Herpes simplex virus infection in normal and mutant human fibroblast cultures

Reproductive efficiency of herpes simplex virus type 1 has been determined in several normal and mutant human skin fibroblast lines. The mutant cell lines were derived from individuals diagnosed as having Duchenne muscular dystrophy, a disease thought to involve genetically determined membrane defects. Yields of infectious virus, determined by plaque assay on rabbit kidney cell monolayers, were consistently lower from dystrophic cells than from normal cells. The yield from dystrophic lines was 3–20% of the normal. The time course of production of infectious particles did not appear to vary between dystrophic and normal host cells. Also, the initiation of replication of the viral genome did not appear to be altered in the dystrophic lines. It is proposed that a late maturation function is involved in the lower virus productivity in dystrophic cells.     

Herpes simplex virus induces the replication of foreign DNA.

Plasmids containing the simian virus 40 (SV40) DNA replication origin and the large T gene are replicated efficiently in Vero monkey cells but not in rabbit skin cells. Efficient replication of the plasmids was observed in rabbit skin cells infected with herpes simplex virus type 1 (HSV-1) and HSV-2. The HSV-induced replication required the large T antigen and the SV40 replication origin. However, it produced concatemeric molecules resembling replicative intermediates of HSV DNA and was sensitive to phosphonoacetate at concentrations known to inhibit the HSV DNA polymerase. Therefore, it involved the HSV DNA polymerase itself or a viral gene product(s) which was expressed following the replication of HSV DNA. Analyses of test plasmids lacking SV40 or HSV DNA sequences showed that, under some conditions, HSV also induced low-level replication of test plasmids containing no known eucaryotic replication origins. Together, these results show that HSV induces a DNA replicative activity which amplifies foreign DNA. The relevance of these findings to the putative transforming potential of HSV is discussed.     

Herpes simplex virus co-infection facilitates rolling circle replication of the adeno-associated virus genome

Adeno-associated virus (AAV) genome replication only occurs in the presence of a co-infecting helper virus such as adenovirus type 5 (AdV5) or herpes simplex virus type 1 (HSV-1). AdV5-supported replication of the AAV genome has been described to occur in a strand-displacement rolling hairpin replication (RHR) mechanism initiated at the AAV 3’ inverted terminal repeat (ITR) end. It has been assumed that the same mechanism applies to HSV-1-supported AAV genome replication. Using Southern analysis and nanopore sequencing as a novel, high-throughput approach to study viral genome replication we demonstrate the formation of double-stranded head-to-tail concatemers of AAV genomes in the presence of HSV-1, thus providing evidence for an unequivocal rolling circle replication (RCR) mechanism. This stands in contrast to the textbook model of AAV genome replication when HSV-1 is the helper virus.     

Herpes simplex virus: selection of origins of DNA replication.

A selection procedure was devised to study the role of cis -acting sequences at origins of DNA replication. Two regions in Herpes simplex virus oriS were examined: an AT-rich spacer sequence and a putative binding site, box III, for the origin binding protein. Plasmid libraries were generated using oligonucleotides with locally random sequences. The library, amplified in Escherichia coli , was used to transfect BHK cells followed by superinfection with HSV-1. Replicated plasmids resistant to Dpn I cleavage were amplified in E. coli. The selection scheme was repeated. Plasmids were isolated at different stages of the procedure and their replication efficiency was determined. Efficiently replicating plasmids had a high AT content in the spacer sequence as well as a low helical stability of this region. In contrast, this was not seen using the box III library. We also noted that the wild type sequence invariably dominated the library after five rounds of selection. These plasmids arose from recombination between plasmids and viral DNA. Our results imply that a large group of sequences can mechanistically serve as origins of DNA replication. In a viral system, however, where the initiation process might be rate-limiting, this potentially large group of sequences would always converge towards the most efficient replicator.     

Herpes simplex virus DNA synthesis at a preformed replication fork in vitro.

Proteins from herpes simplex virus (HSV)-infected cells were used to reconstitute DNA synthesis in vitro on a preformed replication fork. The preformed replication fork consisted of a nicked, double-stranded, circular DNA molecule with a 5' single-strand tail that was noncomplementary to the template. The products of DNA synthesis on this substrate were rolling-circle molecules, as demonstrated by electron microscopy and alkaline agarose gel electrophoresis. The tails contained double-stranded regions, indicating that both leading- and lagging-strand DNA syntheses occurred. Rolling-circle DNA replication was dependent upon HSV DNA polymerase and ATP and was stimulated by a crude fraction containing ICP8 (HSV DNA-binding protein). Similar protein fractions from mock-infected cells were unable to support rolling-circle DNA replication. This in vitro DNA replication system should prove useful in the identification and characterization of the enzymatic activities required at the HSV replication fork.     

