An avirulent ICP34.5 deletion mutant of herpes simplex virus type 1 is capable of in vivo spontaneous reactivation.

The herpes simplex virus type 1 (HSV-1) ICP34.5 gene is a neurovirulence gene in mice. In addition, some ICP34.5 mutants have been reported to have a reduced efficiency of induced reactivation as measured by in vitro explantation of latently infected mouse ganglia. However, since spontaneous reactivation is almost nonexistent in mice, nothing has been reported on the effect of ICP34.5 mutants on spontaneous reactivation in vivo. To examine this, we have deleted both copies of the ICP34.5 neurovirulence gene from a strain of HSV-1 (McKrae) that has a high spontaneous reactivation rate in rabbits and used this mutant to infect rabbit eyes. All rabbits infected with the ICP34.5 mutant virus (d34.5) survived, even at challenge doses greater than 4 x 10(7) PFU per eye. In contrast, a 200-fold-lower challenge dose of 2 x 10(5) PFU per eye was lethal for approximately 50% of rabbits infected with either the wild-type McKrae parental virus or a rescued ICP34.5 mutant in which both copies of the ICP34.5 gene were restored. In mice, the 50% lethal dose of the ICP34.5 mutant was over 10(6) PFU, compared with a value of less than 10 PFU for the rescued virus. The ICP34.5 mutant was restricted for replication in rabbit and mouse eyes and mouse trigeminal ganglia in vivo. The spontaneous reactivation rate in rabbits for the mutant was 1.4% as determined by culturing tear films for the presence of reactivated virus. This was more than 10-fold lower than the spontaneous reactivation rate determined for the rescued virus (19.6%) and was highly significant (P < 0.0001, Fisher exact test). Southern analysis confirmed that the reactivated virus retained both copies of the ICP34.5 deletion. Thus, this report demonstrates that (i) the ICP34.5 gene, known to be a neurovirulence gene in mice, is also important for virulence in rabbits and (ii) in vivo spontaneous reactivation of HSV-1 in the rabbit ocular model, although reduced, can occur in the absence of the ICP34.5 gene.     

p32 Is a Novel Target for Viral Protein ICP34.5 of Herpes Simplex Virus Type 1 and Facilitates Viral Nuclear Egress

As a large double-stranded DNA virus, herpes simplex virus type 1 (HSV-1) assembles capsids in the nucleus where the viral particles exit by budding through the inner nuclear membrane. Although a number of viral and host proteins are involved, the machinery of viral egress is not well understood. In a search for host interacting proteins of ICP34.5, which is a virulence factor of HSV-1, we identified a cellular protein, p32 (gC1qR/HABP1), by mass spectrophotometer analysis. When expressed, ICP34.5 associated with p32 in mammalian cells. Upon HSV-1 infection, p32 was recruited to the inner nuclear membrane by ICP34.5, which paralleled the phosphorylation and rearrangement of nuclear lamina. Knockdown of p32 in HSV-1-infected cells significantly reduced the production of cell-free viruses, suggesting that p32 is a mediator of HSV-1 nuclear egress. These observations suggest that the interaction between HSV-1 ICP34.5 and p32 leads to the disintegration of nuclear lamina and facilitates the nuclear egress of HSV-1 particles.     

The region of the herpes simplex virus type 1 LAT gene that is colinear with the ICP34.5 gene is not involved in spontaneous reactivation.

The goal of this report was to determine if the region of the LAT gene that is colinear with ICP34.5 (kb 6.2 to 7.1 of LAT) is involved in spontaneous reactivation of herpes simplex virus type 1. We inserted one copy of the ICP34.5 gene into the unique long region of a herpes simplex virus type 1 (strain McKrae) mutant lacking both copies of ICP34.5 (one in each viral long repeat) and the corresponding 917-nucleotide colinear portion of LAT (kb 6.2 to 7.1). Rabbits were ocularly infected with this mutant, and spontaneous reactivation relative to that for the wild-type virus and the original mutant was measured. As we previously reported, the original ICP34.5-deleted virus (d34.5) was significantly impaired for spontaneous reactivation and virulence (G. C. Perng, R. L. Thompson, N. M. Sawtell, W. E. Taylor, S. M. Slanina, H. Ghiasi, R. Kaiwar, A. B. Nesburn, and S. L. Wechsler, J. Virol. 69:3033-3041, 1995). In contrast, we report here that restoration of one copy of ICP34.5 at a distant location completely restored the wild-type level of in vivo spontaneous reactivation, despite retention of the deletion in LAT (spontaneous reactivation rate = 0.3 to 1.4% for the ICP34.5 deletion mutant, 7.7 to 19.6% for the wild type, and 9 to 16.1% for virus with one copy of ICP34.5). Thus, the 917-nucleotide region of LAT from kb 6.2 to 7.1 was not involved in the LAT function required for wild-type spontaneous reactivation. We also found that restoration of a single ICP34.5 gene in a novel location did not restore wild-type virulence (rabbit death rate = 0% [0 of 15] for the original ICP34.5 deletion mutant, 8% [2 of 24] for the single-copy IPC34.5 virus, and 52% [14 of 27] for wild-type virus; P < 0.001 for one versus two copies of ICP34.5). It is likely that either two gene doses of ICP34.5 or its location in the long repeat is essential for full functionality of ICP34.5's virulence function. Furthermore, the ability of the single-copy ICP34.5 virus to reactivate at wild-type levels despite being significantly less virulent than wild-type virus separates the spontaneous reactivation phenotype from the virulence phenotype.     

