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 tkgene expression during lytic HSV infections. This finding suggests that cell-associated viral tkgene 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 ^– revenant 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.     

Analysis of complex mutations induced in cells by herpes simplex virus type-1.

The shuttle vector plasmid pZ189 was used as a target for mutagenesis in COS-1 cells. Complex mutations were analyzed in 5 plasmids that were recovered from noninfected cells and 15 plasmids that were recovered from cells that were infected with herpes simplex virus type-1 (HSV-1). Complex mutations in noninfected cells consisted of duplications and rearrangements of plasmid DNA, while those from cells that were infected with HSV-1 included 8 that were enlarged due to insertion of other DNA sequences. Plasmids that contained inserted DNA showed an increase in size of from 118 bp up to around 4500 bp. Maps were constructed based on restriction enzyme digestion, and some or all of each plasmid was examined by DNA sequencing. The inserted DNA was not derived from HSV-1 in any case, since it did not hybridize to DNA from HSV-1 and showed no sequence similarities to the virus. Instead, inserted DNA was found to hybridize to HindIII-digested cellular DNA as a single or double band in 5 plasmids and contained multiple repeat sequences such as alpha satellite, Alu or Kpn repeats in 4 plasmids. In four enlarged plasmids the identity of the inserted sequences could not be determined. The junctions between the shuttle vector and the inserted DNA did not show features of transposable elements and no homology was detected between inserted sequences and the sequence at the insertion site. No preferred site for recombination was detected. Although no similarities were found among the inserted sequences, it is possible that the cellular sequences represent cellular targets for virus-mediated rearrangement. It appears that HSV-1 stimulates nonhomologous recombination between DNA sequences in virus-infected cells.     

DNA sequence of mutations induced in cells by herpes simplex virus type-1

The shuttle vector plasmid pZ189 was used to find the kinds of mutations that are induced in cells by herpes simplex virus type-1 (HSV-1). A significant increase in mutation frequency was detected as early as 2 hr after infection, and reached a peak of two- to sevenfold over background at 4 hr after infection. Several differences were detected between spontaneous mutants and those induced by HSV-1 when they were analyzed by gel electrophoresis and DNA sequencing. Point mutations accounted for 63% of spontaneous mutants but for only 44% of HSV-1-induced mutants (P less than 0.05). In each case the predominant type of point mutation was the G:C to A:T transition, which comprised 51% of point mutations induced by HSV-1, and 32% of spontaneous point mutations. Deletions of DNA were seen in HSV-1-induced mutants at a frequency of 44%, compared with only 29% in spontaneous mutants. HSV-1-induced deletions were less than half the length of spontaneous deletions, and 3 contained short filler sequences. An increase in size was seen in 13% of HSV-1-induced mutants and was due either to duplication of plasmid DNA, or, in 8 instances, to insertion of sequences derived from cellular DNA. Among spontaneous mutants, only 8% were increased in size and none of them had inserted cellular DNA. The proportion of complex mutants increased as infection by the virus progressed and they accounted for 79% of mutants at 24 hr after infection. The observed mutations have implications for understanding the "hit and run" mechanism of malignant transformation of cells by HSV-1.     

A potential andrographolide analogue against the replication of herpes simplex virus type 1 in vero cells

Andrographolide (AD), and 14-deoxyandrographolide (DAD) isolated from Andrographis paniculata Nees, Acanthaceae, and 3,19-isopropylideneandrographolide (IPAD), a semi-synthetic compound of AD, were examined for anti-HSV-1 activity in vitro. The inhibitory effects of these compounds on viral entry and replication steps were determined using pre- and post-infection assays, respectively. All the three compounds exhibited less than 50% inhibitory act against viral entry. In the post-infection, IPAD displayed absolute inhibition, whereas AD and DAD gave moderate activity. IPAD was selected to determine for the stage of anti-replication by time-of-addition and time-of-removal assays. From the time of removal assay, IPAD activity began after 4 h and completed at 16 h post infection which corresponded to the early genes expression. Its ability to inhibit HSV-1 was confirmed by polymerase chain reaction and the expression of viral glycoproteins C and D by western blot analysis. No viral enveloped glycoproteins D and C expressions were found. IPAD exhibited anti-HSV-1 replication relating to the early step of replication. Structure-activity relationships of andrographolide against HSV-1 was proposed, it is the first report of this ent-labdane diterpene.     

