (PRV) and bovine herpesviruses 1 and 5 (BHV-1 and BHV-5). The molecular mechanisms that mediate alphaherpesvirus anterograde axonal transport, anterograde spread, and cell-to-cell spread remain unclear. However, many studies of several alphaherpesviruses have indicated that anterograde transport or anterograde spread involves the viral proteins glycoprotein E (gE), glycoprotein I (gI), and Us9 (2, 5, 7, 9, 11, 13, 16, 26, 30, 31, 41, 46, 51, 52).Glycoproteins E and I are type I membrane proteins that form a heterodimer in the virion membrane and on the surface of infected cells. Although dispensable for the entry of extracellular virus, gE and gI mediate the epithelial cell-to-cell spread of numerous alphaherpesviruses (1, 3, 15, 20, 34, 38, 49, 53, 54). Us9 is a type II nonglycosylated membrane protein with no described biological activity apart from its role in neuronal transport (4, 18, 32). Here, we used several model systems to better characterize the roles of gE, gI, and Us9 in HSV-1 neuronal spread.Animal models to assess alphaherpesvirus neuronal transport (viral movement within a neuron) and spread (viral movement between cells) include the mouse flank and mouse retina models of infection. In the mouse flank model (Fig. ​(Fig.1A),1A), virus is scratch inoculated onto the depilated flank, where it infects the skin and spreads to innervating sensory neurons. The virus travels to the dorsal root ganglia (DRG) in the spinal cord (retrograde direction) and then returns to an entire dermatome of skin (anterograde spread). The virus also is transported in an anterograde direction from the DRG to the dorsal horn of the spinal cord and subsequently spreads to synaptically connected neurons. The production of zosteriform lesions and the presence of viral antigens in the dorsal horn of the spinal cord both are indicators of anterograde spread in this system. PRV gE and Us9 are required for the production of zosteriform disease, while gI is dispensable (7). In the absence of gE, HSV-1 also fails to cause zosteriform disease. However, unlike PRV, HSV-1 gE is required for retrograde spread to the DRG, so the role of gE in HSV-1 anterograde spread could not be evaluated in the mouse flank model (8, 36, 42).Open in a separate windowFIG. 1.Model systems to study HSV-1 neuronal spread. (A) Mouse flank model. Virus was scratch inoculated onto the skin, where it replicates, spreads to innervating neurons, and travels in a retrograde direction to the neuron cell body in the DRG. After replicating in the DRG, the virus travels in an anterograde direction back to the skin and into the dorsal horn of the spinal cord. Motor neurons also innervate the skin, allowing virus to reach the ventral horn of the spinal cord by retrograde transport. (B) Mouse retina model. Virus is injected into the vitreous body, from which it infects the retina as well as other structures of the eye, including the ciliary body, iris, and skeletal muscles of the orbit. From the retina, the virus is transported into the optic nerve and optic tract (OT) (anterograde direction) and then to the brain along visual pathways. Anterograde spread is detected in the lateral geniculate nucleus (LGN) and superior colliculus (SC). From the infected ciliary body, iris, and skeletal muscle, the virus spreads in a retrograde direction along motor and parasympathetic neurons and is detected in the oculomotor and Edinger-Westphal nuclei (OMN/EWN). Only first-order sites of spread to the brain are indicated. (Brain images were modified and reproduced from reference 47 with permission from of the publisher. Copyright Elsevier 1992.) (C) Campenot chamber system. Campenot chambers consist of a Teflon ring that divides the culture into three separate compartments. Neurons are seeded into the S chamber and extend their axons into the M and N chambers. Vero cells are seeded into the N chamber 1 day before infection. Virus is added to the S chamber and detected in the N chamber, a measure of anterograde spread.The mouse retina infection model (Fig. ​(Fig.1B)1B) has the advantage of allowing anterograde and retrograde spread to be studied independently of one another. Virus is delivered to the vitreous body, from which it infects the retina and other structures of the eye. The cell bodies of retinal neurons form the innermost layer of the retina; therefore, the virus infects these neurons directly, and spread from the retina along visual pathways to the brain occurs in an exclusively anterograde direction. In addition, the virus infects the anterior uveal layer of the eye (ciliary body and iris) and skeletal muscles in the orbit. From these tissues, the virus infects innervating parasympathetic and motor neurons and spreads to the brain in a retrograde direction. The localization of viral antigens in specific brain sites indicates whether the virus traveled to the brain along an anterograde or retrograde pathway (21, 25, 26, 39, 44, 51). PRV gE, gI, and Us9 each are essential for anterograde spread to the brain yet are dispensable for retrograde spread (5, 11, 25, 52). Even a strain of PRV lacking all three of these proteins retains retrograde neuronal spread activity (12, 40, 44). In contrast, in the absence of gE, HSV-1 fails to spread to the brain by either the anterograde or retrograde pathway (51).The Campenot chamber system (Fig. ​(Fig.1C)1C) has the advantage of allowing quantitative measurement of anterograde spread. Sympathetic neurons are cultured in a tripartite ring in which neuron cell bodies are contained in a separate compartment from their neurites. Virus is added to neuron cell bodies in one chamber, and anterograde spread to a separate chamber is measured by viral titers (13, 29, 30, 39, 43). Using this system, gEnull, gInull, and Us9null PRV each were shown to have only a partial defect in anterograde spread, while a virus lacking all three proteins was totally defective (13).We sought to quantify the anterograde spread activity of gEnull, gInull, and Us9null HSV-1 using the Campenot chamber system. While gEnull and gInull viruses were completely defective at anterograde spread, we found that a Us9null virus retained wild-type (WT) anterograde spread activity in this system. This observation was unexpected, since others previously had reported that Us9 is required for efficient HSV-1 anterograde transport or spread (26, 41, 46). Therefore, we further characterized the neuronal spread properties of two independent Us9-deleted viruses in the mouse retina and mouse flank models of infection. Our results indicate that gE and gI are essential for HSV-1 anterograde spread, whereas Us9 is dispensable.     