The range and distribution of murine central nervous system cells infected with the gamma(1)34.5- mutant of herpes simplex virus 1.

Wild-type herpes simplex virus 1 (HSV-1) multiplies, spreads, and rapidly destroys cells of the murine central nervous system (CNS). In contrast, mutants lacking both copies of the gamma(1)34.5- gene have been shown to be virtually lacking in virulence even after direct inoculation of high-titered virus into the CNS of susceptible mice (J. Chou, E. R. Kern, R. J. Whitley, and B. Roizman, Science 250:1262-1266, 1990). To investigate the host range and distribution of infected cells in the CNS of mice, 4- to 5-week-old mice were inoculated stereotaxically into the caudate/putamen with 3 x 10(5) PFU of the gamma(1)34.5- virus R3616. Four-micrometer-thick sections of mouse brains removed on day 3, 5, or 7 after infection were reacted with a polyclonal antibody directed primarily to structural proteins of the virus and with antibodies specific for neurons, astrocytes, or oligodendrocytes. This report shows the following: (i) most of the tissue damage caused by R3616 was at the site of injection, (ii) the virus spread by retrograde transport from the site of infection to neuronal cell nuclei at distant sites and to ependymal cells by cerebrospinal fluid, (iii) the virus infected neurons, astrocytes, oligodendrocytes, and ependymal cells and hence did not discriminate among CNS cells, (iv) viral replication in some neurons could be deduced from the observation of infected astrocytes and oligodendrocytes at distant sites, and (v) infected cells were being efficiently cleared from the nervous system by day 7 after infection. We conclude that the gamma(1)34.5- attenuation phenotype is reflected in a gross reduction in the ability of the virus to replicate and spread from cell to cell and is not due to a restricted host range. The block in viral replication appears to be a late event in viral replication.     

Full resistance of herpes simplex virus type 1-infected primary human cells to alpha interferon requires both the Us11 and gamma(1)34.5 gene products

The gamma(1)34.5 gene product is important for the resistance of herpes simplex virus type 1 (HSV-1) to interferon. However, since the inhibition of protein synthesis observed in cells infected with a gamma(1)34.5 mutant virus results from the combined loss of the gamma(1)34.5 gene product and the failure to translate the late Us11 mRNA, we sought to characterize the relative interferon sensitivity of mutants unable to produce either the Us11 or the gamma(1)34.5 polypeptide. We now demonstrate that primary human cells infected with a Us11 mutant virus are hypersensitive to alpha interferon, arresting translation upon entry into the late phase of the viral life cycle. Furthermore, immediate-early expression of Us11 by a gamma(1)34.5 deletion mutant is sufficient to render translation resistant to alpha interferon. Finally, we establish that the Us11 gene product is required for wild-type levels of replication in alpha interferon-treated cells and, along with the gamma(1)34.5 gene, is an HSV-1-encoded interferon resistance determinant.     

Dephosphorylation of eIF-2alpha mediated by the gamma(1)34.5 protein of herpes simplex virus type 1 is required for viral response to interferon but is not sufficient for efficient viral replication

