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.     

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.     

Herpes Simplex Virus: Genome Size and Redundancy Studied by Renaturation Kinetics

Herpes simplex virus subtype 1 deoxyribonucleic acid (DNA) was sheared in a French press to uniform fragments, denatured by heating, then allowed to reassociate. The renaturation reaction followed second-order kinetics with a single rate constant indicating that at least 95% of the genome was unique and that repetitive sequences, if present, were not detectable by this technique. The kinetic complexity of the herpes simplex genome was determined by DNA renaturation kinetics to be (95 ± 1) × 10⁶ daltons. Since this value is in excellent agreement with the molecular weight of viral DNA [(99 ± 5) × 10⁶ daltons] obtained from velocity sedimentation studies, it is concluded that virions contain only one species of double-stranded DNA molecules 95 × 10⁶ to 99 × 10⁶ daltons in molecular weight.     

Electron Microscopy of Herpes Simplex Virus: II. Sequence of Development

Examination of infected cells at sequential intervals after infection revealed that the first viral forms to appear were capsids enclosing cores of low density. Not until the 6th hr were dense cores encountered, and at approximately the same time enveloped virus was seen. Envelopment occurred most frequently in close proximity to the nuclear surface, although the process was also encountered within the nuclear matrix and in the cytoplasm. There was often extensive proliferation of the nuclear membrane. Envelopment of the virus by budding from the cell surface was not observed. It was concluded that enveloped virus consitutes the infectious particle and that the unenveloped capsid is unstable outside the cell. Nevertheless, it is likely that capsids enclosing infectious nucleic acid can pass directly from one cell to another after fusion has taken place.     