Differences in the Regulatory and Functional Effects of the Us3 Protein Kinase Activities of Herpes Simplex Virus 1 and 2

Us3 protein kinases encoded by herpes simplex virus 1 (HSV-1) and 2 (HSV-2) are serine/threonine protein kinases and play critical roles in viral replication and pathogenicity in vivo. In the present study, we investigated differences in the biological properties of HSV-1 and HSV-2 Us3 protein kinases and demonstrated that HSV-2 Us3 did not have some of the HSV-1 Us3 kinase functions, including control of nuclear egress of nucleocapsids, localization of UL31 and UL34, and cell surface expression of viral envelope glycoprotein B. In agreement with the observations that HSV-2 Us3 was less important for these functions, the effect of HSV-2 Us3 kinase activity on virulence in mice following intracerebral inoculation was much lower than that of HSV-1 Us3. Furthermore, we showed that alanine substitution in HSV-2 Us3 at a site (aspartic acid at position 147) corresponding to one that can be autophosphorylated in HSV-1 Us3 abolished HSV-2 Us3 kinase activity. Thus, the regulatory and functional effects of Us3 kinase activity are different between HSV-1 and HSV-2.Us3 protein kinases encoded by herpes simplex virus 1 (HSV-1) and 2 (HSV-2) are serine/threonine protein kinases with amino acid sequences that are conserved in the subfamily Alphaherpesvirinae (6, 24, 36). Based on studies showing that recombinant Us3 mutants of HSV-1 and HSV-2 have significantly impaired viral replication and virulence in mice models, it has been concluded that both HSV-1 and HSV-2 Us3 protein kinases play important roles in viral replication and pathogenicity in vivo (25, 33, 41). In contrast, HSV-1 and HSV-2 Us3 protein kinases are not essential for growth in tissue culture cells (33, 36). Thus, recombinant Us3 mutants grow as well as wild-type viruses in Vero cells, and the mutants exhibit modestly impaired replication in HEp-2 cells (33, 36, 39, 40). The possible functions of Us3 have been extensively studied and gradually elucidated for HSV-1 Us3, but much less is known about HSV-2 Us3. These functions include (i) blocking apoptosis (1, 22, 30, 31, 35); (ii) promoting nuclear egress of progeny nucleocapsids through the nuclear membrane (39, 40, 45); (iii) redistributing and phosphorylating nuclear membrane-associated viral nuclear egress factors UL31 and UL34 (14, 37, 38) and cellular proteins, including lamin A/C and emerin (21, 27, 28); (iv) controlling infected cell morphology (13, 31, 32); and (v) downregulating cell surface expression of viral envelope glycoprotein B (gB) (12).To determine the molecular mechanisms for a viral protein kinase''s effects in infected cells, the kinase''s physiological substrates and its phosphorylation sites must be identified. This can involve studies showing that the altered phenotypes observed in cells infected with a mutant virus lacking the protein kinase activity is also detected in cells infected with a mutant virus in which the substrate''s phosphorylation sites have been modified by mutations. Although more than 15 potential HSV Us3 substrates have been reported, HSV-1 Us3 phosphorylation of only three substrates (Us3 itself, gB, and UL31) has been demonstrated to be linked directly with Us3 functions in infected cells (12, 13, 29, 41) as follows. (i) Us3 has been reported to autophosphorylate serine at position 147 (Ser-147), and this phosphorylation augments Us3''s kinase activity in infected cells (13, 41). Even though only a small fraction of Us3 is autophosphorylated at Ser-147 in infected cells, alanine replacement of Ser-147 in Us3 significantly reduced HSV-1 replication in the mouse cornea and pathogenic manifestations of herpes stroma keratitis and periocular skin disease in mice (41). These results indicated that Us3 kinase activity was, in part, regulated by autophosphorylation of Ser-147, and regulation of Us3 activity by autophosphorylation played a critical role in viral replication in vivo and HSV-1 pathogenesis. (ii) It has been reported that HSV-1 Us3 phosphorylates Thr-887 in the cytoplasmic tail of gB, and this phosphorylation downregulates the cell surface expression of gB (12). Us3 phosphorylation of gB at Thr-887 also has been proposed to be involved in the regulation of fusion of the nascent progeny virion envelope with the cell''s outer nuclear membrane, based on the observation that virions accumulated aberrantly in the perinuclear space in cells infected with mutant viruses carrying the amino acid substitution mutation T887A in gB and lacking the capacity to produce gH (45). The Us3 phosphorylation of gB at Thr-887 appeared to be critical for HSV-1 replication and pathogenesis in vivo, based on studies showing that the T887A substitution in the phosphorylation site in gB significantly reduced viral replication in the mouse cornea and pathogenic manifestations of herpes stroma keratitis and periocular skin disease in mice (Takahiko Imai, Ken Sagou, and Yasushi Kawaguchi, unpublished observations). (iii) It has been shown that Us3 phosphorylated some or all of the six serines in the UL31 N-terminal region, and this phosphorylation regulated the proper localization of UL31 and UL34 at the nuclear membrane and nuclear egress of nucleocapsids (29). Thus, the molecular basis of HSV-1 Us3 effects in infected cells have been gradually elucidated.However, the Us3 phosphorylation sites in Us3 itself and in gB are not conserved between HSV serotypes (12, 13). The amino acid residues in HSV-2 Us3 and gB corresponding to HSV-1 Us3 Ser-147 and gB Thr-887 are aspartic acid (Asp-147) and alanine (Ala-887), respectively. These results suggest that some HSV-1 Us3 functions, such as regulation of its own catalytic activity and control of gB expression on the cell surface, are not regulated by HSV-2 Us3 or are regulated in a manner(s) different from HSV-1 Us3. In agreement with this suggestion, there is a marked difference between HSV-1 and HSV-2 virulence in mice following intracerebral infection, with the HSV-1 Us3 null mutant being >10⁴-fold less virulent than the parent wild-type virus (25), while the HSV-2 Us3 null mutant was only ∼10-fold less virulent (33). Although these results were from different reports and the mouse strains used in the studies were different, they indicate that some HSV-1 Us3 functions are different from those of HSV-2 Us3.Therefore, we investigated differences in the biological properties of HSV-1 and HSV-2 Us3 protein kinases. It was of particular interest to examine whether Asp-147 in HSV-2 Us3 is required for its own kinase activity, since it is well established that acidic amino acids such as Asp or glutamic acid sometimes mimic the negative charges produced by phosphorylation (29, 46). In the present study, using a genetic manipulation system of HSV-2 with our newly constructed HSV-2 bacterial artificial chromosome (BAC) clone, we have shown that HSV-2 Us3 exhibited marked differences from HSV-1 Us3 in its catalytic functions, including the regulation of UL31/UL34 localization, nuclear egress of nucleocapsids, cell surface expression of gB, and virulence in mice. We also found that Asp-147 in HSV-2 Us3 was critical for its kinase activity, raising a possibility that the activity of Us3 kinases was regulated differently in HSV-1 and HSV-2.     