The gamma(1)34.5 protein of herpes simplex virus type 1 (HSV-1) functions to block the shutoff of protein synthesis involving double-stranded RNA-dependent protein kinase (PKR). In this process, the gamma(1)34.5 protein recruits cellular protein phosphatase 1 (PP1) to form a high-molecular-weight complex that dephosphorylates eIF-2alpha. Here we show that the gamma(1)34.5 protein is capable of mediating eIF-2alpha dephosphorylation without any other viral proteins. While deletion of amino acids 1 to 52 from the gamma(1)34.5 protein has no effect on eIF-2alpha dephosphorylation, further truncations up to amino acid 146 dramatically reduce the activity of the gamma(1)34.5 protein. An additional truncation up to amino acid 188 is deleterious, indicating that the carboxyl-terminal domain alone is not functional. Like wild-type HSV-1, the gamma(1)34.5 mutant with a truncation of amino acids 1 to 52 is resistant to interferon, and resistance to interferon is coupled to eIF-2alpha dephosphorylation. Intriguingly, this mutant exhibits a similar growth defect seen for the gamma(1)34.5 null mutant in infected cells. Restoration of the wild-type gamma(1)34.5 gene in the recombinant completely reverses the phenotype. These results indicate that eIF-2alpha dephosphorylation mediated by the gamma(1)34.5 protein is required for HSV response to interferon but is not sufficient for viral replication. Additional functions or activities of the gamma(1)34.5 protein contribute to efficient viral infection.     

ICP34.5 mutants of herpes simplex virus type 1 strain 17syn+ are attenuated for neurovirulence in mice and for replication in confluent primary mouse embryo cell cultures.

In a recent report, the neurovirulence of herpes simplex virus type 1 (HSV-1) was mapped to the ICP34.5 gene (J. Chou, E. R. Kern, R. J. Whitley, and B. Roizman, Science 250:1262-1266, 1990). In this report, specific mutations within ICP34.5 were constructed in HSV-1 strain 17syn+ to determine the effects of these mutations in a fully neurovirulent isolate. It was found that termination of the ICP34.5 gene after the N-terminal 30 amino acids resulted in a mutant, 17termA, which was 25- to 90-fold reduced in neurovirulence. This reduction of neurovirulence was associated with restricted replication of the mutant virus in mouse brain. The reduced replication phenotype was also evident in the trigeminal and dorsal root ganglia following inoculation at the periphery. 17termA was capable of replicating with wild-type kinetics in mouse footpads, and therefore the restriction seen in neural tissues was not due to a generalized replication defect in mouse cells. Significantly, replication of the mutant was also restricted in the mouse cornea in vivo and in confluent primary mouse embryo cells and mouse 10T1/2 cells in vitro. However, 17termA replicated with much greater efficiency in subconfluent mouse embryo cells, suggesting that the physiological state of the cell may be an important factor for productive replication of this mutant. Restoration of the ICP34.5 gene to the mutant resulted in a virus which displayed wild-type neurovirulence and replication kinetics in all cells and tissues tested.     

HSV-1 ICP34.5 confers neurovirulence by targeting the Beclin 1 autophagy protein

Autophagy is postulated to play a role in antiviral innate immunity. However, it is unknown whether viral evasion of autophagy is important in disease pathogenesis. Here we show that the herpes simplex virus type 1 (HSV-1)-encoded neurovirulence protein ICP34.5 binds to the mammalian autophagy protein Beclin 1 and inhibits its autophagy function. A mutant HSV-1 virus lacking the Beclin 1-binding domain of ICP34.5 fails to inhibit autophagy in neurons and demonstrates impaired ability to cause lethal encephalitis in mice. The neurovirulence of this Beclin 1-binding mutant virus is restored in pkr(-/-) mice. Thus, ICP34.5-mediated antagonism of the autophagy function of Beclin 1 is essential for viral neurovirulence, and the antiviral signaling molecule PKR lies genetically upstream of Beclin 1 in host defense against HSV-1. Our findings suggest that autophagy inhibition is a novel molecular mechanism by which viruses evade innate immunity and cause fatal disease.     

Effect of γ34.5 deletions on oncolytic herpes simplex virus activity in brain tumors

