PKR-Dependent Xenophagic Degradation of Herpes Simplex Virus Type 1

The lysosomal pathway of autophagy is the major catabolic mechanism for degrading long-lived cellular proteins and cytoplasmic organelles. Recent studies have also shown that autophagy (xenophagy) may be used to degrade bacterial pathogens that invade intracellularly. However, it is not yet known whether xenophagy is a mechanism for degrading viruses. Previously, we showed that autophagy induction requires the antiviral eIF2alpha kinase signaling pathway (including PKR and eIF2alpha) and that this function ofeIF2alpha kinase signaling is antagonized by the herpes simplex virus (HSV-1) neurovirulence gene product, ICP34.5. Here, we show quantitative morphologic evidence of PKR-dependent xenophagic degradation of herpes simplex virions and biochemical evidence of PKR and eIF2alpha-dependent degradation of HSV-1 proteins, both of which are blocked by ICP34.5. Together, these findings indicate that xenophagy degrades HSV-1 and that this cellular function is antagonized by the HSV-1 neurovirulence gene product, ICP34.5. Thus, autophagy-related pathways are involved in degrading not only cellular constituents and intracellular bacteria, but also viruses.     

The Genome Sequence of Herpes Simplex Virus Type 2

The genomic DNA sequence of herpes simplex virus type 2 (HSV-2) strain HG52 was determined as 154,746 bp with a G+C content of 70.4%. A total of 74 genes encoding distinct proteins was identified; three of these were each present in two copies, within major repeat elements of the genome. The HSV-2 gene set corresponds closely with that of HSV-1, and the HSV-2 sequence prompted several local revisions to the published HSV-1 sequence (D. J. McGeoch, M. A. Dalrymple, A. J. Davison, A. Dolan, M. C. Frame, D. McNab, L. J. Perry, J. E. Scott, and P. Taylor, J. Gen. Virol. 69:1531–1574, 1988). No compelling evidence for the existence of any additional protein-coding genes in HSV-2 was identified.The complete 152-kbp genomic DNA sequence of herpes simplex virus type 1 (HSV-1) was published in 1988 (56) and since then has been very widely employed in a great range of research on HSV-1. Additionally, results from this most studied member of the family Herpesviridae have fed powerfully into research on other herpesviruses. In contrast, although a substantial number of individual gene sequences have been determined for the other HSV serotype, HSV-2, the complete genome sequence for this virus has not been available hitherto. In this paper we report the sequence of the genome of HSV-2, strain HG52.At a gross level the 155-kbp genome of HSV-2 is viewed as consisting of two extended regions of unique sequence (U_L and U_S), each of which is bounded by a pair of inverted repeat elements (TR_L-IR_L and IR_S-TR_S) (17, 66) (Fig. ​(Fig.1).1). There is a directly repeated sequence of some 254 bp at the genome termini (the a sequence), with one or more copies in the opposing orientation (the a′ sequence) at the internal joint between IR_L and IR_S (21). U_L plus its flanking repeats is termed the long (L) region, and U_S with its flanking repeats is termed the short (S) region. In individual molecules of HSV-2 DNA, the L and S components may be linked with each in either orientation, so that DNA preparations contain four sequence-orientation isomers, one of which is defined as the prototype (66). The sequences of the terminal and internal copies of R_L and of R_S are considered to be indistinguishable. Open in a separate windowFIG. 1Overall organization of the genome of HSV-2. The linear double-stranded DNA is represented, with the scale at the top. The unique portions of the genome (U_L and U_S) are shown as heavy solid lines, and the major repeat elements (TR_L, IR_L, IR_S, and TR_S) are shown as open boxes. For each pair of repeats the two copies are in opposing orientations. As indicated, TR_L, U_L, and IR_L are regarded as comprising the L region, and IR_S, U_S, and TR_S are regarded as comprising the S region. Plasmid-cloned fragments used for sequence determination are indicated at the bottom: BamHI and HindIII fragments are indicated by B and H, respectively, followed by individual fragment designations in lowercase; KH and HK indicate KpnI/HindIII fragments as described in the text.This paper presents properties of the HSV-2 DNA sequence and our present understanding of its content of protein-coding genes and other elements. We are also interested in comparative analysis of the HSV-1 and HSV-2 genomes to examine processes of molecular evolution which have occurred since the two species diverged, and we intend to pursue this topic in a separate paper.     