Reevaluating Herpes Simplex Virus Hemifusion

Membrane fusion induced by enveloped viruses proceeds through the actions of viral fusion proteins. Once activated, viral fusion proteins undergo large protein conformational changes to execute membrane fusion. Fusion is thought to proceed through a “hemifusion” intermediate in which the outer membrane leaflets of target and viral membranes mix (lipid mixing) prior to fusion pore formation, enlargement, and completion of fusion. Herpes simplex virus type 1 (HSV-1) requires four glycoproteins—glycoprotein D (gD), glycoprotein B (gB), and a heterodimer of glycoprotein H and L (gH/gL)—to accomplish fusion. gD is primarily thought of as a receptor-binding protein and gB as a fusion protein. The role of gH/gL in fusion has remained enigmatic. Despite experimental evidence that gH/gL may be a fusion protein capable of inducing hemifusion in the absence of gB, the recently solved crystal structure of HSV-2 gH/gL has no structural homology to any known viral fusion protein. We found that in our hands, all HSV entry proteins—gD, gB, and gH/gL—were required to observe lipid mixing in both cell-cell- and virus-cell-based hemifusion assays. To verify that our hemifusion assay was capable of detecting hemifusion, we used glycosylphosphatidylinositol (GPI)-linked hemagglutinin (HA), a variant of the influenza virus fusion protein, HA, known to stall the fusion process before productive fusion pores are formed. Additionally, we found that a mutant carrying an insertion within the short gH cytoplasmic tail, 824L gH, is incapable of executing hemifusion despite normal cell surface expression. Collectively, our findings suggest that HSV gH/gL may not function as a fusion protein and that all HSV entry glycoproteins are required for both hemifusion and fusion. The previously described gH 824L mutation blocks gH/gL function prior to HSV-induced lipid mixing.Membrane fusion is an essential step during the entry process of enveloped viruses, such as herpes simplex virus (HSV), into target cells. The general pathway by which enveloped viruses fuse with target membranes through the action of fusion proteins is fairly well understood. Viral fusion proteins use the free energy liberated during their own protein conformational changes to draw the two membranes—viral and target—together. Fusion is thought to proceed through a “hemifusion” intermediate, in which the proximal leaflets of the two bilayers have merged but a viral pore has not yet formed and viral contents have not yet mixed with the cell cytoplasm (10, 38). Fusion proteins then drive the completion of fusion, which includes fusion pore formation, pore enlargement, and complete content mixing.HSV, an enveloped neurotropic virus, requires four glycoproteins—glycoprotein B (gB), glycoprotein D (gD), glycoprotein H (gH), and glycoprotein L (gL)—to execute fusion (9, 57, 60). gB, gD, and gH are membrane bound; gL is a soluble protein which complexes with gH to form a heterodimer (gH/gL). HSV-1 gH is not trafficked to the cell or virion surface in the absence of gL (32, 52). The requirement of four entry glycoproteins sets HSV apart from other enveloped viruses, most of which induce fusion through the activity of a single fusion protein. Although the specific mode of HSV entry is cell type dependent—fusion with neurons and Vero cells occurs at the plasma membrane at neutral pH; fusion with HeLa and CHO cells involves pH-dependent endocytosis, and fusion with C10 cells involves pH-independent endocytosis (42, 45)—all routes of entry require gD, gB, and gH/gL. Furthermore, although some discrepancies between virus-cell and cell-cell fusion have been observed (8, 44, 55, 58), both generally require the actions of gD, gB, and gH/gL.Much work has gone toward the understanding of how the required HSV entry glycoproteins work together to accomplish fusion, and many questions remain. After viral attachment, mediated by glycoprotein C and/or gB (54), the first step in HSV fusion is thought to be gD binding a host cell receptor (either herpesvirus entry mediator [HVEM], nectin-1, nectin-2, or heparan sulfate modified by specific 3-O-sulfotransferases) (56). The gD-receptor interaction induces a conformational change in gD (39) that is thought to trigger gD-gB and/or gD-gH/gL interactions that are required