The ICP34.5 protein of herpes simplex virus (HSV) is involved in many aspects of viral pathogenesis; promoting neurovirulence, inhibiting interferon-induced shutoff of protein synthesis, interacting with PCNA and TBK1, inhibiting dendritic cell (DC) maturation, and binding to Beclin 1 to interfere with autophagy. Because of its key role in neuropathogenicity, the γ34.5 gene is deleted in all oncolytic HSVs (oHSVs) currently in clinical trial for treating malignant gliomas. Unfortunately, deletion of γ34.5 attenuates virus replication in cancer cells, especially human glioblastoma stem cells (GSCs). To develop new oHSVs for use in the brain and that replicate in GSCs, we explored the effect of deleting the γ34.5 Beclin 1 binding domain (BBD). To ensure cancer selectivity and safety, we inactivated the ICP6 gene (UL39, large subunit of ribonucleotide reductase), constructing ICP6 mutants with different γ34.5 genotypes: Δ68HR-6, intact γ34.5; Δ68H-6, γ34.5 BBD deleted; and 1716-6, γ34.5 deleted. Multimutated Δ68H-6 exhibited minimal neuropathogenicity in HSV-1-susceptible mice, as opposed to Δ68H and Δ68HR-6. It replicated well in human glioma cell lines and GSCs, effectively killing cells in vitro and prolonging survival of mice bearing orthotopic brain tumors. In contrast, 1716 and 1716-6 barely replicated in GSCs. Infection of glioma cells with Δ68H-6 and 1716-6 induced autophagy and increased phosphorylation of eIF2α, while inhibition of autophagy, by Beclin 1 short hairpin RNA (shRNA) knockdown or pharmacological inhibition, had no effect on virus replication or phosphorylated eIF2α (p-eIF2α) levels. Thus, Δ68H-6 represents a new oHSV vector that is safe and effective against a variety of brain tumor models.     

Analysis of the role of autophagy in replication of herpes simplex virus in cell culture

The herpes simplex virus type 1 (HSV-1) neurovirulence gene encoding ICP34.5 controls the autophagy pathway. HSV-1 strains lacking ICP34.5 are attenuated in growth and pathogenesis in animal models and in primary cultured cells. While this growth defect has been attributed to the inability of an ICP34.5-null virus to counteract the induction of translational arrest through the PKR antiviral pathway, the role of autophagy in the regulation of HSV-1 replication is unknown. Here we show that HSV-1 infection induces autophagy in primary murine embryonic fibroblasts and that autophagosome formation is increased to a greater extent following infection with an ICP34.5-deficient virus. Elimination of the autophagic pathway did not significantly alter the replication of wild-type HSV-1 or ICP34.5 mutants. The phosphorylation state of eIF2alpha and viral protein accumulation were unchanged in HSV-1-infected cells unable to undergo autophagy. These data show that while ICP34.5 regulates autophagy, it is the prevention of translational arrest by ICP34.5 rather than its control of autophagy that is the pivotal determinant of efficient HSV-1 replication in primary cell culture.     

Suppression of the phenotype of gamma(1)34.5- herpes simplex virus 1: failure of activated RNA-dependent protein kinase to shut off protein synthesis is associated with a deletion in the domain of the alpha47 gene.

Earlier studies have shown that infection of human cells by herpes simplex virus 1 (HSV-1) results in the activation of RNA-dependent protein kinase (PKR) but that the alpha subunit of eIF-2 is not phosphorylated and that protein synthesis is unaffected. In the absence of the viral gamma(1)34.5 gene, eIF-2alpha is phosphorylated and protein synthesis is prematurely shut off (J. Chou, J. J. Chen, M. Gross, and B. Roizman, Proc. Natl. Acad. Sci. USA 92:10516-10520, 1995). A second recent paper reported the selection of second-site suppressor mutants characterized by near-wild-type protein synthesis in cells infected with gamma(1)34.5- mutants (I. Mohr and Y. Gluzman, EMBO J. 15:4759-4766, 1996). Here, we report the properties of the spontaneous HSV-1 suppressor mutant Sup-1, which is characterized by spontaneous deletion of 503 bp encompassing the domain of the alpha47 gene and junction with the inverted repeats flanking the unique short (U(S)) sequence of the HSV-1 DNA resulting in the juxtaposition of the alpha47 promoter to the coding domain of the U(S)11 gene. This mutant does not exhibit the shutoff of protein synthesis characteristic of the gamma(1)34.5- virus. Specifically, Sup-1 in SK-N-SH human neuroblastoma cells (i) did not exhibit the function of the alpha47 gene characterized by a reduction in the transport of peptides across the endoplasmic reticulum of permealized cells consistent with the absence of alpha47 gene sequences, (ii) accumulated U(S)11 protein at levels analogous to those of the wild-type parent but the protein was made at earlier times after infection, as would be expected from a change in the promoter, and (iii) activated PKR like that of the parent, gamma(1)34.5- virus, but (iv) did not cause premature shutoff of protein synthesis and therefore was similar to the wild-type parent virus rather than the gamma(1)34.5- virus from which it was derived. We conclude that the mechanism by which Sup-1 blocks the shutoff of protein synthesis associated with phosphorylation of eIF-2alpha by the activated PKR is not readily explainable by a secondary mutation characterized by a deletion.     

Identification, transfer, and characterization of cloned herpes simplex virus invasiveness regions.

