Herpes simplex virus immediate-early promoters are responsive to virus and cell trans-acting factors.

The promoters for each of the immediate-early genes from herpes simplex virus type 1 were cloned and fused to a chloramphenicol acetyltransferase cassette. These chimeric genes were used as targets in a transient expression assay to determine how the immediate-early gene products ICP4 and ICP0 and the virion-associated stimulatory protein Vmw65 affected their expression in HeLa and Vero cells. The basal level of expression from these cassettes differed significantly depending on the extent of 5'-flanking sequence and the cell line that served as host. The promoters from IE-4 and IE-0 behaved in a qualitatively similar fashion independent of the host cell. However, the promoter for ICP27 had a unique response pattern: in Vero cells it acted as an alpha gene promoter, whereas in HeLa cells its response was more like that of a beta gene promoter. The promoter sequences for ICP22 and ICP47 behaved as the IE-4 and IE-0 promoters did in HeLa cells, but their response to the effector molecules in Vero cells was unlike that of other alpha gene promoters we have studied. Evidence is also presented for a role for ICP27 in autoregulation.     

Expression of herpes simplex virus ICP0 inhibits the induction of interferon-stimulated genes by viral infection

The herpes simplex virus type 1 (HSV-1) mutant d109 does not express any of the immediate-early (IE) proteins and persists in cells for a prolonged length of time. As has been shown by Nicholl et al. (J. Gen. Virol. 81:2215-2218, 2000) and Mossman et al. (J. Virol. 75:750-758, 2001) using other mutants defective for IE gene expression, infection with d109 induced the expression of a number of interferon-stimulated genes. Induction of these genes was significantly greater at multiplicities of infection (MOI) of 10 PFU/cell or greater, and the resulting antiviral effect was only seen at MOIs greater than 10 PFU/cell. Using mutants defective for sets of IE genes established that the lack of ICP0 expression was necessary for high levels of interferon-stimulated gene expression in HEL cells. The induction of interferon-stimulated genes by d109 could also be inhibited by infection with an E1-:E3-:E4- adenovirus expressing levels of ICP0 that are comparable to those expressed within the first hour of wild-type virus infection. Lastly, the addition of the proteasome inhibitor MG132 to cells infected with a mutant that expresses ICP0, d106, also resulted in the induction of interferon-stimulated genes. Thus, ICP0 may function through the proteasome very early in HSV infection to inhibit a cellular antiviral response induced by the virion.     

Isolation and characterization of deletion mutants of herpes simplex virus type 1 in the gene encoding immediate-early regulatory protein ICP4

Using Vero cells transformed with the wild-type gene for ICP4 as the permissive host cell, we isolated herpes simplex virus type 1 (HSV-1) mutants containing deletions in both copies of the ICP4 gene. The mutants, d120 and d202, contained deletions of 4.1 and 0.5 kilobases, respectively, in each copy of ICP4. ICP4 mRNA synthesized in d202-infected Vero cells was 0.5 kilobases smaller than that synthesized in cells infected with the wild-type virus. No ICP4 mRNA was detected in d120-infected Vero cells. d120 and d202 specified polypeptides that reacted with ICP4 antiserum and were smaller than the wild-type ICP4 polypeptide by 130 and 30 kilodaltons, respectively. The only other HSV-1 gene products detectable on infection of Vero cells with d120 and d202 were ICP6 (of the early kinetic class of HSV-1 polypeptides), ICP0 (immediate early), ICP22 (immediate early), and ICP27 (immediate early). Immediate-early polypeptides ICP0 and ICP27 were expressed to a higher level in Vero cells infected with an ICP4 temperature-sensitive (ts) mutant (tsB32) at 39 degrees C, indicating immediate-early stimulatory activity associated with the ts ICP4 polypeptide. In addition, the patterns of complementation of d120, d202, and tsB32 in ICP4-transformed cells also demonstrated inhibitory activity associated with the ts form of the ICP4 polypeptide.     

Mutants defective in herpes simplex virus type 2 ICP4: isolation and preliminary characterization.