Type-Common CP-1 Antigen of Herpes Simplex Virus Is Associated with a 59,000-Molecular-Weight Envelope Glycoprotein

The CP-1 antigen of herpes simplex virus type 1 (HSV-1) is a glycoprotein found in the soluble portion of infected cells, in detergent extracts of infected cell membranes, and in the envelope of purified virus. Antisera were prepared against a further purified form of CP-1 prepared from HSV soluble antigen mix; a glycoprotein, gp52, isolated from detergent-treated infected cells; and detergent extracts of purified virus. Each of the antisera reacted with CP-1 to give a single immunoprecipitin band of identity, and each antiserum neutralized the infectivity of HSV-1 and HSV-2. Our results suggested that the type-common determinants involved in the stimulation of neutralizing antibody resided on a 52,000-molecular-weight (52K) glycoprotein. The envelope of HSV contains several glycoproteins: one component at 59K and a complex of two or three components at 130K, none of which corresponds in molecular weight to gp52. Using the antisera as immunological probes, we performed pulse-chase experiments with [(35)S]methionine-labeled HSV-1-infected cells and followed the disposition of the glycoproteins during the infectious cycle. Each antiserum immunoprecipitated a (35)S-labeled 52K protein from lysates of cells pulse-labeled at 5 h after infection. By 10 h, the label was chased into a 59K protein also precipitable by each of the three antisera. The results suggest that gp52 is a precursor of gp59 and that the latter corresponds in molecular weight to one of the major glycoproteins of the virion envelope.     

Low pH-Induced Conformational Change in Herpes Simplex Virus Glycoprotein B

Herpesviruses can enter host cells using pH-dependent endocytosis pathways in a cell-specific manner. Envelope glycoprotein B (gB) is conserved among all herpesviruses and is a critical component of the complex that mediates membrane fusion and entry. Here we demonstrate that mildly acidic pH triggers specific conformational changes in herpes simplex virus (HSV) gB. The antigenic structure of gB was specifically altered by exposure to low pH both in vitro and during entry into host cells. The oligomeric conformation of gB was altered at a similar pH range. Exposure to acid pH appeared to convert virion gB into a lower-order oligomer. The detected conformational changes were reversible, similar to those in other class III fusion proteins. Exposure of purified, recombinant gB to mildly acidic pH resulted in similar changes in conformation and caused gB to become more hydrophobic, suggesting that low pH directly affects gB. We propose that intracellular low pH induces alterations in gB conformation that, together with additional triggers such as receptor binding, are essential for virion-cell fusion during herpesviral entry by endocytosis.Herpes simplex virus (HSV) is an important human pathogen, causing significant morbidity and mortality worldwide. HSV enters host cells by fusion of the viral envelope with either an endosomal membrane (38) or the plasma membrane (63). The entry pathway taken is thought to be determined by both virus (17, 45) and host cell (4, 17, 35, 39, 45) factors. Based on experiments with lysosomotropic agents, which elevate the normally low pH of endosomes, acidic pH has been implicated in the endocytic entry of HSV into several cell types, including human epithelial cells (37). Low pH has also recently been implicated in cell infection by several other human and veterinary herpesviruses (1, 21, 26, 47). The mechanistic role of endosomal pH in herpesvirus entry into cells is not known.Herpesviruses are a paradigm for membrane fusion mediated by a complex of several glycoproteins. We have proposed that HSV likely encodes machinery to mediate both pH-dependent and pH-independent membrane fusion reactions. Envelope glycoproteins glycoprotein B (gB) and gD and the heterodimer gH-gL are required for both pH-independent and pH-dependent entry pathways (11, 22, 30, 39, 46). Interaction of gD with one of its cognate receptors is an essential trigger for membrane fusion and entry (13, 52), regardless of the cellular pathway. However, engagement of a gD receptor is not sufficient for fusion, and at least one additional unknown trigger involving gB or gH-gL is likely necessary. gB is conserved among all herpesviruses, and in all cases studied to date, it plays roles in viral entry, including receptor binding and membrane fusion. The crystal structure of an ectodomain fragment of HSV type 1 (HSV-1) gB is an elongated, rod-like structure containing hydrophobic internal fusion loops (28). This structure bears striking architectural homology to the low pH, postfusion form of G glycoprotein from vesicular stomatitis virus (VSV-G) (43). Both the gB and G structures have features of class I and class II fusion proteins and are thus designated class III proteins (57).During entry of the majority of virus families, low pH acts directly on glycoproteins to induce membrane fusion (60). In some cases, the low pH trigger is not sufficient, or it plays an indirect role. For example, host cell proteases, such as cathepsins D and L, require intravesicular low pH to cleave Ebola virus and severe acute respiratory syndrome (SARS) glycoproteins to trigger fusion (14, 51).We investigated the role of low pH in the molecular mechanism of herpesviral entry. The results suggest that mildly acidic pH, similar to that found within endosomes, triggers a conformational change in gB. We propose that, together with other cellular cues such as receptor interaction, intracellular low pH can play a direct activating role in HSV membrane fusion and entry.     

