The Herpes Simplex Virus Type 1 Infected Cell Protein 22

As one of the immediate-early(IE)proteins of herpes simplex virus type 1(HSV-1),ICP22 is a multifunctional viral regulator that localizes in the nucleus of infected cells.It is required in experimental animal systems and some nonhuman cell lines,but not in Vero or HEp-2 cells.ICP22 is extensively phosphorylated by viral and cellular kinases and nucleotidylylated by casein kinase Ⅱ.It has been shown to be required for efficient expression of early(E)genes and a subset of late(L)genes.ICP22,in conjunction wit...     

Reactivation of Herpes Simplex Virus Type 1 in Cells of Latently Infected Mouse Trigeminal Ganglia Cultured in Serum-Free Medium

Herpes simplex virus type 1 (HSV-1) was reactivated more rapidly in cells of latently infected mouse trigeminal ganglia which were cultured in serum-free medium (after 3.7 days of cultivation) than in those cultured in serum-containing Dulbecco's modified Eagle's medium (after 8.5 days of cultivation). The concentration of calcium ion (Ca²⁺) in the medium affected HSV-1 reactivation in ganglionic cultures, and 0.9 mM was the optimum concentration for the reactivation. Reactivation was delayed significantly in ganglia put into culture 4 months or more after infection compared with those cultured 1 month after infection.     

The Probability of In Vivo Reactivation of Herpes Simplex Virus Type 1 Increases with the Number of Latently Infected Neurons in the Ganglia

The purpose of this study was to define the relationship between herpes simplex virus (HSV) latency and in vivo ganglionic reactivation. Groups of mice with numbers of latently infected neurons ranging from 1.9 to 24% were generated by varying the input titer of wild-type HSV type 1 strain 17syn+. Reactivation of the virus in mice from each group was induced by hyperthermic stress. The number of animals that exhibited virus reactivation was positively correlated with the number of latently infected neurons in the ganglia over the entire range examined (r = 0.9852, P < 0.0001 [Pearson correlation]).     

Herpes Simplex Virus Type 1 Renders Infected Cells Resistant to Cytotoxic T-Lymphocyte-Induced Apoptosis

Many viruses interfere with apoptosis of infected cells, presumably preventing cellular apoptosis as a direct response to viral infection. Since cytotoxic T lymphocytes (CTL) induce apoptosis of infected cells as part of the “lethal hit,” inhibition of apoptosis could represent an effective immune evasion strategy. We report here herpes simplex virus type 1 (HSV-1) interference with CTL-induced apoptosis of infected cells and show that HSV-1 inhibits the nuclear manifestations of apoptosis but not the membrane changes. The HL-60 cell line (human promyelocytic leukemia) undergoes apoptosis in response to many stimuli, including incubation with ethanol. After HSV-1 infection (strains E115 and 17+), ethanol-treated cells did not produce oligonucleosomal DNA fragments characteristic of apoptosis, as assayed by gel electrophoresis and enzyme-linked immunosorbent assay. Inhibition was detected 2 h after infection and increased over time. Importantly, HSV-1-infected cells were resistant to apoptosis induced by antigen-specific CD4⁺ CTL, despite the fact that CTL recognition and degranulation in response to infected targets remained intact. Unlike HSV-1, HSV-2 (strains 333 and HG52) did not inhibit DNA fragmentation. In contrast to the inhibition of DNA fragmentation by HSV-1, none of the HSV-1 or -2 strains interfered with the ethanol-induced exposure of surface phosphatidylserine characteristic of apoptosis, as determined by annexin V binding. These results demonstrate that genes of HSV-1 inhibit the nuclear manifestations of apoptosis but not the membrane manifestations, suggesting that these may be mediated via separate pathways. They also suggest that HSV-1 inhibition of CTL-induced apoptosis may be an important mechanism of immune evasion.     