for the progression of fusion (1-4, 13, 18, 23, 49).gB and gH/gL are considered the core fusion machinery of most herpesviruses. The HSV-1 gB structure revealed surprising structural homology to the postfusion structures of two known viral fusion proteins (31, 35, 51). This structural homology indicates that despite not being sufficient for HSV fusion, gB is likely a fusion protein. Although the gB cytoplasmic tail (CT) is not included in the solved structure, it acts as a regulator of fusion, as CT truncations can cause either hyperfusion or fusion-null phenotypes (5, 17). The gB CT has been proposed to bind stably to lipid membranes and negatively regulate membrane fusion (12). Another proposed regulator of gB function is gH/gL. Despite conflicting accounts of whether gD and a gD receptor are required for the interaction of gH/gL and gB (1, 3, 4), a recent study indicates that gH/gL and gB interact prior to fusion and that gB may interact with target membranes prior to an interaction with gH/gL (2). The gB-gH/gL interaction seems to be required for the progression of fusion.Compared to the other required HSV entry glycoproteins, the role of gH/gL during fusion remains enigmatic. Mutational studies have revealed several regions of the gH ectodomain, transmembrane domain (TM), and CT that are required for its function (19, 25, 26, 30, 33). gH/gL of another herpesvirus, Epstein-Barr virus (EBV), have been shown to bind integrins during epithelial cell fusion, and soluble forms of HSV gH/gL have been shown to bind cells and inhibit viral entry in vitro (24, 46). However, the role of gH/gL binding to target cells in regard to the fusion process remains to be determined.There are some lines of evidence that suggest that gH/gL is a fusion protein. The gH/gL complexes of VZV and CMV have been reported to independently execute some level of cell-cell fusion (14, 37). HSV-1 gH/gL has been reported to independently mediate membrane fusion during nuclear egress (15). In silico analyses and studies of synthetic HSV gH peptides have proposed that gH has fusogenic properties (20, 21, 25-28). Finally, of most importance to the work we report here, gH/gL has been shown to be sufficient for induction of hemifusion in the presence of gD and a gD receptor, further promoting the premise that gH/gL is a fusion protein (59). However, the recently solved crystal structure of HSV-2 gH/gL revealed a tight complex of gH/gL in a “boot-like” structure, which bears no structural homology to any known fusion proteins (11). The HSV-2 gH/gL structure and research demonstrating that gH/gL and gB interactions are critical to fusion (2) have together prompted a new model of HSV fusion in which gH/gL is required to either negatively or positively regulate the activity of gB through direct binding.We wanted to investigate the ability of a previously reported gH CT mutant, 824L, to execute hemifusion. 824L gH contains a five-residue insertion at gH residue 824, just C-terminal of the TM domain. 824L is expressed on cell surfaces and incorporated into virions at levels indistinguishable from those of wild-type gH by either cell-based ELISA or immunoblotting, yet it is nonfunctional (33). We relied on a fusion assay capable of detecting hemifusion, developed by Subramanian et al. (59), which we modified to include an additional control for hemifusion or nonenlarging pore formation, glycosylphosphatidylinositol (GPI)-linked hemagglutinin (GPI-HA). GPI-HA is a variant of the influenza virus fusion protein, HA, that is known to stall the fusion process before enlarging fusion pores are formed.We were surprised to find that in our hands, gD, a gD receptor, and gH/gL were insufficient for the induction of hemifusion or lipid mixing in both cell-based and virus-based fusion assays. We found that gD, gB, and gH/gL are all required to observe lipid mixing. Further, we found that gB, gD, gL, and 824L gH are insufficient for lipid mixing. Our findings support the emerging view, based on gH/gL structure, that the gH/gL complex does not function as a fusion protein and does not insert into target membranes to initiate the process of fusion through a hemifusion intermediate. Our findings also further demonstrate that mutations in the CT of gH can have a dramatic effect on the ability of gH/gL to function in fusion.     