Caspase 9 Is Essential for Herpes Simplex Virus Type 2-Induced Apoptosis in T Cells

Herpes simplex virus type 2 (HSV-2) induces apoptosis in T cells by a caspase-dependent mechanism. Apoptosis can occur via extrinsic (death receptor) and/or intrinsic (mitochondrial) pathways. Here, we show that the initiator caspase for the intrinsic pathway is activated in T cells following HSV-2 exposure. To determine the respective contributions of intrinsic and extrinsic pathways, we assessed apoptosis in Jurkat cells that are deficient in caspase 8 or Fas-associating protein with death domain (FADD) for the extrinsic pathway and in cells deficient in caspase 9 for the intrinsic pathway. Our results indicate HSV-2-induced apoptosis in T cells occurs via the intrinsic pathway.Herpes simplex virus (HSV) reactivation in human results in accumulation and persistence of virus-specific CD4⁺ and CD8⁺ cells at the site of reactivation (15, 16). Despite such immune responses, virus reactivation can occur on >75% of days for some individuals (14). Reactivation of the virus in the midst of continued cell-mediated immunity demonstrates the ability of the virus to circumvent the host''s immune system. HSV antigens were readily detected from T cells isolated from human HSV lesions, indicating that T cells are infected with HSV in vivo (1). In vitro, HSV-infected T cells undergo apoptosis (5-7, 10), and we have additionally demonstrated that apoptosis occurs in Jurkat cells, a T-cell leukemia line, and primary CD4⁺ T lymphocytes isolated from human peripheral blood mononuclear cells following exposure to HSV type 2 (HSV-2)-infected human foreskin fibroblasts (5). These results suggest that induction of T-cell death is a mechanism by which the virus limits the effectiveness of local cell-mediated immunity during reactivation.Since T cells are most likely exposed to HSV-2 via infected epithelial cells in vivo, we examined T-cell apoptosis following exposure to infected fibroblasts in vitro. To evaluate whether HSV-2-exposed primary T cells undergo apoptosis by a caspase-dependent mechanism, we examined activation of caspase 3 in human CD4⁺ cells after exposure to HSV-2-infected fibroblasts. Human foreskin fibroblasts (American Type Culture Collection, Manassas, VA) were mock infected or infected with the HSV-2 HG52 strain at a multiplicity of infection of 5. At 6 h postinfection, CD4⁺ cells were exposed to mock-infected or HSV-2-infected fibroblasts at a ratio of approximately 3:1 (lymphocytes to fibroblasts). CD4⁺ cells were isolated to >95% purity using MACS CD4 microbeads (Miltenyi Biotec, Inc., Auburn, CA) immediately prior to experiments with human peripheral mononuclear cells (Memorial Blood Centers, St. Paul, MN) that were stimulated with phytohemagglutinin (Sigma Aldrich, St. Louis, MO) for 48 h and then maintained with interleukin-2 (Invitrogen, Carlsbad, CA) as previously described (5). After 4 h of incubation, CD4⁺ cells were harvested and maintained in RPMI 1640 supplemented with 10% fetal bovine serum and 0.3 ng/ml interleukin-2. At 24 h and 48 h postexposure, CD4⁺ cells were probed with a fluorescence-labeled antibody against activated caspase 3 (BD Biosciences, San Jose, CA). Data were collected on a FACSCanto (BD Biosciences) and analyzed with FlowJo software (Tree Star, Ashland, OR). Side scatter and fluorescence were used as dual parameters to clearly gate the cell population with activated caspase 3, and an identical gate was applied to all samples within each experiment. As shown in Fig. ​Fig.1,1, the percentage of cells with activated caspase 3 was 23% in HSV-2-exposed CD4⁺ cells compared with 5% in mock-exposed cells at 24 h postexposure. At 48 h, the percentage of cells with activated caspase 3 increased to 39% in HSV-2-exposed cells compared to 8% in mock-exposed cells. These percentages are comparable to those for Jurkat cells in our previous report (19% for virus-exposed cells versus 5% for mock-exposed cells at 24 h postexposure) (5).Open in a separate windowFIG. 1.Activation of caspase 3 in CD4⁺ cells following exposure to mock- or HSV-2-infected fibroblasts. CD4⁺ cells were isolated from human peripheral mononuclear cells and exposed to mock- or HSV-2-infected fibroblasts. At 24 h and 48 h postexposure, cells were probed with an antibody for activated caspase 3 and analyzed by flow cytometry. (A) A representative flow cytometry analysis from three independent experiments is shown. Side scatter was used as a second parameter to identify the cell populations. An identical gate was applied to all samples within each experiment. Increased percentages of cells with caspase 3 activation are seen following exposure to HSV-2-infected fibroblasts compared to those following exposure to mock-infected fibroblasts. (B) Mean percentages of CD4⁺ cells with caspase 3 activation from three independent experiments at 24 h and 48 h postexposure are shown with standard errors bars.Efficient infection of T cells by HSV upon exposure to HSV-infected human fibroblasts via cell-to-cell spread has been recently demonstrated (1). To delineate the relationship between virus infection and apoptosis, Jurkat cells were exposed to mock-infected or HSV-2-infected fibroblasts as described above and then coprobed with antibodies for activated caspase 3 and HSV-2 ICP10. As shown in Fig. ​Fig.2,2, two antibodies demonstrated largely mutually exclusive populations in cells exposed to HSV-2-infected fibroblasts. We obtained similar results with antibodies against other HSV-2 antigens, including glycoprotein B, ICP5, and ICP8 (data not shown). The results suggest that infected cells may be inducing apoptosis in uninfected cells via a bystander effect, similar to what has been proposed for HSV-1-infected activated cytotoxic T cells (11). Alternatively, the activation of apoptosis may result in degradation of viral antigens in infected cells.Open in a separate windowFIG. 