Vero cells were biochemically transformed with the gene encoding ICP4 of herpes simplex virus type 2 (HSV-2). These cells were used as permissive hosts to isolate and propagate HSV-2 mutants defective in this gene. Two mutants, designated hr259 and hr79, were isolated. Neither mutant grew in nontransformed Vero cells, but both grew to near wild-type levels in HSV-2 ICP4-expressing cells. hr259 contains a deletion of about 0.6 kilobases which eliminates the mRNA start site of the ICP4 gene. hr79 contains a mutation which maps by marker rescue to the portion of the ICP4 gene encoding the carboxy-terminal half of the protein. Although hr259 failed to generate any detectable ICP4 mRNA in nontransformed Vero cells, hr79 encoded an ICP4 mRNA which is wild type with respect to size. In nontransformed Vero cells infected with hr259, only ICP0, ICP6, ICP22, and ICP27 were readily detectable. In the case of hr79, a truncated form of ICP4 appeared to be made in addition to ICP0, ICP6, ICP22, and ICP27. Both hr259 and hr79 grew efficiently on cell lines transformed with the ICP4 gene of HSV-1 as evidenced by plating efficiencies and single-burst experiments. Similarly, cells transformed with the ICP4 gene of HSV-2 served as efficient hosts for the growth of d120, HSV-1 ICP4 deletion mutant.     

一种1型单纯庖疹病毒载体系统的建立

1型单纯疱疹病毒（HSV-1）作为溶瘤病毒和病毒载体的研究已有很长的历史. 本研究利用细菌人工染色体技术建立了一种HSV-1载体系统. 首先,将HSV-1内部反向重复序列（internal inverted repeat sequences, IR）两侧的片段克隆入pKO5获得穿梭质粒pKO5/BN,其电转含pHSV-BAC的大肠杆菌后筛选获得删除IR区重组DNA的 pHSVΔIR-BAC. pHSVΔIR-BAC转染Vero细胞获得删除IR区的重组病毒HSVΔIR（MH1001）.上述pKO5/BN和含pHSVΔIR BAC的大肠杆菌构成了HSV-1载体系统. 利用该系统获得了表达绿色荧光蛋白EGFP的重组病毒HSVΔIR/EGFP（MH1002）.MH1001和MH1002在感染的Vero细胞中增殖水平略低于野生型HSV-1,但无显著差异|Western印迹检测表明,重组病毒早期蛋白质ICP0、ICP4、ICP8、ICP22、ICP27在感染细胞中的表达水平下降|免疫荧光及激光共聚焦检测表明,重组病毒与野生型病毒均存在于细胞质中.以上结果表明,删除IR区的重组HSV-1保留了复制能力,能够携载并表达外源基因,建立的HSV-1载体系统可用于构建携载外源基因的复制型重组HSV-1.     

Construction and properties of a mutant of herpes simplex virus type 1 with glycoprotein H coding sequences deleted.

A mutant of herpes simplex virus type 1 (HSV-1) in which glycoprotein H (gH) coding sequences were deleted and replaced by the Escherichia coli lacZ gene under the control of the human cytomegalovirus IE-1 gene promoter was constructed. The mutant was propagated in Vero cells which contained multiple copies of the HSV-1 gH gene under the control of the HSV-1 gD promoter and which therefore provide gH in trans following HSV-1 infection. Phenotypically gH-negative virions were obtained by a single growth cycle in Vero cells. These virions were noninfectious, as judged by plaque assay and by expression of beta-galactosidase following high-multiplicity infection, but partial recovery of infectivity was achieved by using the fusogenic agent polyethylene glycol. Adsorption of gH-negative virions to cells blocked the adsorption of superinfecting wild-type virus, a result in contrast to that obtained with gD-negative virions (D. C. Johnson and M. W. Ligas, J. Virol. 62:4605-4612, 1988). The simplest conclusion is that gH is required for membrane fusion but not for receptor binding, a conclusion consistent with the conservation of gH in all herpesviruses.     

Reactivation of latent herpes simplex virus by adenovirus recombinants encoding mutant IE-0 gene products.

We have previously shown that adenovirus recombinants expressing functional ICP0 reactivate latent herpes simplex virus type 2 (HSV-2) in an in vitro latency system. This study demonstrated that ICP0, independent of other HSV gene products, is sufficient to reactivate latent HSV-2 in this in vitro system. To assess the effects of defined mutations in the sequence encoding ICP0 (IE-0) on reactivation, seven in-frame insertion and three in-frame deletion mutants were moved into an adenovirus expression vector. Each recombinant directed the synthesis of stable ICP0 of the correct size. The transactivation activity of the mutated sequences in these recombinants was similar to that when they were tested in plasmids. When these recombinants were examined for their ability to reactivate in the in vitro latency system, mutants with dramatic defects in transactivation (Ad-0/125, Ad-0/89, Ad-0/2/7, and Ad-0/88/93) were unable to reactivate latent HSV-2 independent of the multiplicity of infection. An exception to this correlation was the finding that Ad-0/89, which transactivated poorly, was able to reactivate latent virus after prolonged incubation whereas other transactivation-deficient mutants could not. Moreover, the presence of ICP4 did not compensate for the inability of any of the recombinants tested to reactivate HSV-2. These results show that (i) the transactivation domains of ICP0 are also used in reactivation, (ii) the presence of another essential HSV regulatory protein ICP4 does not alter the pattern of reactivation by ICP0, and (iii) mutations in some regions of IE-0 previously shown to affect viral growth and plaque formation did not alter its ability to reactivate in this in vitro system.     