Phosphorylation of Vesicular Stomatitis Virus In Vivo and In Vitro

The structural protein, NS, of purified vesicular stomatitis virus (VSV) is a phosphoprotein. In infected cells phosphorylated NS is found both free in the cytoplasm and as part of the viral ribonucleoprotein (RNP) complex containing both the 42S RNA and the structural proteins L, N, and NS, indicating that phosphorylation occurs as an early event in viral maturation. VSV contains an endogenous protein kinase activity, probably of host region, which catalyzes the in vitro phosphorylation of the viral proteins NS, M, and L, but not of N or G. The phosphorylated sites on NS appear to be different in the in vivo and in vitro reactions, and are differentially sensitive to alkaline phosphatase. After removal of the membrane components of purified VSV with a dextran-polyethylene glycol two-phase separation, the kinase activity remains tightly associated with the viral RNP. However, viral RNP isolated from infected cells shows only a small amount of kinase activity. The protein kinase enzyme appears to be a cellular contaminant of purified VSV because an activity from the uninfected cell extract can phosphorylate in vitro the dissociated viral proteins NS and M. The virion-associated activity may be derived either from the cytoplasm or the plasma membrane of the host cell since both of these cellular components contain protein kinase activity similar to that found in purified VSV.     

Tetherin Restricts Herpes Simplex Virus 1 and Is Antagonized by Glycoprotein M

Tetherin is a broadly active antiviral effector that works by tethering nascent enveloped virions to a host cell membrane, thus preventing their release. In this study, we demonstrate that herpes simplex virus 1 (HSV-1) is targeted by tetherin. We identify the viral envelope glycoprotein M (gM) as having moderate anti-tetherin activity. We show that gM but not gB or gD efficiently removes tetherin from the plasma membrane and can functionally substitute for the human immunodeficiency virus type 1 (HIV-1) Vpu protein, the prototypic viral tetherin antagonist, in rescuing HIV-1 release from tetherin-expressing cells. Our data emphasize that tetherin is a broadly active antiviral effector and contribute to the emerging hypothesis that viruses must suppress or evade an array of host cell countermeasures in order to establish a productive infection.     

Processing of Lagging-Strand Intermediates In Vitro by Herpes Simplex Virus Type 1 DNA Polymerase