Recovery from herpes simplex virus type-1 hepatitis in a female adult

Summary A study of one case of herpes simplex hepatitis in an adult woman is presented. The clinical feature and laboratory findings were typical for acute hepatitis in a febrile patient without herpetic mucocutaneous lesions. The evidence of high IgM antibody titer in serum against herpes simplex virus and confirmation of the herpes simplex virus hepatitis by immunofluorescent microscopy after liver biopsy helped us establish the diagnosis. After 3-months the patient recovered.Abbreviations HSV Herpes simplex virus - CMV Cytomegalovirus - EBV Epstein Barr virus     

Cellular expression of gH confers resistance to herpes simplex virus type-1 entry

Entry of herpes simplex virus-1 (HSV-1) into cells requires a concerted action of four viral glycoproteins gB, gD, and gH-gL. Previously, cell surface expression of gD had been shown to confer resistance to HSV-1 entry. To investigate any similar effects caused by other entry glycoproteins, gB and gH-gL were coexpressed with Nectin-1 in Chinese hamster ovary (CHO) cells. Interestingly, cellular expression of gB had no effect on HSV-1(KOS) entry. In contrast, entry was significantly reduced in cells expressing gH-gL. This effect was further analyzed by expressing gH and gL separately. Cells expressing gL were normally susceptible, whereas gH-expressing cells were significantly resistant. Further experiments suggested that the gH-mediated interference phenomenon was not specific to any particular gD receptor and was also observed in gH-expressing HeLa cells. Moreover, contrary to a previous report, gL-independent cell surface expression of gH was detected in stably transfected CHO cells, possibly implicating cell surface gH in the interference phenomenon. Thus, taken together these findings indicate that cellular expression of gH interferes with HSV-1 entry.     

Role of the major capsid protein in herpes simplex virus type-1 capsid assembly

Two herpes simplex virus type 1 (HSV-1) temperature-sensitive (ts) mutants with defects in the gene for the major capsid protein, ICP5, were examined for their effects on virion capsid assembly. Polyacrylamide gel electrophoresis revealed that both mutants were able to synthesize wild-type (WT) levels of ICP5 at the nonpermissive temperature. However, the 53 kD capsid protein disappeared concomitant with the appearance of a new, 51 kD species. These results, taken together with ultrastructural and immunological analyses indicate that the processing and assembly of capsid proteins, DNA packaging and thermal stability of HSV-1 virions are dependent upon functional ICP5.     

Differential regulation by varicella-zoster virus (VZV) and herpes simplex virus type-1 trans-activating genes

Transient expression assays were performed in Vero cells in order to compare varicella-zoster virus (VZV)-encoded trans-activating proteins [defined by the products of open reading frames (ORF) 4 and 62] with herpes simplex virus type-1 (HSV-1) trans-activating proteins, ICP4 and ICP0, with respect to activation of gene expression. We demonstrate that the product of VZV ORF4 and ORF62 (which are the HSV-1 analogs of ICP27 and ICP4, respectively) stimulate a variety of viral and cellular gene promoters, including the HSV-1 thymidine kinase (tk) promoter. On the other hand, expression of a recombinant vector containing the VZV tk promoter could not be stimulated, by HSV-1 infection or by the HSV-1 ICP4 or ICP0 proteins expressed during cotransfection experiments. These data suggest different mechanisms of activation of the VZV and the HSV-1 tk gene promoters by "trans-activating" factors.     

Synergistic interactions between herpes simplex virus type-2 and human immunodeficiency virus epidemics

Several factors clearly influence HSV-2 transmission, such as female gender and number of sexual contacts.3,4 Some are less well defined, and may be confounded by other factors, but include time since While the concept linking herpes simplex virus (HSV) shedding to transmission is plausible, few data support shedding as a surrogate marker. If shedding is to be used as a surrogate marker in future clinical studies, it must be clear how far shedding must be reduced, and what the shape of the transmission:viral load curve (or transmission:detection frequency curve) is before transmission is reduced. It remains unclear whether peak virus load, frequency of detection of virus, or the area under curve of the time:virus load plot is the critical parameter in the transmission of HSV. This paper reports on an international meeting of experts and a debate at the International Herpes Management Forum (IHMF) Annual Meeting (2004), convened to examine whether a surrogate marker for HSV transmission is necessary, and whether there is any evidence, either in studies involving HSV or other viral infections, to suggest that viral shedding could be used as such a surrogate.     

Interaction of ultraviolet-irradiated herpes simplex virus type 1 with BSC-1 cells.

Ultraviolet irradiation of herpes simplex virus (HSV) did not affect the transfer of uncoated virus DNA to the nuclei of infected cells but the synthesis of virus DNA was suppressed. The virus-specific DNA polymerase was synthesized in cells infected with the u.v.-irradiated HF strain of HSV. In cells infected with the u.v.-irradiated KOS strain, the virus DNA polymerase activity was hardly detectable. The two strains of HSV differ in the sensitivity of the virus DNA polymerase gene to u.v.-irradiation.     