2.Expression of HSV-2 antigen and caspase 3 activation in Jurkat cells following exposure to mock- or HSV-2-infected fibroblasts. Jurkat cells were first exposed to mock- or HSV-2-infected fibroblasts and then coprobed with antibodies for HSV-2 ICP10 and activated caspase 3 at 24 h postexposure. A representative flow cytometry analysis from three independent experiments is shown. Each antibody detected largely mutually exclusive cell populations following exposure to HSV-2-infected fibroblasts.Our results demonstrate an activation of caspase 3 in HSV-2-exposed T cells, suggesting a caspase-mediated apoptosis. Previous studies have supported such a mechanism, as caspase inhibitors block virus-induced cell death (5, 10). Caspase-dependent apoptosis can occur via extrinsic (death receptor) and/or intrinsic (mitochondrial) pathways (3). Here, we sought to determine the respective contribution of each pathway in HSV-2-induced T-cell death.To investigate the contribution of individual apoptosis pathways in HSV-2-exposed T cells, we first evaluated mitochondrial membrane potential in CD4⁺ and Jurkat cells following virus exposure. Mitochondria play a major role in the intrinsic apoptosis pathway (2, 3). T cells were exposed to mock-infected or HSV-2-infected fibroblasts as described above and then probed with chloromethyl-X-rosamine (CMXRos; Invitrogen) at 24 h and 48 h postexposure. CMXRos is a lipophilic cationic fluorescent dye whose staining of cells is dependent on negative mitochondrial membrane potential, and a loss of staining indicates a loss of mitochondrial potential (2, 9). As shown in Fig. ​Fig.3,3, a loss of mitochondrial membrane potential was observed in increased percentages of HSV-2-exposed CD4⁺ and Jurkat cells compared to those for mock-exposed cells. The percentage of cells with a loss of mitochondrial membrane potential reached 49% for CD4⁺ cells and 92% for Jurkat cells at 48 h postexposure. The changes in mitochondrial membrane potential in HSV-2-exposed T cells suggest that the intrinsic apoptosis pathway is activated in these cells. We previously reported that Jurkat and CD4⁺ cells were equally susceptible to apoptosis induced by anti-Fas monoclonal antibody (5). A recent report suggests that activated primary T cells are less susceptible to HSV infection than are Jurkat cells (1). Thus, we believe that a lower percentage of CD4⁺ cells with a loss of mitochondrial membrane potential than Jurkat cells is due to lower susceptibility to HSV infection.Open in a separate windowFIG. 3.A loss of mitochondrial membrane potential in CD4⁺ and Jurkat cells following exposure to mock- or HSV-2-infected fibroblasts. CD4⁺ and Jurkat cells were exposed to mock- or HSV-2-infected fibroblasts. At 24 h and 48 h postexposure, cells were probed with chloromethyl-X-rosamine (CMXRos). (A) Increased percentages of CD4⁺ cells with a loss of mitochondrial membrane potential are seen following exposure to HSV-2-infected fibroblasts compared to those seen after mock exposure. Shown are mean percentages of cells with a loss of mitochondrial membrane potential at 24 h and 48 h postexposure, with standard error bars, from three independent experiments. (B) Shown are mean percentages of cells with a loss of mitochondrial membrane potential for Jurkat cells at 24 h and 48 h postexposure, with standard error bars, from three independent experiments.To further define the involvement of intrinsic and extrinsic apoptosis pathways in HSV-2-induced T-cell apoptosis, we investigated whether caspase 8, an initiator protease for the extrinsic pathway, and caspase 9, an initiator protease for the intrinsic pathway, are activated in HSV-2-exposed T cells. CD4⁺ and Jurkat cells were exposed to mock-infected or HSV-2-infected fibroblasts as described above. Cell lysates were made at 24 h postexposure, and immunoblots were performed with 30 to 50 μg of protein per well, as described previously (5), with anti-caspase 8 and anti-caspase 9 antibodies (Cell Signaling, Danvers, MA). A 24-h time point was chosen for the immunoblots in order to minimize the detection of baseline apoptotic activity that can be seen with primary CD4⁺ cells in cultures with a longer incubation time.As shown in Fig. ​Fig.4,4, CD4⁺ cells that were exposed to HSV-2 had an increase in cleaved, activated fragments of caspase 9. No clear differences between mock-infected and HSV-2-exposed CD4⁺ cells were seen for caspase 8 cleavage. In contrast, increases in both cleaved caspase 8 and cleaved caspase 9 were evident in Jurkat cells following virus exposure. Together, these results indicate that caspase 9 is activated in HSV-2-exposed T cells and suggest activation of the intrinsic apoptosis pathway. Although activation of caspase 8 was seen with Jurkat cells, the contribution of the extrinsic pathway remained in doubt for virus-induced apoptosis in CD4⁺ cells.Open in a separate windowFIG. 4.Detection of cleaved caspase 8 and caspase 9 in CD4⁺ and Jurkat cells following exposure to mock- or HSV-2-infected fibroblasts. Lysates from cells exposed to mock- or HSV-2-infected fibroblasts were assayed for uncleaved and cleaved forms of caspase 8 and caspase 9 by immunoblotting at 24 h postexposure. Representative immunoblots from more than three independent experiments are shown. Blots demonstrate increases in cleaved caspase 9 for CD4⁺ and Jurkat cells. An increase in cleaved caspase 8 is also seen for Jurkat cells.To determine the respective contributions of each pathway, we assessed apoptosis in Jurkat cells that are deficient in either caspase 8 or Fas-associating protein with death domain (FADD) for the extrinsic pathway and in cells deficient in caspase 9 for the intrinsic pathway. We chose caspase 3 activation to assess apoptosis in HSV-2-exposed cells, because convergence of extrinsic and intrinsic apoptosis pathways occurs at caspase 3.Jurkat cells deficient in caspase 8 or FADD (American Type Culture Collection) have been previously described (8), and we confirmed the absence of caspase 8 and FADD in respective cell lines (Fig. ​(Fig.5A).5A). We also investigated the parental Jurkat cell A3 subclone and verified that both caspase 8 and FADD were expressed as expected (Fig. ​(Fig.5A).5A). Parental and deficient Jurkat cells were exposed to mock-infected or HSV-2-infected fibroblasts as described above. At 24 h postexposure, cells were probed with fluorescence-labeled antibody against activated caspase 3. As shown in Fig. ​Fig.5B,5B, cells deficient in caspase 8 or FADD underwent apoptosis at levels similar to those of parental cells as determined by caspase 3 activation. The results indicate that neither caspase 8 nor FADD is required for HSV-2-induced apoptosis in Jurkat cells. We additionally evaluated for cleavage products of caspase 8 and caspase 9 in these cells. As shown in Fig. ​Fig.5C,5C, cleaved fragments of caspase 9 were seen in all three cell types following HSV-2 exposure. Interestingly, cleaved fragments of caspase 8 were detected in both parental and FADD-deficient cells, suggesting that FADD is not required for cleavage of caspase 8 following HSV-2 exposure.Open in a separate windowFIG. 