The processing of lagging-strand intermediates has not been demonstrated in vitro for herpes simplex virus type 1 (HSV-1). Human flap endonuclease-1 (Fen-1) was examined for its ability to produce ligatable products with model lagging-strand intermediates in the presence of the wild-type or exonuclease-deficient (exo⁻) HSV-1 DNA polymerase (pol). Primer/templates were composed of a minicircle single-stranded DNA template annealed to primers that contained 5′ DNA flaps or 5′ annealed DNA or RNA sequences. Gapped DNA primer/templates were extended but not significantly strand displaced by the wild-type HSV-1 pol, although significant strand displacement was observed with exo⁻ HSV-1 pol. Nevertheless, the incubation of primer/templates containing 5′ flaps with either wild-type or exo⁻ HSV-1 pol and Fen-1 led to the efficient production of nicks that could be sealed with DNA ligase I. Both polymerases stimulated the nick translation activity of Fen-1 on DNA- or RNA-containing primer/templates, indicating that the activities were coordinated. Further evidence for Fen-1 involvement in HSV-1 DNA synthesis is suggested by the ability of a transiently expressed green fluorescent protein fusion with Fen-1 to accumulate in viral DNA replication compartments in infected cells and by the ability of endogenous Fen-1 to coimmunoprecipitate with an essential viral DNA replication protein in HSV-1-infected cells.Herpes simplex virus type 1 (HSV-1), the prototypic member of the family of Herpesviridae and that of the alphaherpesviridae subfamily, has served as the model for understanding the replication of herpesvirus genomes during lytic virus replication (29). The 152-kbp genome of herpes simplex virus type 1 (HSV-1) possesses approximately 85 genes, 7 of which have been shown to be necessary and sufficient for viral DNA replication within host cells (reviewed in references 5 and 38). These seven genes encode a DNA polymerase (pol) and its processivity factor (UL42), a heterotrimeric complex containing a DNA helicase (UL5), primase (UL52), and noncatalytic accessory protein (UL8), a single-stranded DNA binding protein (infected cell protein 8 [ICP-8]), and an origin binding protein with DNA helicase activity (UL9). There is strong evidence in support of the circularization of the linear virion DNA shortly after entry, and DNA replication then is thought to initiate at one or more of the three redundant origins of replication (29, 38). At least in the earliest stages of viral DNA replication, UL9 protein is required, presumably to bind to and unwind the DNA and to attract the other DNA replication proteins (29, 38). The electron microscopic examination of pulse-labeled replicating HSV-1 DNA indicates the presence of lariats, eye-forms, and D-forms (21), which is consistent with bidirectional theta-like replication from origins. To date, however, no biochemical assay has demonstrated origin-dependent DNA replication in vitro. However, in the absence of UL9, the other six HSV DNA replication proteins can support initiation and replication from a circular single-stranded DNA (ssDNA) template in an origin-independent fashion (15, 26), resembling the rolling-circle mode of replication thought to occur during the later stages of viral replication.Although nicks and small gaps have been observed in isolated replicating and virion DNA (38), the evidence for bidirectional duplex synthesis, the rapid rate of viral DNA replication, and the absence of long stretches of ssDNA in replicating and mature DNA isolated from HSV-1-infected cells suggest that leading- and lagging-strand synthesis are closely coordinated in vivo. Falkenberg et al. (15) used a minicircle DNA template with a strand bias and the six essential HSV-1 DNA replication proteins needed for rolling circle replication to demonstrate lagging-strand synthesis in vitro. However, replication from the parental strand template (leading-strand synthesis) was more efficient than synthesis from the complementary-strand template (lagging-strand synthesis). These results suggest the possibility that one or more host functions required for efficient lagging-strand synthesis or for its close coordination with leading-strand synthesis is missing in such in vitro systems.Although leading- and lagging-strand syntheses share many of the same requirements for bulk DNA synthesis, lagging-strand synthesis is a more complex process. Because the direction of polymerization of lagging-strand intermediates is opposite the direction of replication fork movement, lagging-strand synthesis requires that priming and extension occur many times to produce discontinuous segments called Okazaki fragments (reviewed in reference 25). Okazaki fragments need to be processed to remove the RNA primer, to fill in the area previously occupied by the RNA, and to seal the remaining nick between fragments, all of which must occur efficiently, accurately, and completely. Failure to do so would result in the accumulation of DNA breaks, multiple mutations, delayed DNA replication, and/or cell death (16, 61).In eukaryotes, what is currently known regarding the process of lagging-strand synthesis is based on genetic and biochemical studies with Saccharomyces cerevisiae and on in vitro reconstitution studies to define the mammalian enzymes required for simian virus 40 (SV40) T-antigen-dependent DNA replication (17, 37, 44, 57, 58). These studies have revealed that the extension of a newly synthesized Okazaki fragment DNA with pol δ causes the strand displacement of the preceding fragment to produce a 5′ flap (25). Results suggest that flap endonuclease 1 (Fen-1) is the activity responsible for the removal of the bulk of the 5′ flaps generated (1, 44, 48), although dna2 protein may facilitate the removal of longer flaps coated with the ssDNA binding protein complex (2, 44). In addition, the overexpression of exonuclease I can partially compensate for the loss of Fen-1 function in yeast (24, 51). For the proper processing of lagging-strand intermediates, the entire 5′ flap and all of the RNA primer need to be removed, and the gap must be filled to achieve a ligatable nick. DNA ligase I has been shown to be the enzyme involved in sealing Okazaki fragments in yeast and in humans (3, 31, 50, 56, 57). DNA ligase I requires a nick in which there is a 5′ phosphate on one end and a 3′ hydroxyl linked to a deoxyribose sugar entity on the other, and it works poorly in the presence of mismatches (54). The close coordination of Fen-1 and DNA ligase I activities for Okazaki fragment processing is facilitated by the interactions of these proteins with proliferating cell nuclear antigen (PCNA), the processivity factor for pol δ and ɛ (6, 30, 32, 46, 52, 53).HSV-1 does not appear to encode a protein with DNA ligase activity or one that can specifically cleave 5′ flaps, although it does encode a 5′-to-3′ exonuclease activity (UL12 [10, 20]) and a 3′-to-5′ exonuclease activity that is part of the HSV-1 pol catalytic subunit (27). As for most eukaryotes, RNA primers are essential for HSV-1 DNA synthesis, as demonstrated by the presence of oligoribonucleotides in replicating DNA in vivo (4), by the well-characterized ability of the UL52 protein in complex with the UL5 helicase activity to synthesize oligoribonucleotide primers on ssDNA in vitro (11, 13), and by the requirement of the conserved catalytic residues in the UL52 primase in vitro and in HSV-1-infected cells (14, 26). It is the strand displacement activity of pol δ that produces the 5′ flaps that are key to the removal of RNA primers during Okazaki fragment processing (6, 25). However, we previously demonstrated that wild-type HSV-1 DNA polymerase possesses poor strand displacement activity (62), in contrast to mammalian DNA pol δ (25). Thus, it is not apparent how RNA primers would be removed when encountered by HSV-1 pol during HSV-1 lagging-strand synthesis or how such intermediates would be processed.We wished to test the hypothesis that the nick translation activity of mammalian Fen-1 could function in collaboration with HSV-1 pol to facilitate the proper removal of RNA primers and/or short flaps to produce the ligatable products required for Okazaki fragment processing. In this report, we have examined the ability of wild-type and exonuclease-deficient (exo⁻) HSV-1 pol, which differ in their respective strand displacement activities, to extend model lagging-strand substrates in the presence or absence of mammalian Fen-1. Our results demonstrate that both wild-type and exo⁻ HSV-1 pol can cooperate with and enhance Fen-1 activity to achieve a ligatable nick in vitro. Moreover, colocalization and coimmunoprecipitation studies reveal a physical association of Fen-1 with HSV-1 DNA replication proteins, supporting a model for the involvement of Fen-1 in HSV-1 DNA replication.     

