ICP0 is required for efficient reactivation of herpes simplex virus type 1 from neuronal latency

Relative to wild-type herpes simplex virus type 1 (HSV-1), ICP0-null mutant viruses reactivate inefficiently from explanted, latently infected mouse trigeminal ganglia (TG), indicating that ICP0 is not essential for reactivation but plays a central role in enhancing the efficiency of reactivation. The validity of these findings has been questioned, however, because the replication of ICP0-null mutants is impaired in animal models during the establishment of latency, such that fewer mutant genomes than wild-type genomes are present in latently infected mouse TG. Therefore, the reduced number of mutant viral genomes available to reactivate, rather than mutations in the ICP0 gene per se, may be responsible for the reduced reactivation efficiency of ICP0-null mutants. We have recently demonstrated that optimization of the size of the ICP0 mutant virus inoculum and transient immunosuppression of mutant-infected mice with cyclophosphamide can be used to establish wild-type levels of ICP0-null mutant genomes in latently infected TG (W. P. Halford and P. A. Schaffer, J. Virol. 74:5957-5967, 2000). Using this procedure to equalize mutant and wild-type genome numbers, the goal of the present study was to determine if, relative to wild-type virus, the absence of ICP0 function in two ICP0-null mutants, n212 and 7134, affects reactivation efficiency from (i) explants of latently infected TG and (ii) primary cultures of latently infected TG cells. Although equivalent numbers of viral genomes were present in TG of mice latently infected with either wild-type or mutant viruses, reactivation of n212 and 7134 from heat-stressed TG explants was inefficient (31 and 37% reactivation, respectively) relative to reactivation of wild-type virus (KOS) (95%). Similarly, n212 and 7134 reactivated inefficiently from primary cultures of dissociated TG cells plated directly after removal from the mouse (7 and 4% reactivation, respectively), relative to KOS (60% reactivation). The efficiency and kinetics of reactivation of KOS, n212, and 7134 from cultured TG cells (treated with acyclovir to facilitate the establishment of latency) in response to heat stress or superinfection with a nonreplicating HSV-1 ICP4(-) mutant, n12, were compared. Whereas heat stress induced reactivation of KOS from 69% of latently infected TG cell cultures, reactivation of n212 and 7134 was detected in only 1 and 7% of cultures, respectively. In contrast, superinfection with the ICP4(-) virus, which expresses high levels of ICP0, resulted in the production of infectious virus in nearly 100% of cultures latently infected with KOS, n212, or 7134 within 72 h. Thus, although latent mutant viral genome loads were equivalent to that of wild-type virus, in the absence of ICP0, n212 and 7134 reactivated inefficiently from latently infected TG cells during culture establishment and following heat stress. Collectively, these findings demonstrate that ICP0 is required to induce efficient reactivation of HSV-1 from neuronal latency.     

Glutamine deprivation causes enhanced plating efficiency of a herpes simplex virus type 1 ICP0-null mutant

Isoleucine deprivation of cellular monolayers prior to infection has been reported to result in partial complementation of a herpes simplex virus type 1 (HSV-1) ICP0 null (ICP0⁻) mutant. We now report that glutamine deprivation alone is able to enhance the plating efficiency of an ICP0⁻ virus and that isoleucine deprivation has little or no effect. Because a low glutamine level is associated with stress and because stress is known to induce reactivation, low levels of glutamine may be relevant to the reactivation of HSV-1 from latency. Additionally, we demonstrate that arginine and methionine deprivation result in partial complementation of the ICP0⁻ virus.     

Herpes Simplex Virus 2 ICP0? Mutant Viruses Are Avirulent and Immunogenic: Implications for a Genital Herpes Vaccine

Herpes simplex virus 1 (HSV-1) ICP0 ⁻ mutants are interferon-sensitive, avirulent, and elicit protective immunity against HSV-1 (Virol J, 2006, 3:44). If an ICP0 ⁻ mutant of herpes simplex virus 2 (HSV-2) exhibited similar properties, such a virus might be used to vaccinate against genital herpes. The current study was initiated to explore this possibility. Several HSV-2 ICP0 ⁻ mutant viruses were constructed and evaluated in terms of three parameters: i. interferon-sensitivity; ii. virulence in mice; and iii. capacity to elicit protective immunity against HSV-2. One ICP0 ⁻ mutant virus in particular, HSV-2 0ΔNLS, achieved an optimal balance between avirulence and immunogenicity. HSV-2 0ΔNLS was interferon-sensitive in cultured cells. HSV-2 0ΔNLS replicated to low levels in the eyes of inoculated mice, but was rapidly repressed by an innate, Stat 1-dependent host immune response. HSV-2 0ΔNLS failed to spread from sites of inoculation, and hence produced only inapparent infections. Mice inoculated with HSV-2 0ΔNLS consistently mounted an HSV-specific IgG antibody response, and were consistently protected against lethal challenge with wild-type HSV-2. Based on their avirulence and immunogenicity, we propose that HSV-2 ICP0 ⁻ mutant viruses merit consideration for their potential to prevent the spread of HSV-2 and genital herpes.     