5.Loss of caspase 8 or FADD expression does not impede HSV-2-induced apoptosis in Jurkat cells. (A) Immunoblots of I2.1, I9.2, and A3 cell lysates were probed with antibodies for procaspase 8, FADD, and β-actin. FADD is not expressed in I2.1 cells, while caspase 8 is not expressed in I9.2 cells. The parental cell line A3 is a subclone of Jurkat cells that expresses both caspase 8 and FADD. β-Actin expression is shown as a control. (B) Percentages of cells with caspase 3 activation are comparable in cells with caspase 8 or FADD deficiency and in parental A3 cells following exposure to HSV-2. Mean percentages of cells with caspase 3 activation, with standard error bars, are shown. The graph represents results from three independent experiments. (C) Lysates from cells exposed to mock- or HSV-2-infected fibroblasts were probed for cleaved forms of caspase 8 and caspase 9 by immunoblotting at 24 h postexposure. Representative immunoblots from three independent experiments are shown. Blots demonstrate increases in cleaved caspase 8 for I2.1 and A3 cells that were exposed to HSV-2. Increases in cleaved caspase 9 are seen for all three cell types.Jurkat cells deficient in caspase 9 expression and the corresponding caspase 9-reconstituted clones (12, 13) were a gift of Ingo Schmitz (University of Dusseldorf, Dusseldorf, Germany). Immunoblot analysis confirmed the presence or absence of caspase 9 in both cell lines (Fig. ​(Fig.6A).6A). Cells were exposed to mock- or HSV-2-infected fibroblasts and analyzed for activation of caspase 3 as described above. Following exposure to HSV-2, cells deficient in caspase 9 were protected from apoptosis as determined by caspase 3 activation (Fig. ​(Fig.6B).6B). In contrast, cells with reconstituted caspase 9 showed an increased percentage of caspase 3 activation. An evaluation of caspase 8 and caspase 9 cleavage in these cells showed that cleaved fragments were seen only in the cells reconstituted with caspase 9 (Fig. ​(Fig.6C).6C). To confirm that the findings were applicable to the parental Jurkat cell line, we evaluated the effect of a caspase 9 inhibitor, Z-LEHD-fmk (R&D Biosystems, Minneapolis, MN) on Jurkat cells that were exposed to mock- or HSV-2-infected fibroblasts. Jurkat cells were incubated with 100 μM Z-LEHD-fmk or a dimethyl sulfoxide (DMSO) solvent control for 1 h before exposure to fibroblasts, throughout the exposure, and postexposure. At 24 h postexposure, the percentage of virus-exposed cells with activated caspase 3 was inhibited by Z-LEHD-fmk to 3%, identical to the baseline percentage seen with mock-exposed cells with DMSO solvent control (Fig. ​(Fig.6D).6D). Together, these findings indicate that caspase 9 is required for HSV-2-induced apoptosis and cleavage of both caspase 8 and caspase 9 in Jurkat cells.Open in a separate windowFIG. 6.Caspase 9 is required for HSV-2-induced apoptosis in Jurkat cells. (A) Immunoblots of JMR and F9 cell lysates probed with antibodies for procaspase 9 and β-actin. Caspase 9 is not expressed in JMR cells, a subclone of Jurkat cells. Caspase 9 expression is reconstituted in F9 cells, which are JMR cells stably transfected with a plasmid expressing caspase 9. (B) JMR cells with caspase 9 show protection from HSV-2-induced apoptosis, while F9 cells with reconstituted caspase 9 are susceptible to apoptosis induced by the virus. Mean percentages of cells with caspase 3 activation, with standard error bars, are shown. The graph represents results from three independent experiments. (C) Lysates from JMR and F9 cells were exposed to mock- or HSV-2-infected fibroblasts and then probed for cleaved forms of caspase 8 and caspase 9 by immunoblotting at 24 h postexposure. Representative immunoblots from three independent experiments are shown. Blots demonstrate an increase in cleaved caspase 8 and caspase 9 for F9 cells but not JMR cells. (D) Jurkat cells that were incubated with Z-LEHD-fmk show protection from HSV-2-induced apoptosis compared to results with the DMSO solvent control at 24 h postexposure. Mean percentages of cells with caspase 3 activation, with standard error bars, are shown. The graph represents results from three independent experiments.Our results provide evidence that exposure to HSV-2-infected fibroblasts leads to apoptosis in T cells. It is important to note that our method of activation and maintenance of human peripheral blood mononuclear cells enriches for CD4⁺ cells. Phytohemagglutinin and interleukin-2 nonspecifically activate these cells, and no specific immune response to HSV-2 is expected. Further studies are necessary to determine if our observations with human CD4⁺ cells are applicable to other primary lymphocyte cell types and to virus-specific immune cells. Despite these limitations, the similarity in the pattern of change in apoptotic markers in Jurkat and primary human CD4⁺ cells suggests that apoptosis occurs by a similar mechanism in the two cell types.It is our prediction that the ability of HSV-2 to kill T cells plays a role in mitigating the cell-mediated immune response in vivo. In the present study, we have begun to elucidate the mechanism behind apoptosis in T cells following exposure to HSV-2-infected fibroblasts. Viral antigens could be detected in virus-exposed Jurkat cells, suggesting infection that occurs by cell-to-cell spread. We previously reported that expression of HSV-1 ICP0 and HSV-2 ICP10 results in apoptosis of transfected Jurkat cells (4). Therefore, we predicted that infected cells would be positive for apoptotic markers in the present study. However, viral antigens and activated caspase 3 were largely mutually exclusive in virus-exposed cells. We are currently undertaking studies to further decipher the relationship between viral infection and apoptosis to determine whether apoptosis occurs by a bystander effect in T cells.Our results revealed the requirement of caspase 9 in HSV-2-induced apoptosis of Jurkat cells. The finding that neither caspase 8 nor FADD was required suggests that the extrinsic pathway is not involved in HSV-2-induced apoptosis in Jurkat cells. Consistent with this finding, cleavage of caspase 9 was clearly demonstrated in virus-exposed CD4⁺ cells compared to mock-exposed cells, while cleavage of caspase 8 was not evident. Although similarities between HSV-2-induced apoptosis in CD4⁺ and Jurkat cells predict that similar mechanisms are likely to be responsible in both cell types, the applicability of the finding to other types of T cells remains to be elucidated.In conclusion, the present study demonstrated activation of intrinsic apoptosis pathways in Jurkat and primary human CD4⁺ cells following exposure to HSV-2. Further understanding of virus-induced apoptosis in T cells, which is predicted to be an immune-evasion mechanism during reactivation, is expected to provide means to prevent HSV-2 reactivation in infected individuals.     