Varicella-zoster virus (VZV) open reading frame 61 protein transactivates VZV gene promoters and enhances the infectivity of VZV DNA.

The varicella-zoster virus (VZV) open reading frame 61 (ORF61) protein is the homolog of herpes simplex virus type 1 (HSV-1) ICP0. Both genes are located in similar parts of the genome, their predicted products share a cysteine-rich motif, and cell lines expressing VZV ORF61 are able to complement an HSV-1 ICP0 deletion mutant (H. Moriuchi, M. Moriuchi, H. A. Smith, S. E. Straus, and J. I. Cohen, J. Virol. 66:7303-7308, 1992). In transient expression assays, HSV-1 ICP0 is a transactivator alone and transactivates in synergy with another viral transactivator, ICP4. However, VZV ORF61 represses the activation by VZV-encoded proteins ORF62 (the homolog of ICP4) and ORF4. To further characterize the function of VZV ORF61 and its role(s) in regulation of viral gene expression, we performed transient expression assays using target promoters from VZV, HSV-1, and unrelated viruses. In the absence of other viral activators, VZV ORF61 transactivated most promoters tested. In addition, a cell line stably expressing VZV ORF61 complemented the HSV-1 mutant in 1814, which lacks the transactivating function of VP16. The cell line expressing VZV ORF61 enhanced the infectivity of HSV-1 virion DNA. Moreover, transient expression of VZV ORF61 also enhanced the infectivity of VZV DNA. These results indicate that VZV ORF61 can stimulate expression of HSV-1 and VZV genes at an early stage in the viral replicative cycle and that ORF61 has an important role in VZV gene regulation.     

A cellular function can enhance gene expression and plating efficiency of a mutant defective in the gene for ICP0, a transactivating protein of herpes simplex virus type 1.

ICP0 transactivates herpes simplex virus type 1 genes of all classes as well as a number of heterologous viral and cellular genes, yet it is not essential for virus replication in vitro or in vivo. Stocks of ICP0 deletion mutants, however, exhibit significantly lower plating efficiencies on standard 24-h-old Vero cell monolayers than do stocks of wild-type virus. In an attempt to determine whether the growth status of cells in the monolayer affects the ability of ICP0 mutants to initiate plaque formation, the plating efficiencies and abilities of an ICP0 null mutant (7134) and of wild-type virus (KOS) to express selected viral proteins were determined on Vero cell monolayers whose growth had been arrested either by contact inhibition-trypsinization or by isoleucine deprivation and had then been released from growth arrest. The proportion of cells cycling synchronously after release from growth arrest was assessed by flow cytometry. The results of these studies indicate that the plating efficiency of 7134 was greatest on Vero cell monolayers 8 h after release from growth arrest induced by either treatment. Monolayers of both types released from growth arrest at other times supported 7134 plaque formation less efficiently. In contrast, the plating efficiency of KOS was nearly equal on monolayers at all times after release from growth arrest. Notably, both KOS and 7134 were equally efficient in entering cells and inducing expression of the immediate-early protein ICP4 in either 8- or 24-h monolayers. Relative to KOS, however, 7134 was significantly impaired in the expression of selected early and late genes in cells at 24 h postrelease. When the plating efficiencies of 7134 and KOS were examined in 0-28 cells (Vero cells that are stably transformed with the ICP0 gene) whose growth had been arrested and then released, no differences in the plating efficiencies of the two viruses as a function of growth status were noted. These findings suggest that a cellular function expressed maximally in cells 8 h after release from growth arrest can substitute operationally for ICP0 to enhance plaque formation and viral gene expression by 7134. They further suggest that one role of ICP0 in viral infection is to facilitate virus replication in cells that do not express this function.     

Induction of immunoglobulin G Fc receptors by recombinant vaccinia viruses expressing glycoproteins E and I of herpes simplex virus type 1.