Interleukin-18 Protects Mice against Acute Herpes Simplex Virus Type 1 Infection

We examined the effects of interleukin-18 (IL-18) in a mouse model of acute intraperitoneal infection with herpes simplex virus type 1 (HSV-1). Four days of treatment with IL-18 (from 2 days before infection to 1 day after infection) improved the survival rate of BALB/c, BALB/c nude, and BALB/c SCID mice, suggesting innate immunity. One day after infection, HSV-1 titers were higher in the peritoneal washing fluid of control BALB/c mice than in that of IL-18-treated mice. A genetic deficiency of gamma interferon (IFN-γ), however, diminished the survival rate and the inhibition of HSV-1 growth at the injection site in the mice. Anti-asialo GM1 treatment had no influence on the protective effect of IL-18 in infected mice. IL-18 augmented IFN-γ release in vitro by peritoneal cells from uninfected mice, while no appreciable IFN-γ production was found in uninfected mice administered IL-18. Although IFN-γ has the ability to induce nitric oxide (NO) production by various types of cells, administration of the NO synthase inhibitor N^G-monomethyl-l-arginine resulted in superficial loss of the improved survival, but there was no influence on the inhibition of HSV-1 replication at the injection site in IL-18-treated mice. Based on these results, we propose that IFN-γ produced before HSV-1 infection plays a key role as one of the IL-18-promoted protection mechanisms and that neither NK cells nor NO plays this role.Interleukin-18 (IL-18) is a newly cloned murine and human cytokine (28, 36) previously called gamma interferon (IFN-γ)-inducing factor. It is synthesized by activated macrophages and has a structural relationship to the IL-1 family (5). Precursor IL-18 is processed by IL-1β-converting enzyme and is cleaved into mature IL-18 (11). IL-18 induces IFN-γ production by murine helper T cells and NK cells and stimulates T-cell proliferation and NK activation (18, 28). Moreover, IL-18 augments the Fas ligand-mediated cytotoxic activity of the Th1 clone and the NK cell clone (8, 35). Thus, IL-18 shares some biological activities with IL-12, although no significant homology between the two cytokines has been detected at the protein level (34). Furthermore, treatment with IL-12 and IL-18 has a synergistic effect on IFN-γ production (2, 14, 38, 40).According to a review by Nash (27), not only nonspecific or innate immunity, such as that from IFN, NK cells, or macrophages, but also specific or adaptive immunity is important in protection against herpesvirus infection. Herpes simplex virus is known to be an IFN inducer (13). IFN is produced at an early stage of virus infection. In addition to the direct inhibition of viral replication, it enhances the efficiency of the adaptive (specific) immune response by stimulating increased expression of major histocompatibility complex class I and II or by activating macrophages and NK cells. In protection from infection by herpesviruses, especially cytomegalovirus, NK cells have been major effector cells because of the correlation of increased susceptibility to cytomegalovirus infection with the absence or reduction of NK cell activity, as seen in Chediak-Higashi syndrome patients and beige mice (27). Upon target cell disruption, NK and cytotoxic T cells share not only the perforin but also the Fas ligand as an effector molecule (4, 20, 37). Recently, nitric oxide (NO) was reported to be involved in host defense against bacteria, fungi, parasites, and viruses (10, 16, 19, 39). NO produced by herpes simplex virus type 1 (HSV-1)-infected macrophages is reported to inhibit viral replication (7). CD4⁺ T cells, macrophages, IFN-γ, and tumor necrosis factor (TNF) are important in adaptive immunity against HSV-1 infection. The Th2 response exacerbates HSV-1-induced disease (25).Recently a protective role of IL-18 was reported in microbial infections (6, 17). Here, we demonstrate that IL-18 treatment protects mice from acute viral infection via both IFN-γ-dependent and -independent pathways. Although IFN-γ has the ability to induce NO production by a variety of cells, including macrophages (9), it is not likely to be important, at least in the inhibition of HSV-1 proliferation at the injection site of IL-18-treated mice. Furthermore, the protective effect of IL-18 on HSV-1 infection also does not seem to require complete NK cell activity in our experimental system, whereas our colleagues have already reported that deletion of NK cells by administration of anti-asialo GM1 antibody resulted in lowering of the improved survival rate of tumor-bearing mice treated with IL-18 (23).     

Identification of Rep-Associated Factors in Herpes Simplex Virus Type 1-Induced Adeno-Associated Virus Type 2 Replication Compartments