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes during initial entry and establishes a primary infection in the epithelium, which is followed by latent infection of neurons. After reactivation, viruses can become evident at mucocutaneous sites that appear as skin vesicles or mucosal ulcers. How HSV-1 invades skin or mucosa and reaches its receptors is poorly understood. To investigate the invasion route of HSV-1 into epidermal tissue at the cellular level, we established an ex vivo infection model of murine epidermis, which represents the site of primary and recurrent infection in skin. The assay includes the preparation of murine skin. The epidermis is separated from the dermis by dispase II treatment. After floating the epidermal sheets on virus-containing medium, the tissue is fixed and infection can be visualized at various times postinfection by staining infected cells with an antibody against the HSV-1 immediate early protein ICP0. ICP0-expressing cells can be observed in the basal keratinocyte layer already at 1.5 hr postinfection. With longer infection times, infected cells are detected in suprabasal layers, indicating that infection is not restricted to the basal keratinocytes, but the virus spreads to other layers in the tissue. Using epidermal sheets of various mouse models, the infection protocol allows determining the involvement of cellular components that contribute to HSV-1 invasion into tissue. In addition, the assay is suitable to test inhibitors in tissue that interfere with the initial entry steps, cell-to-cell spread and virus production. Here, we describe the ex vivo infection protocol in detail and present our results using nectin-1- or HVEM-deficient mice.     

