Uncoating the herpes simplex virus genome

Initiation of infection by herpes simplex virus (HSV-1) involves a step in which the parental virus capsid docks at a nuclear pore and injects its DNA into the nucleus. Once "uncoated" in this way, the virus DNA can be transcribed and replicated. In an effort to clarify the mechanism of DNA injection, we examined DNA release as it occurs in purified capsids incubated in vitro. DNA ejection was observed following two different treatments, trypsin digestion of capsids in solution, and heating of capsids after attachment to a solid surface. In both cases, electron microscopic analysis revealed that DNA was ejected as a single double helix with ejection occurring at one vertex presumed to be the portal. In the case of trypsin-treated capsids, DNA release was found to correlate with cleavage of a small proportion of the portal protein, UL6, suggesting that UL6 cleavage may be involved in making the capsid permissive for DNA ejection. In capsids bound to a solid surface, DNA ejection was observed only when capsids were warmed above 4 degrees C. The proportion of capsids releasing their DNA increased as a function of incubation temperature with nearly all capsids ejecting their DNA when incubation was at 37 degrees C. The results demonstrate heterogeneity among HSV-1 capsids with respect to their sensitivity to heat-induced DNA ejection. Such heterogeneity may indicate a similar heterogeneity in the ease with which capsids are able to deliver DNA to the infected cell nucleus.     

Nuclear delivery mechanism of herpes simplex virus type 1 genome

Herpes simplex virus type 1 (HSV-1) is a widespread human pathogen infecting more than 80% of the population worldwide. Its replication involves an essential, poorly understood multistep process, referred to as uncoating. Uncoating steps are as follows: (1) The incoming capsid pinpoints the nuclear pore complex (NPC). (2) It opens up at the NPC and releases the highly pressurized viral genome. (3) The viral genome translocates through the NPC. In the present review, we highlight recent advances in this field and propose mechanisms underlying the individual steps of uncoating. We presume that the incoming HSV-1 capsid pinpoints the NPC by hydrophobic interactions and opens up upon binding to NPC proteins. Genome translocation is initially pressure-driven.     

Structural variability in the genome of the Thermoproteus tenax virus TTV1

Summary Six variants of the TTV1 genome, including the primary isolate, have been characterized. DNA sequence comparison of wild-type virus (WT) and one of the variants (VT3) showed that differences are due to insertions and deletions that were confined to contiguous portions of two distinctClaI fragments. Seven similar short DNA sequences (30–102 bp) were involved in the variation. The deletions and insertions of these short DNA sequences occurred in every case adjacent to the 8 by consensus sequence 5-ACXCCTAC-3 which formed the 5 flank of the segments involved.     

Cloning of herpes simplex virus type 1 sequences representing the whole genome.

Sequences representative of the whole genome of herpes simplex virus type 1 (HSV-1) strain KOS were cloned in the plasmid vector pBR325 in the form of EcoRI-generated DNA fragments. The cloned fragments were identified by digestion of the chimeric plasmid DNA with restriction enzymes EcoRI or EcoRI and BglII followed by comparison of their electrophoretic mobilities in agarose gels with that of similarly digested HSV-1 virion DNA. The cloned fragments showed the same migration patterns as the corresponding fragments from restricted virion DNA, indicating that no major insertions or deletions were present. The presence of HSV-1 sequences in the chimeric plasmids was confirmed by hybridization of plasmid DNA to HSV-1 virion DNA. Additionally, some of the cloned fragments were shown to be biologicaly active in that they efficiently rescued three HSV-1 temperature-sensitive mutants in cotransfection marker rescue experiments.     

The genome nucleotide sequence of herpes simplex virus 1 strain L2

The genome nucleotide sequence of the reference strain of herpes simplex virus type 1 was obtained using the technique of full size sequencing. For the virus genome structure determination, 402444 reads with an average length of 202 bp were performed, which corresponded to the 542-fold genome coverage. The data were collected to 52 contigs with N50-4518 and the total contig length of 120929 bp. The sequence obtained was deposited into the GenBank database.     

In vitro nuclear egress of herpes simplex virus type 1 capsids

During their life cycles, viruses typically undergo many transport events throughout the cell. These events depend on a variety of both viral and host proteins and are often not fully understood. Such studies are often complicated by asynchronous infections and the concurrent presence of various viral intermediates in the cells, making it difficult to molecularly define each step. In the case of the herpes simplex virus type 1, the etiological agent of cold sores and many other illnesses, the viral particles undergo an intricate series of transport steps during its life cycle. Upon entry by fusion with a cellular membrane, they travel to the host cell nucleus where the virus replicates and assembles new viral particles. These particles then travel across the two nuclear envelopes and transit through the trans-Golgi network before finally being transported to and released at the cell surface. Though viral components and some host proteins modulating these numerous transport events have been identified, the details of these processes remain to be elucidated. To specifically address how the virus escapes the nucleus, we set up an in vitro model that reproduces the unconventional route used by herpes simplex type 1 virus to leave nuclei. This has not only allowed us to clarify the route of capsid egress of the virus but is now useful to define it at the molecular level.     