Glycoprotein E (gE) of herpes simplex virus type 1 (HSV-1) will bind immunoglobulin G (IgG) (Fc) affinity columns (R. B. Bauke and P. G. Spear, J. Virol. 32:779-789, 1979), but recent evidence suggests that the HSV-1 Fc receptor is composed of a complex of gE and glycoprotein I (gI) and that both gI and gE are required for Fc receptor activity (D. C. Johnson and V. Feenstra, J. Virol. 61:2208-2216, 1987; D. C. Johnson, M. C. Frame, M. W. Ligas, A. M. Cross, and N. D. Stow, J. Virol. 62:1347-1354, 1988). We have expressed gE and gI, either alone or in combination, on the surface of HeLa cells by using recombinant vaccinia viruses and have measured Fc receptor activity by Fc-rosetting or IgG-binding assays. Expression of gE alone resulted in the induction of Fc receptor activity, while expression of gI alone gave no detectable Fc binding. Coexpression of gE and gI resulted in higher levels of IgG binding than did expression of gE alone, despite the fact that under conditions of coexpression, the levels of surface gE were reduced. We propose that gE and gI together form a receptor of higher affinity than gE alone and that HSV-1 therefore has the potential to induce two Fc receptors of different affinities.     

An ICP6::lacZ insertional mutagen is used to demonstrate that the UL52 gene of herpes simplex virus type 1 is required for virus growth and DNA synthesis.

An insertional mutagen was developed which consists of the lacZ gene of Escherichia coli under the control of the regulatory elements of the large subunit of ribonucleotide reductase (ICP6). This ICP6::lacZ cassette was used to create a mutation in a gene designated UL52 (D. J. McGeoch, M. A. Dalrymple, A. Dolan, D. McNab, L. J. Perry, P. Taylor, and M. D. Challberg, J. Virol. 62:444-453, 1988), which is predicted to encode a 114,000-molecular-weight protein. To isolate and propagate this mutant, we generated a cell line, BL-1, by cotransfection of Vero cells with pSV2neo and a plasmid containing the herpes simplex virus type 1 KOS strain BamHI L fragment (coordinates 0.708 to 0.745). An ICP6::lacZ insertion mutant, hr114, was capable of growing in BL-1 cells but not in normal Vero cells. In addition, hr114 was defective in the synthesis of viral DNA and late proteins; however, this mutant appeared to exhibit normal early gene expression. Thus, the results presented in this report show that the UL52 gene product is required for viral DNA synthesis. Furthermore, our studies indicate that the ICP6::lacZ cassette will provide a useful tool for obtaining mutants of other herpes simplex virus genes.     

Herpes simplex virus 1 ICP0 phosphorylation site mutants are attenuated for viral replication and impaired for explant-induced reactivation

In cell culture experiments, phosphorylation appears to be a critical regulator of the herpes simplex virus 1 (HSV-1) immediate-early (IE) protein, ICP0, which is an E3 ubiquitin ligase that transactivates viral gene expression. Three major regions of phosphorylation in ICP0 (amino acids 224 to 232, 365 to 371, and 508 to 518) have been identified, and mutant viruses that block phosphorylation sites within each region (termed Phos 1, 2, and 3, respectively) have been constructed. Previous studies indicated that replication of Phos 1 is significantly reduced compared to that of wild-type virus in cell culture (C. Boutell, et al., J. Virol. 82:10647-10656, 2008). To determine the effects these phosphorylation site mutations have on the viral life cycle in vivo, mice were ocularly infected with wild-type HSV-1, the Phos mutants, or their marker rescue counterparts. Subsequently, viral replication, establishment of latency, and viral explant-induced reactivation of these viruses were examined. Relative to wild-type virus, Phos 1 eye titers were reduced as much as 7- and 18-fold on days 1 and 5 postinfection, respectively. Phos 2 eye titers showed a decrease of 6-fold on day 1 postinfection. Titers of Phos 1 and 2 trigeminal ganglia were reduced as much as 16- and 20-fold, respectively, on day 5 postinfection. Additionally, the reactivation efficiencies of Phos 1 and 2 were impaired relative to wild-type HSV-1, although both viruses established wild-type levels of latency in vivo. The acute replication, latency, and reactivation phenotypes of Phos 3 were similar to those of wild-type HSV-1. We conclude from these studies that phosphorylation is likely a key modulator of ICP0's biological activities in a mouse ocular model of HSV-1 infection.