Adeno-associated virus (AAV) is a human parvovirus that replicates only in cells coinfected with a helper virus, such as adenovirus or herpes simplex virus type 1 (HSV-1). We previously showed that nine HSV-1 factors are able to support AAV rep gene expression and genome replication. To elucidate the strategy of AAV replication in the presence of HSV-1, we undertook a proteomic analysis of cellular and HSV-1 factors associated with Rep proteins and thus potentially recruited within AAV replication compartments (AAV RCs). This study resulted in the identification of approximately 60 cellular proteins, among which factors involved in DNA and RNA metabolism represented the largest functional categories. Validation analyses indicated that the cellular DNA replication enzymes RPA, RFC, and PCNA were recruited within HSV-1-induced AAV RCs. Polymerase δ was not identified but subsequently was shown to colocalize with Rep within AAV RCs even in the presence of the HSV-1 polymerase complex. In addition, we found that AAV replication is associated with the recruitment of components of the Mre11/Rad50/Nbs1 complex, Ku70 and -86, and the mismatch repair proteins MSH2, -3, and -6. Finally, several HSV-1 factors were also found to be associated with Rep, including UL12. We demonstrated for the first time that this protein plays a role during AAV replication by enhancing the resolution of AAV replicative forms and AAV particle production. Altogether, these analyses provide the basis to understand how AAV adapts its replication strategy to the nuclear environment induced by the helper virus.Adeno-associated virus (AAV) is a human parvovirus that is currently used as a gene transfer vector (14). AAV particles consist of a small icosahedral capsid protecting a single 4.7-kb single-stranded DNA (ssDNA) genome with two open reading frames, rep and cap, surrounded by inverted terminal repeats (ITRs). The ITRs are the only sequences required in cis for genome replication and packaging. The rep gene encodes four nonstructural Rep proteins: Rep78, -68, -52, and -40. The two larger isoforms, Rep78 and -68, have origin binding, helicase, and site-specific endonuclease activities and are involved in AAV gene expression and genome processing, including replication and site-specific integration (39). The two smaller Rep isoforms are not required for AAV DNA replication but are involved in the control of viral gene expression and packaging of viral DNA (30).When wild-type (wt) AAV infects a cell in the absence of a helper virus, it enters latency. Latent AAV genomes persist in cells either as episomes or as integrated genomes, preferentially at a specific locus (named AAVS1) on human chromosome 19. In most instances, no detectable viral gene expression or genome replication occurs unless the cell is co- or superinfected by a helper virus, such as adenovirus, herpes simplex virus type 1 (HSV-1), or HSV-2. Under these conditions, AAV replication and assembly take place in large intranuclear domains called replication compartments (RCs) that frequently colocalize with replication domains formed by the helper virus itself (81). The viral genome replicates by leading-strand synthesis and generates new ssDNA molecules by a strand displacement mechanism that occurs after strand- and site-specific cleavage of viral DNA by Rep78/68 within the ITRs (39).Studies conducted on the relationship between AAV and its helper viruses are important not only to identify helper activities that can be used to produce recombinant AAV vectors but also to understand how AAV adapts its replication strategy to the helper virus and to the nuclear environment in general. Adenovirus helper functions have historically been the first and most extensively studied functions. These studies have shown that adenovirus helps AAV by stimulating viral gene expression and by enhancing AAV genome replication, mostly indirectly (19). Indeed, early studies showed that the adenovirus polymerase (E2b) is dispensable for AAV replication (8) and that the viral DNA-binding protein (DBP), the product of the E2a gene, is able to modestly enhance the processivity of AAV genome replication in vitro (77). More recently, the adenovirus proteins E1b55k and E4orf6 were shown to stimulate AAV genome replication by degrading the cellular Mre11/Rad50/Nbs1 (MRN) complex that restricts AAV genome replication during adenovirus coinfection (32). The concept that AAV genome replication can rely mostly, if not uniquely, on direct help from cellular factors was further strengthened by the demonstration that purified proteins such as replication protein A (RPA), replication factor C (RFC), proliferating cell nuclear antigen (PCNA), minichromosome maintenance (MCM) proteins, and DNA polymerase δ (Pol δ) were sufficient to replicate the AAV genome in vitro in the presence of Rep (40-41, 43). The involvement of these cellular proteins during AAV genome replication was also confirmed by the proteomic analysis of factors associated with Rep proteins during adenovirus-induced AAV replication (42).Interestingly, studies conducted on HSV-1 helper activities suggest that the strategy of AAV replication may vary depending on the helper virus. Indeed, previous studies showed that the HSV-1 helicase-primase (HP) complex (UL5/8/52) and DBP (ICP8) could replicate transfected AAV-2 plasmids (80) and that the helicase activity, but not primase activity, of the HP complex was required for this effect (62, 66). More recently, a comprehensive study of HSV-1 helper activities demonstrated that the HSV-1 immediate-early proteins ICP0, ICP4, and ICP22 could stimulate rep gene expression, probably by diminishing intrinsic antiviral effects (1, 18). In addition, the HSV-1 DNA polymerase encoded by UL30, along with its associated processivity factor (UL42), although not strictly required, was demonstrated to significantly increase AAV replication levels induced in the presence of the HP complex and ICP8. Interestingly, the HSV-1 HP complex, DBP, and polymerase were also shown to be sufficient to replicate AAV DNA in vitro in the presence of Rep proteins without any cellular protein (78). Altogether, these observations indicate that in the context of an HSV-1 coinfection, AAV relies extensively on viral activities provided by the helper that directly participate in AAV genome replication.To further elucidate the strategy of AAV replication in the presence of HSV-1, we undertook a proteomic analysis to identify the cellular and HSV-1 factors associated with Rep proteins and, consequently, potentially recruited within AAV RCs. To analyze Rep-associated proteins in the presence and absence of HSV-1 DNA replication, this analysis was performed using wt HSV-1 and an HSV-1 mutant in which the DNA polymerase encoded by the UL30 gene is absent (HSVΔUL30). This study resulted in the identification of approximately 60 cellular proteins, among which the largest functional categories corresponded to factors involved in DNA and RNA metabolism. Immunofluorescence analyses confirmed that in the presence of HSV-1, a basal set of cellular DNA replication enzymes, including RPA, RFC, and PCNA, was recruited within AAV RCs, with the exception of the MCM helicases. The cellular DNA polymerases, in particular Pol δ, were not identified by this analysis but subsequently were shown to be recruited in AAV RCs even in the presence of the HSV-1 polymerase complex. In addition, our results indicate that AAV replication induced by HSV-1 is associated with the recruitment of DNA repair factors, including components of the MRN complex, the Ku proteins, PARP-1, and factors of the mismatch repair (MMR) pathway. Finally, several HSV-1 proteins, most notably the UL12 protein, were also identified within AAV RCs. Our analyses confirmed the association between UL12 and Rep and demonstrated for the first time that this viral exonuclease plays a critical role during AAV replication by enhancing the formation of discrete AAV replicative forms and the production of AAV particles.Altogether, these results indicate that in the presence of HSV-1, AAV may replicate by using a basal set of cellular DNA replication enzymes but also relies extensively on HSV-1-derived proteins for its replication, including UL12, a newly discovered helper factor. These results suggest that AAV may be able to differentially adapt its replication strategy to the nuclear environment induced by the helper virus.     