Herpes Simplex Virus 1 Protein Kinase Us3 Phosphorylates Viral dUTPase and Regulates Its Catalytic Activity in Infected Cells

Us3 is a serine-threonine protein kinase encoded by herpes simplex virus 1 (HSV-1). In this study, a large-scale phosphoproteomic analysis of titanium dioxide affinity chromatography-enriched phosphopeptides from HSV-1-infected cells using high-accuracy mass spectrometry (MS) and subsequent analyses showed that Us3 phosphorylated HSV-1-encoded dUTPase (vdUTPase) at serine 187 (Ser-187) in HSV-1-infected cells. Thus, the following observations were made. (i) In in vitro kinase assays, Ser-187 in the vdUTPase domain was specifically phosphorylated by Us3. (ii) Phosphorylation of vdUTPase Ser-187 in HSV-1-infected cells was detected by phosphate-affinity polyacrylamide gel electrophoresis analyses and was dependent on the kinase activity of Us3. (iii) Replacement of Ser-187 with alanine (S187A) in vdUTPase and an amino acid substitution in Us3 that inactivated its kinase activity significantly downregulated the enzymatic activity of vdUTPase in HSV-1-infected cells, whereas a phosphomimetic substitution at vdUTPase Ser-187 restored the wild-type enzymatic activity of vdUTPase. (iv) The vdUTPase S187A mutation as well as the kinase-dead mutation in Us3 significantly reduced HSV-1 replication in human neuroblastoma SK-N-SH cells at a multiplicity of infection (MOI) of 5 but not at an MOI of 0.01, whereas the phosphomimetic substitution at vdUTPase Ser-187 restored the wild-type viral replication at an MOI of 5. In contrast, these mutations had no effect on HSV-1 replication in Vero and HEp-2 cells. Collectively, our results suggested that Us3 phosphorylation of vdUTPase Ser-187 promoted HSV-1 replication in a manner dependent on cell types and MOIs by regulating optimal enzymatic activity of vdUTPase.     

Contributions of Herpes Simplex Virus 1 Envelope Proteins to Entry by Endocytosis

Herpes simplex virus (HSV) proteins specifically required for endocytic entry but not direct penetration have not been identified. HSVs deleted of gE, gG, gI, gJ, gM, UL45, or Us9 entered cells via either pH-dependent or pH-independent endocytosis and were inactivated by mildly acidic pH. Thus, the required HSV glycoproteins, gB, gD, and gH-gL, may be sufficient for entry regardless of entry route taken. This may be distinct from entry mechanisms employed by other human herpesviruses.     

单纯疱疹病毒Ⅰ型糖蛋白D在酵母中的表达

从提取的HSV-1基因组中扩增得到编码gD蛋白胞外区1～314aa的基因gDt,将其插入毕赤酵母表达质粒pPIC9K的醇氧化酶(AOX1)启动子下游,构建携带gDt的重组载体,经电转化GS115菌株和G418筛选,得到了高效分泌表达gD蛋白的毕赤酵母菌株,表达量达到250mg/L,该目的蛋白可被gD单抗(1-I-9)特异性识别.表达产物经离子交换、金属螯合、分子筛柱层析纯化后得到纯度较高的重组蛋白.重组gD蛋白免疫BALB/c小鼠可诱生一定水平的特异性抗体,表明该蛋白具有较好的免疫原性,能够诱导小鼠产生体液免疫应答.     

单纯疱疹病毒Ⅰ型糖蛋白D在酵母中的表达

从提取的HSV-1基因组中扩增得到编码gD蛋白胞外区1～314aa的基因gDt,将其插入毕赤酵母表达质粒pPIC9K的醇氧化酶(AOX1)启动子下游,构建携带gDt的重组载体,经电转化GS115菌株和G418筛选,得到了高效分泌表达gD蛋白的毕赤酵母菌株,表达量达到250mg/L,该目的蛋白可被gD单抗(1-I-9)特异性识别。表达产物经离子交换、金属螯合、分子筛柱层析纯化后得到纯度较高的重组蛋白。重组gD蛋白免疫BALB/c小鼠可诱生一定水平的特异性抗体,表明该蛋白具有较好的免疫原性,能够诱导小鼠产生体液免疫应答。     

In Vivo Immune Evasion Mediated by the Herpes Simplex Virus Type 1 Immunoglobulin G Fc Receptor

Herpes simplex virus (HSV) glycoproteins gE and gI form an immunoglobulin G (IgG) Fc receptor (FcγR) that binds the Fc domain of human anti-HSV IgG and inhibits Fc-mediated immune functions in vitro. gE or gI deletion mutant viruses are avirulent, probably because gE and gI are also involved in cell-to-cell spread. In an effort to modify FcγR activity without affecting other gE functions, we constructed a mutant virus, NS-gE₃₃₉, that has four amino acids inserted into gE within the domain homologous to mammalian IgG FcγRs. NS-gE₃₃₉ expresses gE and gI, is FcγR⁻, and does not participate in antibody bipolar bridging since it does not block activities mediated by the Fc domain of anti-HSV IgG. In vivo studies were performed with mice because the HSV-1 FcγR does not bind murine IgG; therefore, the absence of an FcγR should not affect virulence in mice. NS-gE₃₃₉ causes disease at the skin inoculation site comparably to wild-type and rescued viruses, indicating that the FcγR⁻ mutant virus is pathogenic in animals. Mice were passively immunized with human anti-HSV IgG and then infected with mutant or wild-type virus. We postulated that the HSV-1 FcγR should protect wild-type virus from antibody attack. Human anti-HSV IgG greatly reduced viral titers and disease severity in NS-gE₃₃₉-infected animals while having little effect on wild-type or rescued virus. We conclude that the HSV-1 FcγR enables the virus to evade antibody attack in vivo, which likely explains why antibodies are relatively ineffective against HSV infection.     