Lactoferrin inhibits herpes simplex virus type-1 (HSV-1) infection to mouse cornea

Summary Lactoferrin inhibits bacterial growth in the conjunctival sac. However, its antiviral function particularly in ocular tissue has not been reported in the literature. We studied whether lactoferrin inhibits infection of herpes simplex virus type-1 (HSV-1) in vitro using Vero cell monolayer. We also tested the inhibitory effect of lactoferrin on HSV-1 infection in the mouse cornea. Lactoferrin prevented HSV-1 plaque formation. Administration of topical 1% lactoferrin prior to the virus inoculation suppressed infection on ocular tissue, however it did not inhibit propagation of the virus.     

Blocked herpes simplex virus type 2-specific DNA synthesis in simian virus 40-transformed hamster cells permissive for herpes simplex virus type 1.

Simian virus 40-transformed hamster cells (LL-1) permissive to herpes simplex virus type 1 (HSV-1) were shown to be relatively nonpermissive to HSV-2. When LL-1 cells were infected with HSV-2, there was a 3- to 4-log reduction in infectious viral progeny at 24 h postinfection as compared with HSV-1 under identical cultured conditions. HSV-2 could be carried in the LL-1 cell line for up to 12 passages without any appreciable cytopathology. Various early functions of the replicative cycle of HSV-2 appeared to be normal. Experiments demonstrated that early enzyme activity, HSV-2 thymidine kinase, and DNA polymerase appeared at permissive levels in extracts of HSV-2-infected LL-1 cells. However, DNA analysis of HSV-2 infected LL-1 cells demonstrated a block in HSV-2-specific DNA synthesis, although HSV-2 was capable of inhibiting DNA synthesis in LL-1 cells. Furthermore, indirect immunofluorescence studies indicate that late HSV-2 structural protein synthesis was inhibited in infected LL-1 cells. Thus, the inability of HSV-2 to replicate in LL-1 cells is due to a block at or before HSV-specific DNA synthesis, resulting in a reduction of the structural protein synthesis required for viral maturation.     

Effects of antimetabolites on the production of tubular structures in vero cells infected with herpes simplex virus type 2

Summary Treatment of Vero cells infected with herpes simplex virus type 2 with the proper concentrations of hydroxyurea (HU) reduced the production of infectious virus and markedly increased the accumulation of tubular structures in the nuclei when the drug was added within 6 hours after infection. Similar accumulation of tubular structures in the infected nuclei was also observed when infected cells were treated with phosphonoacetic acid at proper concentrations. The release of HU-treated, herpes simplex virus type 2-infected cells from the drug-induced blocking of synthesis of infectious virus resulted in the marked decrease of tubular structures coincidentally with the beginning of production of infectious virus. The data suggest the possibility that the disappearance of tubular structures may be related to the active production of infectious virus.With 12 Figures     

Expression of the herpes simplex virus type-2 major DNA-binding protein (ICP8) gene in COS-1 cells

By the use of an SV40 origin of replication plasmid vector and the COS-1 cell system, expression of the gene encoding the herpes simplex virus type-2 (HSV-2) major DNA binding protein (ICP8) has been achieved. The HSV-2 4.5 kb BgIII 0 DNA fragment containing the ICP8 coding region was inserted into the plasmid vector pSVOd containing the SV40 origin of replication. Transfection of COS-1 cells by the resultant recombinant plasmid (pSVOd20), and subsequent multiplication of this plasmid, led to the production of the HSV-2 major DNA binding protein in sufficient quantities to allow its detection with either monoclonal or polyclonal antibodies as well as sera taken from HSV-2 patients.     

Cross-reacting herpes simplex virus antigens in hamster and mouse cells transformed by ultraviolet light-inactivated herpes simplex virus type 2.

Murine and hamster cell lines, each transformed with a different strain of herpes simplex virus (HSV), were examined for cross-reacting antigens by in vitro and in vivo assays. A comparative study by the indirect immunofluorescence technique detected common cross-reacting viral antigens. Cytoplasmic fluorescence patterns were observed in the 333-8-9 hamster line, the H238 murine line, and the H238 clonal lines; these patterns were identical to the fluorescence pattern of HSV -2-infected controls when reacted with HSV antiserum. Tumor rejection studies in the BALB/c host indicated that each cell line provided immunity against a tumorigenic challenge of transformed mouse cells. The H238 clone EC1 3 provided a 53% immunity against itself at an inoculum of 10(6); the 333-8-9 line supported a 26% immunity. These data demonstrate a common HSV antigenicity between the murine and hamster transformed lines and further indicate that the HSV genome is involved in transformation.     

Polyamine metabolism in cells infected with herpes simplex virus.

Ifection of LS cells with HSV-1 resulted in an inhibition of spermidine and spermine synthesis from putrescine, possibly through inhibition of host cell protein synthesis. The rate of putrescine uptake increased soon after infection, and later, polyamines were lost from the cells. Inhibition of spermidine and spermine synthesis by methylglyoxal bis (amidinohydrazone) did not affect virus replication.