Alterations of Neutral Glycolipids in Cells Infected with Syncytium-Producing Mutants of Herpes Simplex Virus Type 1

The isolation of syncytium-producing mutants of herpes simplex virus type 1 (KOS strain), which cause extensive cell fusion during otherwise normal infections, has been reported previously (S. Person, R. W. Knowles, G. S. Read, S. C. Warner, and V. C. Bond, J. Virol. 17:183-190, 1976). Seven of these mutants, plus two syncytial strains obtained elsewhere, were used to compare the incorporation of labeled galactose into neutral glycolipids of mock-infected, wild-type-infected, and syncytially infected human embryonic lung cells. Five predominant cellular glycolipid species were observed, denoted GL-1 through GL-5 in order of increasing oligosaccharide chain length; for example, GL-1 and GL-2 correspond to glycolipids that contain mono- and disaccharide units, respectively. Wild-type virus infection caused an increase in galactose incorporation into GL-1 and GL-2 relative to GL-3 through GL-5. For a single labeling interval from 4 to 10 h after adsorption, syncytial infections generally resulted in a relatively greater incorporation into more complex glycolipids than did wild-type infections. One mutant, syn 20, was compared with wild-type virus throughout infection by using a series of shorter labeling pulses and appeared to delay by at least 2 h the alterations observed during wild-type infections. These alterations are apparently due to defects in synthesis, since prelabeled cellular glycolipids were not differentially degraded during mock or virus infection.     

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.     

Reactivation of Herpes Simplex Virus Type 1 in the Mouse Trigeminal Ganglion: an In Vivo Study of Virus Antigen and Cytokines

Reactivation of herpes simplex virus type 1 (HSV-1) in the trigeminal ganglion (TG) was induced by UV irradiation of the corneas of latently infected mice. Immunocytochemistry was used to monitor the dynamics of cytokine (interleukin-2 [IL-2], IL-4, IL-6, IL-10, gamma interferon [IFN-γ], and tumor necrosis factor alpha [TNF-α]) and viral antigen production in the TG and the adjacent central nervous system on days 1 to 4, 6, 7, and 10 after irradiation. UV irradiation induced increased expression of IL-6 and TNF-α from satellite cells in uninfected TG. In latently infected TG, prior to reactivation, all satellite cells were TNF-α⁺ and most were also IL-6⁺. Reactivation, evidenced by HSV-1 antigens and/or infiltrating immune cells, occurred in 28 of 45 (62%) TG samples. Viral antigens were present in the TG in neurons, often disintegrating on days 2 to 6 after irradiation. Infected neurons were usually surrounded by satellite cells and the foci of immune cells producing TNF-α and/or IL-6. IL-4⁺ cells were detected as early as day 3 and were more numerous by day 10 (a very few IL-2⁺ and/or IFN-γ⁺ cells were seen at this time). No IL-10 was detected at any time. Our observations indicate that UV irradiation of the cornea may modulate cytokine production by satellite cells. We confirm that neurons are the site of reactivation and that they probably do not survive this event. The predominance of TNF-α and IL-6 following reactivation parallels primary infection in the TG and suggests a role in viral clearance. The presence of Th2-type cytokines (IL-4 and IL-6) indicates a role for antibody. Thus, several clearance mechanisms may be at work.     