The effect of mycoplasmas on replication and plaquing ability of Herpes simplex virus.

M. arginini, an arginine utilizer, can decrease the yield of Herpes simplex virus, type 1 grown in Vero cells. M. arginini can also cause a reduction in number and size of plaques produced by HSV. The reduction in titer and plaque size produced in M. arginini-infected cells can be reversed by supplementing medium with additional arginine. A. laidlawii, a nonarginine utilizing mycoplasma, had no effect on the growth of HSV.     

Herpes simplex virus glycoprotein D mediates interference with herpes simplex virus infection.

We showed that the expression of a single protein, glycoprotein D (gD-1), specified by herpes simplex virus type 1 (HSV-1) renders cells resistant to infection by HSV but not to infection by other viruses. Mouse (LMtk-) and human (HEp-2) cell lines containing the gene for gD-1 under control of the human metallothionein promoter II expressed various levels of gD-1 constitutively and could be induced to express higher levels with heavy metal ions. Radiolabeled viruses bound equally well to gD-1-expressing and control cell lines. Adsorbed viruses were unable to penetrate cells expressing sufficient levels of gD-1, based on lack of any cytopathic effects of the challenge virus and on failure to detect either the induction of viral protein synthesis or the shutoff of host protein synthesis normally mediated by a virion-associated factor. The resistance to HSV infection conferred by gD-1 expression was not absolute and depended on several variables, including the amount of gD-1 expressed, the dosage of the challenge virus, the serotype of the challenge virus, and the properties of the cells themselves. The interference activity of gD-1 is discussed in relation to the role of gD-1 in virion infectivity and its possible role in permitting escape of progeny HSV from infected cells.     

Inhibition of herpes simplex virus type 1 replication in fibroblast cultures by human blood mononuclear cells.

An assay was developed to test the effect of human blood mononuclear cells (MNCs) on herpes simplex virus (HSV) replication. In this assay, human fibroblast monolayers were inoculated with HSV and then cultured with or without blood MNCs. Fewer HSV-infected cells were recovered from human fibroblasts cultured in the presence than in the absence of blood MNCs. This inhibition of viral replication by MNCs was independent of HLA matching between the MNCs and fibroblasts and persisted even when T cells were depleted by antibody and complement. However, depletion of Leu11+ MNCs either by panning or with antibody and complement reduced the ability of the cells to suppress HSV infection, whereas enrichment of Leu11+ cells by fluorescence-activated cell sorting increased the viral suppression. Depletion of OKM1+ MNCs also reduced the viral suppression. After coculturing of MNCs and HSV-infected fibroblasts for 3 days, alpha interferon (IFN) and gamma IFN were detected in the supernatants. Predepletion of Leu11+ MNCs reduced the amount of gamma IFN produced in these cultures. Incubation of the MNCs and HSV-infected fibroblasts with antibody specific for either alpha or gamma IFN resulted in reduced viral suppression. Preincubation of MNCs for 18 h with either interleukin 2 or alpha IFN or for 7 days with antigen increased the suppression of HSV infection. These results suggest that natural killer cells with the Leu11+ phenotype participate in the recognition of HSV-infected cells at a point sufficiently early to interfere with the spread of infection in vitro and may inhibit viral replication by natural killer cell cytotoxicity, by generation of interferon, and by lymphokine-activated killing.     

Ribonuclease H activities associated with Herpes simplex virus

Ribonuclease H activities were detected in purified particles of herpes simplex virus type 1 and 2. The RNase H specifically degrades the ribo moiety of the synthetic homopolymet [³H]-poly rA·dT as well as the RNA part of the hybrid [³H]-RNA·DNA duplex. Single-stranded polymers were not degraded under the conditions used. p-Hydroxy-mercuribenzoate inhibits the RNase H activities which co-purify with HSV type 1 and 2.     

The effects of crude and purified human gonadotropin on in vitro stimulated human lymphocyte cultures.