一种可切除包装信号的重组1型单纯疱疹病毒的产生

A new kind of recombinant herpes simplex virus type 1 (HSV 1) was constructed. This recombinant, named HSV1 LaL, contained an unique packaging signal (“a sequence) flanked by two loxP sites in parallel orientation, named LaL, while the original packaging signals of HSV 1 were deleted. Based on a set of cosmids containing the entire HSV 1 genome except the “a sequence, the LaL was inserted into HSV 1 UL44 gene on one of the cosmids, cos56, generating cos56/LaL. By co transfecting cos56/LaL with the other cosmids, HSV1 LaL was generated in the cells by recombination. By introducing cos56/LaL or HSV1 LaL respectively into E.coli or BHK cells that expressed Cre recombinase, LaLs on both of them were excised by Cre, which was proved by PCR detection. To study the potential use as helper virus in packaging amplicon vector, HSV1 LaL was compared with a control virus HSV1 lacZ that contained a lacZ gene in the UL44 gene. The titer of amplicon virus generated from HSV1 LaL infected BHK/Cre cells was basically the same as that from HSV1 lacZ infected cells, however,the former contained about 10 fold less helper virus than the later, while HSV1 LaL showed the same replication rate as HSV1 lacZ on standard cells, like BHK 21.     

Electron Microscopy of Herpes Simplex Virus: I. Entry

Although capsids of herpes simplex virus were encountered within phagocytic vesicles, they were more commonly observed free within the cytoplasm. Stages in the release of virus from vesicles were not seen. There appeared to be five distinct steps in the process whereby the virus initiates infection: attachment, digestion of the viral envelope, digestion of the cell wall, passage of the capsid directly into the cytoplasm, and digestion of the capsid with release of the core. Antibody probably interferes with the first two stages.     