Reconstitution of herpes simplex virus type 1 nuclear capsid egress in vitro

Newly assembled herpesvirus capsids travel from the nucleus to the plasma membrane by a mechanism that is poorly understood. Furthermore, the contribution of cellular proteins to this egress has yet to be clarified. To address these issues, an in vitro nuclear egress assay that reproduces the exit of herpes simplex virus type 1 (HSV-1) capsids from nuclei isolated from infected cells was established. As expected, the assay has all the hallmarks of intracellular transport assays, namely, a dependence on time, energy, and temperature. Surprisingly, it is also dependent on cytosol and was slightly enhanced by infected cytosol, suggesting an implication of both host and viral proteins in the process. The capsids escaped these nuclei by budding through the inner nuclear membrane, accumulated as enveloped capsids between the two nuclear membranes, and were released in cytosol exclusively as naked capsids, exactly as in intact cells. This is most consistent with the view that the virus escapes by crossing the two nuclear membranes rather than through nuclear pores. Unexpectedly, nuclei isolated at the nonpermissive temperature from cells infected with a U(L)26 thermosensitive protease mutant (V701) supported capsid egress. Although electron microscopy, biochemical, and PCR analyses hinted at a likely reconstitution of capsid maturation, DNA encapsidation could not be confirmed by a traditional SQ test. This assay should prove very useful for identification of the molecular players involved in HSV-1 nuclear egress.     

Reconstitution of herpes simplex virus microtubule-dependent trafficking in vitro

Microtubule-mediated anterograde transport of herpes simplex virus (HSV) from the neuronal cell body to the axon terminal is crucial for the spread and transmission of the virus. It is therefore of central importance to identify the cellular and viral factors responsible for this trafficking event. In previous studies, we isolated HSV-containing cytoplasmic organelles from infected cells and showed that they represent the first and only destination for HSV capsids after they emerge from the nucleus. In the present study, we tested whether these cytoplasmic compartments were capable of microtubule-dependent traffic. Organelles containing green fluorescent protein-labeled HSV capsids were isolated and found to be able to bind rhodamine-labeled microtubules polymerized in vitro. Following the addition of ATP, the HSV-associated organelles trafficked along the microtubules, as visualized by time lapse microscopy in an imaging microchamber. The velocity and processivity of trafficking resembled those seen for neurotropic herpesvirus traffic in living axons. The use of motor-specific inhibitors indicated that traffic was predominantly kinesin mediated, consistent with the reconstitution of anterograde traffic. Immunocytochemical studies revealed that the majority of HSV-containing organelles attached to the microtubules contained the trans-Golgi network marker TGN46. This simple, minimal reconstitution of microtubule-mediated anterograde traffic should facilitate and complement molecular analysis of HSV egress in vivo.     

Influencing of ionizing radiation on herpes simplex virus and its genome

The effects of cobalt-60 gamma-rays, 10 MeV electrons and 52 MeV deutrons on the survival of plaque-forming ability has been studied in various strains of herpes simplex virus (HSV). The results show that the D0 for the loss of plaque-forming ability in different HSV strains lies in the range 1-3 kGy. Irradiation of isolated HSV-1 DNA with cobalt-60 gamma-rays resulted in damage, as indicated by electrophoresis of purified viral DNA and by restriction endonuclease analysis, at doses of 1 kGy, with complete loss of structure at doses above 4 kGy. The infectivity of the irradiated naked DNA was lost at doses above 4 kGy, but after irradiation of the intact virus some plaque-forming ability was retained after doses of 10 or even 40 kGy. Thus the organization within the viral capsid may play a protective role by modifying the severity of the radiation damage, and preserving at least some degree of infectivity.     

Mapping of the herpes simplex virus DNA sequences present in herpes simplex virus type-1 thymidine kinase-transformed cells

Analyses of the herpes simplex virus (HSV) DNA sequences which are present in three HSV thymidine kinase-transformed (HSVtk+) mouse cell lines have revealed that these cells contain relatively large and variable portions of the viral genome. Two of these cell lines do not contain the viral DNA sequences known to encode the early viral genes normally responsible for regulating tk gene expression during lytic HSV infections. This finding suggests that cell-associated viral tk gene expression may be regulated by cellular rather than viral control mechanisms. In addition, we have compared the viral DNA sequences present in one unstable HSVtk+ cell line to those present in tk- revertant and tk+ rerevertant cell lines sequentially derived from it. Our results have shown that within the limits of sensitivity of our mapping approach, these three related cell lines contain the same set of viral DNA sequences. Thus, gross changes in viral DNA content do not appear to be responsible for the different tk phenotypes of these cells.     