Pathogenicity of Glycoprotein C-Deficient Herpes Simplex Virus 1 Strain TN-1 Which Encodes Truncated Glycoprotein C

A clinical isolate of herpes simplex virus 1 (TN-1) from a stromal keratitis patient was found to be defective in the glycoprotein C (gC) gene (UL44), thus resulting in the production of truncated gC upon infection. To study the pathogenetic role of truncated gC, we prepared a recombinant LTN-8 derived from TN-1 with deletions of the 1.5 kilobase pairs of the gC gene including the initiation codon. A penetration assay revealed LTN-8 to be less efficient in its penetration ability than TN-1, the laboratory strain KOS and RTN-1-20-3, a recombinant derived from TN-1 with the KOS gC gene. The penetration of LTN-8 was facilitated by the addition of TN-1-infected culture medium. TN-1 virus preparations had no hemagglutinating activity. However, the animals infected with TN-1 did develop hemagglutination inhibition (HI) antibodies. The LTN-8-infected animals did not develop HI antibodies. The pathogenicity in BALB/c mice following either corneal, intraperitoneal or intracerebral inoculation did not significantly differ among TN-1, RTN-1-20-3 or LTN-8. Our results indicate that truncated gC was sufficient for the induction of HI antibodies and was also able to facilitate penetration in vitro. Although truncated gC might be a virulence factor acting as a decoy, both truncated gC and intact gC had little effect on the outcome following intracerebral, intraperitoneal or corneal inoculation.     

The Herpes Simplex Virus 1 Us11 Protein Inhibits Autophagy through Its Interaction with the Protein Kinase PKR

Autophagy is now known to be an essential component of host innate and adaptive immunity. Several herpesviruses have developed various strategies to evade this antiviral host defense. Herpes simplex virus 1 (HSV-1) blocks autophagy in fibroblasts and in neurons, and the ICP34.5 protein is important for the resistance of HSV-1 to autophagy because of its interaction with the autophagy machinery protein Beclin 1. ICP34.5 also counteracts the shutoff of protein synthesis mediated by the double-stranded RNA (dsRNA)-dependent protein kinase PKR by inhibiting phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α) in the PKR/eIF2α signaling pathway. Us11 is a late gene product of HSV-1, which is also able to preclude the host shutoff by direct inhibition of PKR. In the present study, we unveil a previously uncharacterized function of Us11 by demonstrating its antiautophagic activity. We show that the expression of Us11 is able to block autophagy and autophagosome formation in both HeLa cells and fibroblasts. Furthermore, immediate-early expression of Us11 by an ICP34.5 deletion mutant virus is sufficient to render the cells resistant to PKR-induced and virus-induced autophagy. PKR expression and the PKR binding domain of Us11 are required for the antiautophagic activity of Us11. However, unlike ICP34.5, Us11 did not interact with Beclin 1. We suggest that the inhibition of autophagy observed in cells infected with HSV-1 results from the activity of not only ICP34.5 on Beclin 1 but also Us11 by direct interaction with PKR.     

Fusion-Deficient Insertion Mutants of Herpes Simplex Virus Type 1 Glycoprotein B Adopt the Trimeric Postfusion Conformation