Kinetics of Cell Fusion Induced by a Syncytia-Producing Mutant of Herpes Simplex Virus Type I

We have isolated a number of plaque-morphology mutants from a strain of herpes simplex virus type I which, unlike the wild type, cause extensive cell fusion during a productive viral infection. After the onset of fusion, there is an exponential decrease in the number of single cells as a function of time after infection. At a multiplicity of infection (MOI) of 3.8 plaque-forming units per cell, fusion begins 5.3 h after infection with the number of single cells decreasing to 10% of the original number 10.2 h after infection. As the MOI is gradually increased from 0.4 to 8, the onset of fusion occurs earlier during infection. However, when the MOI is increased from 8 to 86, the onset of fusion does not occur any earlier. The rate of fusion is independent of the MOI for an MOI greater than 1. The rate of fusion varies linearly with initial cell density up to 3.5 × 10⁴ cells/cm² and is independent of initial cell density at higher cell concentrations. To assay cell fusion we have developed a simple quantitative assay using a Coulter counter to measure the number of single cells as a function of time after infection. Data obtained using a Coulter counter are similar to those obtained with a microscope assay.     

Suppression of PACT-Induced Type I Interferon Production by Herpes Simplex Virus 1 Us11 Protein

Herpes simplex virus 1 (HSV-1) Us11 protein is a double-stranded RNA-binding protein that suppresses type I interferon production through the inhibition of the cytoplasmic RNA sensor RIG-I. Whether additional cellular mediators are involved in this suppression remains to be determined. In this study, we report on the requirement of cellular double-stranded RNA-binding protein PACT for Us11-mediated perturbation of type I interferon production. Us11 associates with PACT tightly to prevent it from binding with and activating RIG-I. The Us11-deficient HSV-1 was indistinguishable from the Us11-proficient virus in the suppression of interferon production when PACT was compromised. More importantly, HSV-1-induced activation of interferon production was abrogated in PACT knockout murine embryonic fibroblasts. Our findings suggest a new mechanism for viral evasion of innate immunity through which a viral double-stranded RNA-binding protein interacts with PACT to circumvent type I interferon production. This mechanism might also be used by other PACT-binding viral interferon-antagonizing proteins such as Ebola virus VP35 and influenza A virus NS1.     

Herpes Simplex Virus Glycoproteins: Participation of Individual Herpes Simplex Virus Type 1 Glycoprotein Antigens in Immunocytolysis and Their Correlation with Previously Identified Glycopolypeptides

Tissue culture cells infected with herpes simplex type 1 virus express virus-specified glycoprotein antigens on the plasma membrane. Three of these have been previously identified and have been designated as Ag-11, Ag-8, and Ag-6. In the present study, immunoglobulins to each of the antigens were shown to be capable of mediating immunocytolysis in the presence of either complement (antibody-dependent complement-mediated cytotoxicity) or peripheral blood mononuclear cells (antibody-dependent cell-mediated cytotoxicity [ADCC]). Two herpes simplex virus type 1 strains, VR-3 and F, reacted similarly in the ADCC test in the presence of immunoglobulins to Ag-11, Ag-8, and Ag-6 in both infected Chang liver cells and HEp-2 cells. Anti-Ag-6, however, produced a lower ADCC reaction in HEp-2 cells than in Chang liver cells, suggesting differences in the Ag-6 surface expression in, or release from, these cells. Chang liver and HEp-2 cells infected with the MP mutant strain of herpes simplex virus type 1 showed reduced ADCC in the presence of anti-Ag-11 and anti-Ag-8, but no reactivity at all with anti-Ag-6. Crossed immunoelectrophoretic analysis showed that MP-infected cell extracts contain Ag-11 and Ag-8, but lack Ag-6. Polypeptide analysis of herpes simplex virus type 1 strains F, VR-3, and MP showed that Ag-11 consists of the glycoproteins gA and gB, that Ag-8 consists of gD, and that Ag-6 consists of gC. In conclusion, the present study demonstrates that either one of the glycoproteins (gC, gD, and a mixture of gA and gB) can function as a target for immunocytolysis and that the antibody preparation to gC (Ag-6) does not cross-react with any of the other glycoproteins.     

The First Immunoglobulin-Like Domain of HveC Is Sufficient To Bind Herpes Simplex Virus gD with Full Affinity, While the Third Domain Is Involved in Oligomerization of HveC