Adeno-Associated Virus Type 2 Rep68 Can Bind to Consensus Rep-Binding Sites on the Herpes Simplex Virus 1 Genome

Adeno-associated virus type 2 is known to inhibit replication of herpes simplex virus 1 (HSV-1). This activity has been linked to the helicase- and DNA-binding domains of the Rep68/Rep78 proteins. Here, we show that Rep68 can bind to consensus Rep-binding sites on the HSV-1 genome and that the Rep helicase activity can inhibit replication of any DNA if binding is facilitated. Therefore, we hypothesize that inhibition of HSV-1 replication involves direct binding of Rep68/Rep78 to the HSV-1 genome.     

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.     

Analysis of Herpes Simplex Virus Type 1 DNA Packaging Signal Mutations in the Context of the Viral Genome

The minimal signal required for the cleavage and packaging of replicated concatemeric herpes simplex virus type 1 (HSV-1) DNA corresponds to an approximately 200-bp fragment, Uc-DR1-Ub, spanning the junction of the genomic L and S segments. Uc and Ub occupy positions adjacent to the L and S termini and contain motifs (pac2 and pac1, respectively) that are conserved near the ends of other herpesvirus genomes. We have used homologous Red/ET recombination in Escherichia coli to introduce wild-type and specifically mutated Uc-DR1-Ub fragments into an ectopic site of a cloned HSV-1 genome from which the resident packaging signals had been previously deleted. The resulting constructs were transfected into mammalian cells, and their abilities to replicate and become encapsidated, generate Uc- and Ub-containing terminal fragments, and give rise to progeny virus were assessed. In general, the results obtained agree well with previous observations made using amplicons and confirm roles for the pac2 T element in the initiation of DNA packaging and for the GC-rich motifs flanking the pac1 T element in termination. In contrast to a previous report, the sequence of the DR1 element was also crucial for DNA packaging. Following repair of the resident packaging signals in mammalian cells, recombination occurred at high frequency in progeny virus between the repaired sequences and mutated Uc-DR1-Ub inserts. This restored the ability of mutated Uc-DR1-Ub inserts to generate terminal fragments, although these were frequently larger than expected from simple repair of the original lesion.Herpesviruses possess linear double-stranded DNA genomes that are circularized early after infection and upon replication generate concatemeric structures. During progeny particle assembly, the cleavage of concatemers at specific sites, corresponding to the genomic termini, is tightly coupled to the insertion of the viral DNA into a preformed structure referred to as the procapsid (reviewed in references 2, 4, and 11). In the case of herpes simplex virus type 1 (HSV-1), a terminally redundant region of the genome, known as the a sequence (Fig. ​(Fig.1a),1a), contains all the cis-acting sequences required for DNA packaging (24, 27). This region, which is 250 to 500 bp in length depending on the virus strain, is present as a single copy at the S terminus and as one or more tandem copies at the L terminus. In addition, one or more copies are present in inverted orientation at the junction between the L and S segments (30, 31).Open in a separate windowFIG. 1.Structure of the HSV-1 Uc-DR1-Ub element. (a) Structure of the HSV-1 genome showing the positions and relative orientations (horizontal arrows) of copies of the a sequence. (b) Circularization of linear genomes by direct ligation brings together two copies of the a sequence separated by a single DR1 repeat. The site of ligation, and of cleavage of concatemers, is shown by the vertical arrow. (c) Motifs and regions within the 194-bp Uc-DR1-Ub fragment. To facilitate naming of mutants, component regions of Uc, Ub, and DR1 were also referred to as c1 to c4, b1 to b4, and R, respectively, as indicated in parentheses.The structure of the HSV-1 a sequence is depicted in Fig. ​Fig.1b.1b. Each a sequence is flanked by direct repeats (DR1) of 17 to 20 bp, with single copies of DR1 separating tandem a sequences. Genomic termini are generated by a cleavage event toward one end of DR1, and circularization of infecting genomes restores a complete a sequence. The central portion of the a sequence comprises multiple repeats of one or two other short sequences (DR2 and sometimes DR4), while quasi-unique sequences are located between DR1 and either side of the DR2/DR4 repeats. These regions are termed Ub and Uc, and in virion DNA they lie adjacent to the S and L termini, respectively (6, 17, 18).An approximately 200-bp fragment (Uc-DR1-Ub) spanning the junction between tandem a sequences, such as is generated upon fusion of the genomic ends (Fig. ​(Fig.1b),1b), has been shown to contain all the essential cis-acting sequences necessary for DNA packaging (10, 20). Within the Ub and Uc regions are two domains, pac1 and pac2, respectively, which contain several characteristic sequence motifs that are conserved near the ends of other herpesvirus genomes (3, 8, 15). These motifs, as originally defined by Deiss et al. (8), are illustrated in Fig. ​Fig.1c.1c. It is now recognized that the major conserved motif within the pac1 region comprises the T-rich element flanked on each side by short G tracts (from the proximal and distal GC-rich regions). In the case of pac2, the T-rich