Crude human chorionic gonadotropin (hCG) was found to be several fold more immunosuppressive than purified hCG in human peripheral blood lymphocyte cultures stimulated by phytohemagglutinin, pokeweed, purified protein derivative and allogeneic cells in vitro. Immunosuppression by crude hCG was consistently noted at levels less than 1000 IU/ml and usually 80% inhibition was achieved with doses of 5000–10,000 IU/ml, whereas 40–50% inhibition or less was observed by purified hCG at 10,000 IU/ml. In two crude hCG preparations subjected to Sephadex G-100 chromatography, the fractions that inhibited lymphocyte cultures appeared in the eluate after the major peak of hCG activity. These data indicate that inhibitory substance(s) other than hCG are responsible for most of the immunosuppressive properties of first trimester pregnancy urine. Both crude and purified hCG were stimulatory to human lymphocytes when used alone without mitogens when cultured in fetal calf serum.     

Herpes simplex virus glycoprotein D bound to the human receptor HveA

Herpes simplex virus (HSV) infection requires binding of the viral envelope glycoprotein D (gD) to cell surface receptors. We report the X-ray structures of a soluble, truncated ectodomain of gD both alone and in complex with the ectodomain of its cellular receptor HveA. Two bound anions suggest possible binding sites for another gD receptor, a 3-O-sulfonated heparan sulfate. Unexpectedly, the structures reveal a V-like immunoglobulin (Ig) fold at the core of gD that is closely related to cellular adhesion molecules and flanked by large N- and C-terminal extensions. The receptor binding segment of gD, an N-terminal hairpin, appears conformationally flexible, suggesting that a conformational change accompanying binding might be part of the viral entry mechanism.     

Herpes simplex virus interferes with amyloid precursor protein processing

Background  

The early events underlying Alzheimer's disease (AD) remain uncertain, although environmental factors may be involved. Work in this laboratory has shown that the combination of herpes simplex virus type 1 (HSV1) in brain and carriage of the APOE-ε4 allele of the APOE gene strongly increases the risk of developing AD. The development of AD is thought to involve abnormal aggregation or deposition of a 39–43 amino acid protein - β amyloid (Aβ) - within the brain. This is cleaved from the much larger transmembranal protein 'amyloid precursor protein' (APP). Any agent able to interfere directly with Aβ or APP metabolism may therefore have the capacity to contribute towards AD. One recent report showed that certain HSV1 glycoprotein peptides may aggregate like Aβ; a second study described a role for APP in transport of virus in squid axons. However to date the effects of acute herpesvirus infection on metabolism of APP in human neuronal-type cells have not been investigated. In order to find if HSV1 directly affects APP and its degradation, we have examined this protein from human neuroblastoma cells (normal and transfected with APP 695) infected with the virus, using Western blotting.     

Herpes simplex virus type 1 gene products required for DNA replication: identification and overexpression.

Seven herpes simplex virus (HSV) genes have been shown recently to be necessary and sufficient to support the replication of origin-containing plasmids. Two of these genes (pol and dbp) encode well-known DNA replication proteins (the DNA polymerase and the major single-stranded DNA binding protein), and a third gene (UL42) encodes a previously identified infected-cell protein which binds tightly to double-stranded DNA. The products of the four remaining genes have not previously been identified. Using the predicted amino acid sequence data (D.J. McGeoch, M.A. Dalrymple, A. Dolan, D. McNab, L.J. Perry, P. Taylor, and M.D. Challberg, J. Virol. 62:444-453; D.J. McGeoch and J.P. Quinn, Nucleic Acids Res. 13:8143-8163), we have raised rabbit antisera against the products of all seven genes. We report here the use of these reagents to identify these proteins in infected cells. All seven proteins localized to the nucleus and were expressed in a manner consistent with the idea that they are the products of early genes. Various immunological assays suggest that four of these proteins (UL5, UL8, UL9, and UL52) are made in infected cells in very low abundance relative to the other three. To improve our ability to study these proteins, we have expressed UL5, UL8, UL9, and UL52 in insect cells by using the baculovirus expression system. The HSV protein made in insect cells were immunoprecipitable with the appropriate antisera, and the size of each protein was indistinguishable from the size of the corresponding protein made in HSV-infected Vero cells. Our data offer strong support for the accuracy of open reading frames proposed by McGeoch et al. In addition, the antisera and the overproduced HSV replication proteins should be useful reagents with which to analyze the biochemistry of HSV DNA replication.