The human herpesvirus entry mediator C (HveC/PRR1) is a member of the immunoglobulin family used as a cellular receptor by the alphaherpesviruses herpes simplex virus (HSV), pseudorabies virus, and bovine herpesvirus type 1. We previously demonstrated direct binding of the purified HveC ectodomain to purified HSV type 1 (HSV-1) and HSV-2 glycoprotein D (gD). Here, using a baculovirus expression system, we constructed and purified truncated forms of the receptor containing one [HveC(143t)], two [HveC(245t)], or all three immunoglobulin-like domains [HveC(346t)] of the extracellular region. All three constructs were equally able to compete with HveC(346t) for gD binding. The variable domain bound to virions and blocked HSV infection as well as HveC(346t). Thus, all of the binding to the receptor occurs within the first immunoglobulin-like domain, or V-domain, of HveC. These data confirm and extend those of Cocchi et al. (F. Cocchi, M. Lopez, L. Menotti, M. Aoubala, P. Dubreuil, and G. Campadelli-Fiume, Proc. Natl. Acad. Sci. USA 95:15700, 1998). Using biosensor analysis, we measured the affinity of binding of gD from HSV strains KOS and rid1 to two forms of HveC. Soluble gDs from the KOS strain of HSV-1 had the same affinity for HveC(346t) and HveC(143t). The mutant gD(rid1t) had an increased affinity for HveC(346t) and HveC(143t) due to a faster rate of complex formation. Interestingly, we found that HveC(346t) was a tetramer in solution, whereas HveC(143t) and HveC(245t) formed dimers, suggesting a role for the third immunoglobulin-like domain of HveC in oligomerization. In addition, the stoichiometry between gD and HveC appeared to be influenced by the level of HveC oligomerization.     

Herpes Simplex Virus Membrane Proteins gE/gI and US9 Act Cooperatively To Promote Transport of Capsids and Glycoproteins from Neuron Cell Bodies into Initial Axon Segments

Herpes simplex virus (HSV) and other alphaherpesviruses must move from sites of latency in ganglia to peripheral epithelial cells. How HSV navigates in neuronal axons is not well understood. Two HSV membrane proteins, gE/gI and US9, are key to understanding the processes by which viral glycoproteins, unenveloped capsids, and enveloped virions are transported toward axon tips. Whether gE/gI and US9 function to promote the loading of viral proteins onto microtubule motors in neuron cell bodies or to tether viral proteins onto microtubule motors within axons is not clear. One impediment to understanding how HSV gE/gI and US9 function in axonal transport relates to observations that gE⁻, gI⁻, or US9⁻ mutants are not absolutely blocked in axonal transport. Mutants are significantly reduced in numbers of capsids and glycoproteins in distal axons, but there are less extensive effects in proximal axons. We constructed HSV recombinants lacking both gE and US9 that transported no detectable capsids and glycoproteins to distal axons and failed to spread from axon tips to adjacent cells. Live-cell imaging of a gE⁻/US9⁻ double mutant that expressed fluorescent capsids and gB demonstrated >90% diminished capsids and gB in medial axons and no evidence for decreased rates of transport, stalling, or increased retrograde transport. Instead, capsids, gB, and enveloped virions failed to enter proximal axons. We concluded that gE/gI and US9 function in neuron cell bodies, in a cooperative fashion, to promote the loading of HSV capsids and vesicles containing glycoproteins and enveloped virions onto microtubule motors or their transport into proximal axons.     

O-Linked Glycosylation of the Mucin Domain of the Herpes Simplex Virus Type 1-specific Glycoprotein gC-1 Is Temporally Regulated in a Seed-and-spread Manner

The herpes simplex virus type 1 (HSV-1) glycoprotein gC-1, participating in viral receptor interactions and immunity interference, harbors a mucin-like domain with multiple clustered O-linked glycans. Using HSV-1-infected diploid human fibroblasts, an authentic target for HSV-1 infection, and a protein immunoaffinity procedure, we enriched fully glycosylated gC-1 and a series of its biosynthetic intermediates. This fraction was subjected to trypsin digestion and a LC-MS/MS glycoproteomics approach. In parallel, we characterized the expression patterns of the 20 isoforms of human GalNAc transferases responsible for initiation of O-linked glycosylation. The gC-1 O-glycosylation was regulated in an orderly manner initiated by synchronous addition of one GalNAc unit each to Thr-87 and Thr-91 and one GalNAc unit to either Thr-99 or Thr-101, forming a core glycopeptide for subsequent additions of in all 11 GalNAc residues to selected Ser and Thr residues of the Thr-76–Lys-107 stretch of the mucin domain. The expression patterns of GalNAc transferases in the infected cells suggested that initial additions of GalNAc were carried out by initiating GalNAc transferases, in particular GalNAc-T2, whereas subsequent GalNAc additions were carried out by followup transferases, in particular GalNAc-T10. Essentially all of the susceptible Ser or Thr residues had to acquire their GalNAc units before any elongation to longer O-linked glycans of the gC-1-associated GalNAc units was permitted. Because the GalNAc occupancy pattern is of relevance for receptor binding of gC-1, the data provide a model to delineate biosynthetic steps of O-linked glycosylation of the gC-1 mucin domain in HSV-1-infected target cells.