Genetic variability of herpes simplex virus: development of a pathogenic variant during passaging of a nonpathogenic herpes simplex virus type 1 virus strain in mouse brain.

Herpes simplex virus type 1 ANG (HSV-1 ANG) is originally nonpathogenic for inbred mice upon intraperitoneal intravenous, or intravaginal inoculation. In contrast, mice died of encephalitis within 4 to 5 days after intracerebral inoculation with this strain. HSV-1 ANG was serially passaged in mouse brains. In two independent series, peripherally pathogenic virus variants had developed and accumulated in the virus progeny after 12 to 15 intracerebral passages. In mixed infections both nonpathogenic and pathogenic viruses replicated at the primary site of infection and spread to various organs. However, only the pathogenic phenotype could be recovered from the spinal cord and the brain. Comparison of the restriction enzyme cleavage patterns of pathogenic ANG and nonpathogenic ANG virus DNAs revealed distinct alterations in the S-segment (US) sequences bounded by coordinates 0.953 and 0.958 in the prototype orientation and by coordinates 0.862 to 0.867 in the IS orientation of the viral genome. However, it is not known whether these alterations are physiologically relevant to the observed changes in pathogenicity. When coinjected intraperitoneally at 50 to 100-fold excess, the nonpathogenic HSV-1 ANG protected mice against its own pathogenic variant as well as against other pathogenic HSV-1 strains. Pathogenic HSV-1 ANG proved to be genetically and phenotypically stable for at least 25 serial passages in tissue culture at either high or low multiplicity of infection.     

Structural characterization of the UL25 DNA-packaging protein from herpes simplex virus type 1

Herpesviruses replicate their double stranded DNA genomes as high-molecular-weight concatemers which are subsequently cleaved into unit-length genomes by a complex mechanism that is tightly coupled to DNA insertion into a preformed capsid structure, the procapsid. The herpes simplex virus type 1 UL25 protein is incorporated into the capsid during DNA packaging, and previous studies of a null mutant have demonstrated that its function is essential at the late stages of the head-filling process, either to allow packaging to proceed to completion or for retention of the viral genome within the capsid. We have expressed and purified an N-terminally truncated form of the 580-residue UL25 protein and have determined the crystallographic structure of the region corresponding to amino acids 134 to 580 at 2.1-Angstroms resolution. This structure, the first for any herpesvirus protein involved in processing and packaging of viral DNA, reveals a novel fold, a distinctive electrostatic distribution, and a unique "flexible" architecture in which numerous flexible loops emanate from a stable core. Evolutionary trace analysis of UL25 and its homologues in other herpesviruses was used to locate potentially important amino acids on the surface of the protein, leading to the identification of four putative docking regions for protein partners.     

Herpes simplex virus type 1 DNA is immunostimulatory in vitro and in vivo

Recently, prokaryotic DNAs containing unmethylated CpG motifs have been shown to be intrinsically immunostimulatory both in vitro and in vivo, tending to promote Th1-like responses. In contrast, CpG dinucleotides in mammalian DNAs are extensively methylated on cytosines and hence immunologically inert. Since the herpes simplex virus (HSV) genome is unmethylated and G+C rich, we predicted that CpG motifs would be highly prevalent in the HSV genome; hence, we examined the immunostimulatory potential of purified HSV DNA in vitro and in vivo. Mouse splenocyte cultures treated with HSV DNA or HSV-derived oligodeoxyribonucleotides (ODNs) showed strong proliferative responses and production of inflammatory cytokines (gamma interferon [IFN-γ], tumor necrosis factor [TNF], and interleukin-6 [IL-6]) in vitro, whereas splenocytes treated with mammalian CV-1 DNA or non-CpG ODN did not. After immunization with ovalbumin (OVA), only splenocytes from mice immunized with HSV DNA or HSV-ODN as the adjuvants proliferated strongly and produced typical Th1 responses, including CD8⁺ cytotoxic T-lymphocyte responses, upon restimulation with OVA. Furthermore, HSV-ODN synergized with IFN-γ to induce nitric oxide (NO), IL-6, and TNF production from macrophages. These results demonstrate that HSV DNA and HSV-ODN are immunostimulatory, driving potent Th1 responses both in vitro and in vivo. Considering that HSV DNA has been found to persist in nonneuronal cells, these results fuel speculation that HSV DNA might play a role in pathogenesis, in particular, in diseases like herpes stromal keratitis (HSK) that involve chronic inflammatory responses in the absence of virus or viral antigens.     

Analysis of the herpes simplex virus genome during in vitro latency in human diploid fibroblasts and rat sensory neurons.