Effect of Heparin on Hemagglutinin of Herpes Simplex Virus Type 1

Heparin inhibited the hemagglutinin activity of herpes simplex virus (HSV) type 1. The minimal inhibitory concentration of heparin required to inhibit 8 hemagglutination (HA) U of HSV ranged from 0.005 to 0.01 U/ml. Mouse erythrocytes failed to combine with the HA inhibitory factor of heparin. On the other hand, mouse erythrocytes treated with heparinase had greatly reduced agglutinability by HSV. Virus-heparin complex formation was observed by sedimenting heparin with the virus particles.     

Persistence of Herpes Simplex Virus Type 1 DNA in Chronic Conjunctival and Eyelid Lesions of Mice

Herpes simplex virus type 1 (HSV-1) causes chronic blepharitis and conjunctivitis as well as keratitis in humans. The pathogenesis of these inflammatory ocular and dermal lesions is not well understood. We have examined the persistence of HSV-1 DNA and its relationship to inflammatory lesions in the conjunctiva and eyelid skin of mice which were inoculated with HSV-1 by the corneal route. Viral DNA was detected by in situ PCR in the conjunctiva and eyelid tissue of infected mice at 5, 11, 23, and 37 days postinfection (p.i.). This DNA was localized in the epithelial cells of the conjunctiva and hair follicles and in the epidermal cells of the eyelid skin. Viral proteins were not detected in the conjunctiva or the eyelid skin after 5 days p.i., even though histopathological lesions were found at 23 and 37 days p.i. in both tissues. The DNA-containing cells were adjacent to sites of inflammation in the chronic lesions in both the conjunctiva and the eyelid skin. A similar temporal and spatial relationship between HSV-1 DNA and inflammatory lesions has been previously reported for the cornea. Our data suggest that the lesions in the cornea, conjunctiva, and eyelid skin progress similarly. Further studies are required to determine whether the long-term presence of HSV-1 is involved in the mechanism by which these chronic inflammatory lesions develop. The presence of HSV-1 DNA in these extraocular tissues for extended periods may constitute persistent viral infection of nonneuronal cells.     

疱疹病毒Ⅱ型感染SH-SY5Y细胞的生物学效应

目的：探讨疱疹病毒Ⅱ型（HSV-2）感染人神经母细胞瘤细胞株SH-SY5Y的生物学效应。方法：病毒液接种SH-SY5Y细胞后,用相差和电子显微镜观察感染细胞的形态变化,RT-PCR检测病毒在细胞中的增殖,MTT法检测病毒感染对细胞增殖的影响,流式细胞仪测定感染后的细胞凋亡状况。结果：相差显微镜显示细胞病变,从24～72h,细胞变性、坏死的程度和数量随感染时间延长而增加;电镜结果显示感染24h后,细胞核染色质固缩,出现多核巨细胞,线粒体内嵴紊乱、断裂,出现不同程度的自噬化、溶酶体化、空泡化,并可见大量鹰眼样已包装成熟的病毒颗粒及正在包装的病毒粒子;HSV-2LAT基因RT-PCR扩增表明,病毒能在SH-SY5Y细胞中增殖;凋亡检测显示HSV-2在体外细胞感染中并未使细胞出现凋亡现象;感染后24、48及72h,SH-SY5Y细胞的抑制率分别为11.3%、31.2%和63.1%,与对照组相比均存在显著性差异（P〈0.05）;分别用0.1、1、10MOI的病毒感染SH-SY5Y细胞,上述不同组在24、48、72h时细胞形态变化基本一致,感染结果相似,各组之间病毒毒力无明显差异（P〉0.05）。结论：初步在人神经母细胞瘤细胞株SH—SY5Y中建立了HSV-2感染的细胞模型,并研究了感染对细胞生物性状的影响,为探讨HSV-2的潜伏与激发机制、了解HSV-2的致病机制打下基础。     

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.