Glycoprotein B (gB) enables the fusion of viral and cell membranes during entry of herpesviruses. However, gB alone is insufficient for membrane fusion; the gH/gL heterodimer is also required. The crystal structure of the herpes simplex virus type 1 (HSV-1) gB ectodomain, gB730, has demonstrated similarities between gB and other viral fusion proteins, leading to the hypothesis that gB is a fusogen, presumably directly involved in bringing the membranes together by refolding from its initial or prefusion form to its final or postfusion form. The only available crystal structure likely represents the postfusion form of gB; the prefusion form has not yet been determined. Previously, a panel of HSV-1 gB mutants was generated by using random 5-amino-acid-linker insertion mutagenesis. Several mutants were unable to mediate cell-cell fusion despite being expressed on the cell surface. Mapping of the insertion sites onto the crystal structure of gB730 suggested that several insertions might not be accommodated in the postfusion form. Thus, we hypothesized that some insertion mutants were nonfunctional due to being “trapped” in a prefusion form. Here, we generated five insertion mutants as soluble ectodomains and characterized them biochemically. We show that the ectodomains of all five mutants assume conformations similar to that of the wild-type gB730. Four mutants have biochemical properties and overall structures that are indistinguishable from those of the wild-type gB730. We conclude that these mutants undergo only minor local conformational changes to relieve the steric strain resulting from the presence of 5 extra amino acids. Interestingly, one mutant, while able to adopt the overall postfusion structure, displays significant conformational differences in the vicinity of fusion loops, relative to wild-type gB730. Moreover, this mutant has a diminished ability to associate with liposomes, suggesting that the fusion loops in this mutant have decreased functional activity. We propose that these insertions cause a fusion-deficient phenotype not by preventing conversion of gB to a postfusion-like conformation but rather by interfering with other gB functions.Herpes simplex virus type 1 (HSV-1) is the prototype of the diverse herpesvirus family that includes many notable human pathogens (26). In addition to the icosahedral capsid and the tegument that surround its double-stranded DNA genome, herpesviruses have an envelope—an outer lipid bilayer—bearing a number of surface glycoproteins. During infection, HSV-1 must fuse its envelope with a cellular membrane in order to deliver the capsid into a target host cell. Among its viral glycoproteins, only glycoprotein C (gC), gB, gD, gH, and gL participate in this entry process, and only the last four are required for fusion (28). Although gD is found only in alphaherpesviruses, all herpesviruses encode gB, gH, and gL, which constitute their core fusion machinery. Of these three proteins, gB is the most highly conserved.We recently determined the crystal structure of a nearly full-length ectodomain of HSV-1 gB, gB730 (18). The crystal structure of the ectodomain of gB from Epstein-Barr virus, another herpesvirus, has also been subsequently determined (4). The two structures showed similarities between gB and other viral fusion proteins, in particular, G from an unrelated vesicular stomatitis virus (VSV) (25), leading to the hypothesis that gB is a fusogen, presumably directly involved in bringing the viral and host cell membranes together to enable their fusion. However, gB alone is known to be insufficient for membrane fusion; the gH/gL heterodimer is also required. This insufficiency raises the question of exactly how gB functions during viral entry. Answering this question is critical for understanding the complex mechanism that herpesviruses use to enter their host cells.In acting as a viral fusogen, gB must undergo dramatic conformational changes, refolding through a series of conformational intermediates from its initial, or prefusion form, to its final, or postfusion form (15). These conformational changes are not only necessary to bring the two membranes into proximity; they are also thought to provide the energy for the fusion process. The prefusion form corresponds to the protein present on the viral surface prior to initiation of fusion. The postfusion form represents the protein after fusion of the viral and host cell membranes. The available gB structure likely represents its postfusion form, since it shares more in common with the postfusion rather than the prefusion structure of vesicular stomatitis virus (VSV) G (3, 17). However, the prefusion form has not yet been characterized.Recently, a panel of gB mutants was generated by using random linker-insertion mutagenesis (21). Of these mutants, 16 were particularly interesting because they were nonfunctional in cell-cell fusion assays despite being expressed on the cell surface at levels that indicate proper folding for transport. These observations suggested that each insertion somehow interfered with gB function. Insertions in 12 of these mutants are located within the available structure of the gB ectodomain, which allowed Lin and Spear to analyze their locations (21).The most prominent examples of such nonfunctional mutants are two mutants with insertions after residues I185 or E187, henceforth referred to as “cavity mutants” because both I185 and E187 point into a cavity inside the gB trimer (Fig. 1B and D). Although this cavity might accommodate a single 5-amino-acid insertion, it “is not large enough to accommodate three 5-amino-acid insertions” (21) that would be present in the trimer (one insertion per protomer).Open in a separate windowFIG. 1.Location of the insertion sites in the sequence of gB and the structure of the postfusion form of its ectodomain. (A) Linear diagram of the full-length gB with functional domains highlighted (as in reference 18). Domain I is shown in cyan, domain II in green, domain III in yellow, domain IV in orange, domain V in red, and the disordered region between domains II and III in purple. Regions absent from the crystal structure of gB730 are shown in gray. Sequences in the region of 5-amino-acid insertions (residues 181 to 190 and residues 661 to 680) are shown in black. Arrows mark the locations of 5-amino-acid insertions, shown as red text. (B) Crystal structure of gB730 (18). Residues preceding the 5-amino-acid insertions in mutants studied here are shown as spheres colored by domain, consistent with panel A. Boxes delineate the hinge region, enlarged in panel C, and the cavity region, enlarged in panel D. (C) Close-up view of the hinge region shown in molecular surface representation, with residues 663 to 675 displayed as sticks. Hydrophobic residues are colored orange. Residues preceding the 5-amino-acid insertions in mutants studied here are labeled with asterisks; remaining labels correspond to additional hydrophobic residues in the 663-675 region. (D) Enlarged view of the cavity region. Residues that line the cavity and are not solvent exposed are colored magenta. Residue E187 of each protomer is colored teal and shown as spheres. Fusion loops for two protomers are marked with asterisks; the third pair of fusion loops lies behind the crystal structure and is not visible. Panels B, C, and D were made by using Pymol (http://www.pymol.org/).Five other nonfunctional mutants have insertions after residues D663, T665, V667, I671, or L673, respectively. We refer to them as “hinge mutants.” These residues lie in the region located between domains IV and V, which has been termed the hinge region because it may play an important role during the conformational transition from the prefusion to the postfusion form (17). Lin and Spear proposed that insertions following these residues “would likely affect hinge regions” (21), with the implication that they may prevent gB from refolding into the postfusion conformation. Our analysis suggested that insertions after these residues could, perhaps, be sterically accommodated in the structure but would probably be energetically unfavorable by causing several buried hydrophobic side chains in the 665-673 region, such as F670, I671, and L673, to become exposed (Fig. 1B and C).In light of these observations, we hypothesized that the insertion mutants are “trapped” in a prefusion form. We decided to test this hypothesis by determining whether the ectodomain of gB containing one of these insertion mutations is able to assume the conformation seen in the crystal structure of the wild-type gB ectodomain, which we are referring to as the likely postfusion conformation. For this purpose, we chose one cavity mutant, containing an insertion after E187, and four hinge mutants, containing insertions after T665, V667, I671, or L673, respectively. We chose to test four hinge mutants because structure analysis suggested to us that insertions following the respective residues might not affect the structure in precisely the same way. We expressed the soluble ectodomain of each mutant by using a baculovirus expression system and characterized the purified proteins by using biochemical and biophysical methods. Surprisingly, we found that the ectodomains of all five mutants assume a conformation similar to that of the wild-type gB ectodomain. The four hinge mutants had biochemical properties and overall three-dimensional structures that were indistinguishable from those of the wild-type gB ectodomain. We conclude that these mutants undergo only minor local conformational changes to relieve the steric strain resulting from the presence of 5 extra amino acids. Interestingly, the cavity mutant, while able to adopt the overall postfusion structure, still displayed significant conformational differences relative to wild-type gB. Because these conformational differences are in the vicinity of fusion loops, we conclude that the fusion loops in this mutant have decreased functional activity.