We have previously designed in vitro model systems to characterize the herpes simplex virus type 1 (HSV-1) genome during in vitro virus latency. Latency was established by treatment of infected human embryo lung fibroblast (HEL-F) cells or rat fetal neurons with (E)-5-(2-bromovinyl)-2'-deoxyuridine and human leukocyte interferon and was maintained by increasing the incubation temperature after inhibitor removal. Virus was reactivated by reducing the incubation temperature. We have now examined the HSV-1-specific DNA content of latently infected HEL-F cells and rat fetal neurons treated with (E)-5-(2-bromovinyl)-2'-deoxyuridine and human leukocyte interferon and increased temperature. The HEL-F cell population contained, on an average, between 0.25 and 0.5 copies of most, if not all, HSV-1 HindIII and XbaI DNA fragments per haploid cell genome equivalent. In contrast, the latently infected neurons contained, on an average, 8 to 10 copies per haploid cell genome equivalent of most HSV-1 BamHI DNA fragments. There was no detectable alteration in size or molarity of the HSV-1 terminal or junction DNA fragments obtained by HindIII, XbaI, or BamHI digestion of the latently infected neuron or HEL-F cell DNA, as compared with digestion of a reconstruction mixture of purified HSV-1 virion and HEL-F cell DNAs. These data suggest that the predominant form of the HSV-1 genome in either latently infected cell population is nonintegrated, linear, and nonconcatameric.     

The a sequence is dispensable for isomerization of the herpes simplex virus type 1 genome.

The herpes simplex virus type 1 (HSV-1) genome consists of two components, L (long) and S (short), that invert relative to each other during productive infection to generate four equimolar isomeric forms of viral DNA. Recent studies have indicated that this genome isomerization is the result of DNA replication-mediated homologous recombination between the large inverted repeat sequences that exist in the genome, rather than site-specific recombination through the terminal repeat a sequences present at the L-S junctions. However, there has never been an unequivocal demonstration of the dispensability of the latter element for this process using a recombinant virus whose genome lacks a sequences at its L-S junctions. This is because the genetic manipulations required to generate such a viral mutant are not possible using simple marker transfer, since the cleavage and encapsidation signals of the a sequence represent essential cis-acting elements which cannot be deleted outright from the viral DNA. To circumvent this problem, a simple two-step strategy was devised by which essential cis-acting sites like the a sequence can be readily deleted from their natural loci in large viral DNA genomes. This method involved initial duplication of the element at a neutral site in the viral DNA and subsequent deletion of the element from its native site. By using this approach, the a sequence at the L-S junction was rendered dispensable for virus replication through the insertion of a second copy into the thymidine kinase (TK) gene of the viral DNA; the original copies at the L-S junctions were then successfully deleted from this virus by conventional marker transfer. The final recombinant virus, HSV-1::L-S(delta)a, was found to be capable of undergoing normal levels of genome isomerization on the basis of the presence of equimolar concentrations of restriction fragments unique to each of the four isomeric forms of the viral DNA. Interestingly, only two of these genomic isomers could be packaged into virions. This restriction was the result of inversion of the L component during isomerization, which prevented two of the four isomers from having the cleavage and encapsidation signals of the a sequence in the TK gene in a packageable orientation. This phenomenon was exploited as a means of directly measuring the kinetics of HSV-1::L-S(delta)a genome isomerization. Following infection with virions containing just the two packaged genomic isomers, all four isomers were readily detected at a stage in infection coincident with the onset of DNA replication, indicating that the loss of the a sequence at the L-S junction had no adverse effect on the frequency of isomerization events in this virus. These results therefore validate the homologous recombination model of HSV-1 genome isomerization by directly demonstrating that the a sequence at the L-S junction is dispensable for this process. The strategy used to remove the a sequence from the HSV-1 genome in this work should be broadly applicable to studies of essential cis-acting elements in other large viral DNA molecules.     

Expression of an early, nonstructural antigen of herpes simplex virus in cell transformed in vitro by herpes simplex virus.

Hyperimmune rabbit antiserum to an early, nonstructural herpes simplex virus type 2 (HSV-2)-induced polypeptide (VP143) reacted in immunofluorescence tests with a variety of cell lines transformed by HSV-2. Cytoplasmic fluorescence was observed in 10 to 50% of HSV-2-transformed cells, whereas no fluorescence was observed in cells transformed by other oncogenic DNA viruses or by a chemical carcinogen. VP143-specific reactivity could be absorbed from anti-VP143 serum with HSV-2-transformed cells but not with cells transformed by other agents. When HSV-2-transformed cells were synchronized in mitosis and examined at various times postmitosis for VP143-specific fluorescence, the expression of VP143 was shown to be cell cycle dependent.