Repression of gene expression upon infection of cells with herpes simplex virus type 1 mutants impaired for immediate-early protein synthesis.

Herpes simplex virus type 1 (HSV-1) mutants defective in immediate-early (IE) gene expression do not readily enter productive replication after infection of tissue culture cells. Instead, their genomes are retained in a quiescent, nonreplicating state in which the production of viral gene products cannot be detected. To investigate the block to virus replication, we used the HSV-1 triple mutant in1820K, which, under appropriate conditions, is effectively devoid of the transactivators VP16 (a virion protein), ICP0, and ICP4 (both IE proteins). Promoters for the HSV-1 IE ICP0 gene or the human cytomegalovirus (HCMV) major IE gene, cloned upstream of the Escherichia coli lacZ coding sequences, were introduced into the in1820K genome. The regulation of these promoters and of the endogenous HSV-1 IE promoters was investigated upon conversion of the virus to a quiescent state. Within 24 h of infection, the ICP0 promoter became much less sensitive to transactivation by VP16 whereas the same element, when used to transform Vero cells, retained its responsiveness. The HCMV IE promoter, which is not activated by VP16, also became less sensitive to the HCMV functional homolog of VP16. Both elements remained available for transactivation by HSV-1 IE proteins at 24 h postinfection, showing that the in1820K genome was not irreversibly inactivated. The promoters controlling the HSV-1 ICP4, ICP22, and ICP27 genes also became essentially unresponsive to transactivation by VP16. The ICP0 promoter was induced when hexamethylene bisacetamide was added to cultures at the time of infection, but the response to this agent was also lost by 24 h after infection. Therefore, promoter elements within the HSV-1 genome are actively repressed in the absence of IE gene expression, and repression is not restricted specifically to HSV-1 IE promoters.     

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 open reading frame 10 protein, the herpes simplex virus VP16 homolog, transactivates herpesvirus immediate-early gene promoters.

The varicella-zoster virus (VZV) open reading frame 10 (ORF10) protein is the homolog of the herpes simplex virus type 1 (HSV-1) protein VP16. These are two virion tegument proteins that have extensive amino acid sequence identity in their amino-terminal and middle domains. ORF10, however, lacks the acidic carboxy terminus which is critical for transactivation by VP16. Earlier studies showed that VZV ORF10 does not form a tertiary complex with the TAATGARAT regulatory element (where R is a purine) with which HSV-1 VP16 interacts, suggesting that ORF10 may not have transactivating ability. Using transient-expression assays, we show that VZV ORF10 is able to transactivate VZV immediate-early (IE) gene (ORF62) and HSV-1 IE gene (ICP4 and ICP0) promoters. Furthermore, cell lines stably expressing ORF10 complement the HSV-1 mutant in1814, which lacks the transactivating function of VP16, and enhance the de novo synthesis of infectious virus following transfection of HSV-1 virion DNA. These results indicate that ORF10, like its HSV-1 homolog VP16, is a transactivating protein despite the absence of sequences similar to the VP16 carboxy-terminal domain. The transactivating function of the VZV ORF10 tegument protein may be critical for efficient initiation of viral infection.     

Inhibition of Major Histocompatibility Complex Class I Antigen Presentation in Pig and Primate Cells by Herpes Simplex Virus Type 1 and 2 ICP47

Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) express an immediate-early protein, ICP47, that effectively inhibits the human transporter associated with antigen presentation (TAP), blocking major histocompatibility complex (MHC) class I antigen presentation to CD8⁺ T cells. Previous work indicated that the mouse TAP is relatively resistant to inhibition by the HSV-1 and HSV-2 ICP47 proteins (ICP47-1 and ICP47-2) and that mouse cells infected with HSV-1 are lysed by anti-HSV CD8⁺ cytotoxic T lymphocytes (CTL). Therefore, mice are apparently not suitable animals in which to study the in vivo effects of ICP47. In order to find an animal model, we introduced ICP47-1 and ICP47-2 into cells from various animal species—mice, rats, guinea pigs, rabbits, dogs, pigs, cows, monkeys, and humans—and measured TAP activity in the cells. Both proteins were unable to inhibit TAP in mouse, rat, guinea pig, and rabbit cells. In contrast, ICP47-1 and ICP47-2 inhibited TAP in pig, dog, cow, and monkey cells, and the TAP in pig and dog fibroblasts was often more sensitive to both proteins than TAP in human fibroblasts. These results were extended by measuring CD8⁺-T-cell recognition (CTL lysis) of cells from various species. Cells were infected with recombinant HSV-1 constructed to express murine MHC class I proteins so that the cells would be recognized and lysed by well-characterized murine anti-HSV CTL unless antigen presentation was blocked by ICP47. Anti-HSV CD8⁺ CTL effectively lysed pig and primate cells infected with a recombinant HSV-1 ICP47⁻ mutant but were unable to lyse pig or primate cells infected with a recombinant HSV-1 that expressed ICP47. Therefore, pigs, dogs, and monkeys may be useful animal models in which to test the effects of ICP47 on HSV pathogenesis or the use of ICP47 as a selective immunosuppressive agent.Herpes simplex virus (HSV) infection of human fibroblasts leads to inhibition of antigen presentation to CD8⁺ T cells so that the virus-infected fibroblasts are not lysed by cytotoxic T lymphocytes (CTL) (10, 12, 14). The principal reason for this resistance to CTL appears to be the expression of an HSV immediate-early protein, ICP47, which causes major histocompatibility complex (MHC) class I proteins to accumulate in infected cells in a peptide-empty form (19). ICP47 was subsequently shown to inhibit the transporter associated with antigen presentation (TAP), which functions to translocate antigenic peptides across the membrane of the endoplasmic reticulum (ER) (3, 8), and without antigenic peptides, MHC class I proteins accumulate in the ER. More recent results demonstrated that ICP47 blocks peptide binding to TAP by binding with high affinity to a domain of TAP that includes the peptide binding site (1, 15).Although HSV type 1 (HSV-1) ICP47 (ICP47-1) effectively blocks TAP in human fibroblasts, it inhibits TAP little, if at all, in a variety of mouse cells unless applied in high concentrations (1, 3, 15, 19). Similarly, HSV-2 ICP47 (ICP47-2), which has only 42% amino acid identity with ICP47-1 (4), effectively blocks human TAP but inhibits murine TAP less effectively (16). Inhibition of murine TAP with these proteins occurs at ICP47-1 and ICP47-2 concentrations 50- to 100-fold higher than those required to inhibit human TAP. ICP47-1 and ICP47-2 bind poorly to mouse TAP (15, 16), which explains their inability to block peptide transport and antigen presentation in mouse cells.We were interested in extending the study of the species specificity of ICP47 for several reasons. Firstly, we wanted to find an animal model with which to assess the effects of ICP47 in vivo, both to assess its role in virus-host interactions and to provide a model for the use of ICP47 in autoimmunity, in transplantation, and in gene therapy vectors. Secondly, we wanted to determine whether ICP47 was functional in the species currently widely used for HSV pathogenesis and vaccine studies—mice, rabbits, and guinea pigs. Thirdly, we were interested in the mechanism of the extraordinary virulence of HSV in owl monkeys (aotus), speculating that the TAP in this New World primate might be exceptionally susceptible to ICP47.In order to assess the effects of ICP47 on the TAPs of various species, cells were permeabilized, recombinant ICP47-1 and ICP47-2 were introduced into the cells, and assays of TAP activity were performed. To examine the effects of ICP47 on antigen presentation and recognition by CD8⁺ T cells, fibroblasts were infected with recombinant HSV-1 that expresses mouse class I proteins and not ICP47, and lysis of the cells by mouse anti-HSV CTL was tested. We found that ICP47-1 and ICP47-2 did not block TAP in mouse, rat, guinea pig, or rabbit skin fibroblasts but effectively inhibited TAP and antigen presentation in pig, dog, cow, and monkey fibroblasts. Therefore, pigs, dogs, and monkeys can be used to study the in vivo effects of ICP47, though for several reasons, the use of pigs might be a practical starting point.     

Impact of the interaction between herpes simplex virus type 1 regulatory protein ICP0 and ubiquitin-specific protease USP7 on activation of adeno-associated virus type 2 rep gene expression

Expression of the herpes simplex virus type 1 (HSV-1) regulatory protein ICP0 in transfected cells reactivates rep gene expression from integrated adeno-associated virus (AAV) type 2 genomes via a mechanism that requires both its RING finger and USP7 interaction domains. In this study, we found that the rep reactivation defect of USP7-binding-negative ICP0 mutants can be overcome by further deletion of sequences in the C-terminal domain of ICP0, indicating that binding of USP7 to ICP0 is not directly required. Unlike the case in transfected cells, only the RING finger domain of ICP0 was essential for rep gene reactivation during HSV-1 infection. However, mutants unable to bind to USP7 activate HSV-1 gene expression and reactivate rep gene expression with reduced efficiencies. These results further elucidate the role of ICP0 as a helper factor for AAV replication and illustrate that care is required when extrapolating from the properties of ICP0 in transfection assays to events occurring during HSV-1 infection.