element is most highly conserved with a consensus CGCGGCG motif also frequently being present (32).Detailed studies, employing primarily HSV-1 and murine cytomegalovirus (MCMV), have highlighted the roles of the major conserved motifs and suggested the following general mechanism by which concatemers are cleaved and packaged (1, 10, 13, 15, 16, 23, 25, 29, 32). Within Uc the most critical sequence is the pac2 T element, which is essential for cleavage to initiate DNA packaging. Cleavage occurs at a fixed distance from the pac2 T element, and the resulting Uc-containing end is inserted into the procapsid. Additional important cis-acting sequences are present further from the cleavage site, possibly including the pac2 consensus motif. Deletion, but not substitution, of the pac2 GC element and unconserved region impaired DNA packaging, suggesting that the relative spacing of the cleavage site, T element, and distal motifs is crucial. Packaging proceeds from pac2 toward the pac1 terminus, and a second cleavage event terminates DNA packaging. This cleavage appears to be directed by, and occurs at a fixed distance from, a single region comprising the pac1 T element and the flanking G tracts. Surprisingly, substitutions within the highly conserved T element are tolerated, but it remains unclear whether this region functions as a spacer element. The UL28 component of the HSV-1 terminase enzyme binds to a specific conformation adopted by the region comprising the T element and G tracts, and this interaction is likely to be crucial for cleavage.The functional analysis of herpesvirus DNA packaging signals has employed two major approaches. In the first, amplicons (i.e., bacterial plasmids containing a viral DNA replication origin and packaging signal) are transfected into mammalian cells and their ability to be replicated and packaged is assessed following the provision of viral helper functions, either by superinfection with virus particles or by cotransfection of virion DNA (7, 20, 24, 27, 29, 35). The second assay introduces an additional copy of the packaging signal under test at an ectopic site within the viral genome and determines whether it functions as a site for the cleavage of concatemeric DNA and the generation of novel terminal fragments of virion DNA (5, 15, 18, 23, 29, 32). Both these approaches, however, suffer from the disadvantage that recombination occurs between the test packaging signal and the wild-type (wt) signal present either in the helper virus or in its normal location within an ectopic-site recombinant (5, 8, 15, 23, 32). Additionally, concatemers generated following replication of amplicons have a significantly different structure from standard herpesviral genomes in that multiple copies of the packaging signal are present, spaced at regular intervals corresponding to the size of the input plasmid. This raises the possibility that the activity of wt or mutated packaging signals in the amplicon assay may not accurately reflect their behavior in a standard genome.To avoid these difficulties and allow analysis of mutated packaging signals in the context of the viral genome, we have used a cloned full-length HSV-1 genome, fHSVΔpac, which is complete with the exception that all copies of the a sequence have been deleted (22). This molecule is propagated as a bacterial artificial chromosome (BAC), and specific sequences can be inserted via homologous recombination either in mammalian cells or in the bacterial host. We previously demonstrated that a single copy of the minimal packaging signal Uc-DR1-Ub introduced into the viral thymidine kinase (TK) locus of fHSVΔpac by recombination in mammalian cells was sufficient to allow the products of replication to be packaged in mammalian cells and to allow the generation of viable progeny (28). Here, we describe the introduction of the packaging signal into fHSVΔpac by Red/ET recombination in Escherichia coli (19, 34), allowing previously described (10) and new Uc-DR1-Ub mutants to be screened for their ability to direct encapsidation, generate Uc- and Ub-containing terminal fragments, and give rise to progeny virus.     

Ribonucleotides Linked to DNA of Herpes Simplex Virus Type 1

Cells of a continuous cell line derived from rabbit embryo fibroblasts were infected with herpes simplex type 1 virus (HSV-1) and maintained in the presence of either [5-(3)H]uridine or [methyl-(3)H]thymidine or (32)PO(4) (3-). Nucleocapsids were isolated from the cytoplasmic fraction, partially purified, and treated with DNase and RNase. From the pelleted nucleocapsids, DNA was extracted and purified by centrifugation in sucrose and cesium sulfate gradients. The acid-precipitable radioactivity of [5-(3)H]uridine-labeled DNA was partially susceptible to pancreatic RNase and alkaline treatment; the susceptibility to the enzyme decreased with increasing salt concentration. No drop of activity of DNA labeled with [(3)H]thymidine was observed either after RNase or alkali treatment. Base composition analysis of [5-(3)H]uridine-labeled DNA showed that the radioactivity was recovered as uracil and cytosine. In the cesium sulfate gradient, the purified [5-(3)H]uridine-labeled DNA banded at the same position as the (32)P-labeled DNA. The present data tend to suggest that ribonucleotide sequences are present in HSV DNA, that they are covalently attached to the viral DNA, and that they can form double-stranded structures.