Following peripheral inoculation of experimental animals, herpes simplex virus type 2 (HSV-2) strains are more virulent than HSV-1 strains, and clinical studies suggest that they possess enhanced virulence in humans. One dramatic type-specific difference in virulence is observed following inoculation of the chorioallantoic membrane (CAM) of the chicken embryo: HSV-2, but not HSV-1, makes large pocks on the CAM, invades the mesoderm, generalizes in the embryo, and kills the chicken. These properties have been believed to be specific for HSV-2, and their molecular basis is unknown. We now report that an HSV-1 strain, ANG, behaves even more efficiently than HSV-2. In addition, we have transferred restriction fragments of ANG DNA to another HSV-1 strain, 17 syn+, conferring the CAM virulence phenotype on the normally CAM-avirulent 17 syn+. Like ANG, these recombinant viruses are 10(6)-fold more virulent (PFU/50%) lethal dose [LD50] ratio, less than or equal to 10(2)) than the parental 17 syn+ strain (PFU/LD50 ratio, greater than or equal to 10(8)). A molecularly cloned library of ANG DNA was used to identify two distinct regions containing the virulence functions. Transfer of sequences contained in either cloned ANG EcoRI fragment A (0.49 to 0.64 map units) or F (0.32 to 0.42 map units) DNA to 17 syn+ confers CAM virulence, whereas other cloned regions of the ANG genome do not. Using cloned DNA, we derived and plaque purified several virulent recombinant viruses with inserts from either the ANG EcoRI fragment A (INV-I) or F (INV-II) areas. In each instance, the transfer of the cloned INV-I or INV-II sequences enhanced virulence for the chicken embryo 10(6)-fold (PFU/LD50 ratio, less than or equal to 10(2]. In addition, the transfer of the cloned ANG EcoRI-F INV-II sequences resulted in a 10(3)-fold enhancement of neuroinvasiveness and virulence for mice. Following footpad inoculation, these recombinants kill mice with a PFU/LD50 ratio of approximately 10(3) (similar to HSV-2 strains) compared with 10(6) for 17 syn+. Thus, we have identified, cloned, and transferred two DNA regions from HSV-1 ANG which contain virulence genes (INV-I and INV-II) important in mesodermal invasiveness on the CAM and, in the case of INV-II, neuroinvasiveness in the mouse. In each instance, the recombinant HSV-1 viruses have attained enhanced virulence beyond that described for HSV-1 strains and similar to that seen with HSV-2.(ABSTRACT TRUNCATED AT 400 WORDS)     

Strain-dependent structural variants of herpes simplex virus type 1 ICP34.5 determine viral plaque size,efficiency of glycoprotein processing,and viral release and neuroinvasive disease potential

The ability of certain strains of herpes simplex virus type 1 (HSV-1) to cause encephalitis or neuroinvasive disease in the mouse upon peripheral infection is dependent on a combination of activities of specific forms of viral proteins. The importance of specific variants of ICP34.5 to neuroinvasive disease potential and its correlation with small-plaque production, inefficient glycoprotein processing, and virus release were suggested by comparison of ICP34.5 from the SP7 virus, originally obtained from the brain of a neonate with disseminated disease, and the tissue culture-passaged progeny of SP7 (SLP5 and SLP10) and the KOS321 virus. SLP5, SLP10, and KOS321 are attenuated and exhibit a large-plaque phenotype, including efficient glycoprotein processing and viral release. We show that expression of the KOS321 ICP34.5 protein in cells infected with SP7 or ICP34.5 deletion mutants promotes large plaque formation and efficient viral glycoprotein processing, while expression of the SP7 ICP34.5 protein decreases efficiency of viral glycoprotein processing. In addition, a recombinant virus, 4hS1, with the SP7 ICP34.5 gene replacing the KOS321-like ICP34.5 gene in the SLP10a background, rescues the small-plaque phenotype and neuroinvasive disease. The major difference in the ICP34.5 gene product is the number of Pro-Ala-Thr repeats in the middle region of the protein, with 18 for SP7 and 3 for KOS321. Strain-dependent differences in the ICP34.5 protein can therefore alter the tissue culture behavior and the virulence of HSV-1.     

Complementary lethal invasion of the central nervous system by nonneuroinvasive herpes simplex virus types 1 and 2.

It is well-known that viral thymidine kinase (TK) expression is important for the maximum demonstration of virulence of herpes simplex virus (HSV). In this study, we have investigated interactions of a TK- mutant of virulent HSV type 2 (HSV-2) (syn+) and a nonneuroinvasive HSV-1 (syn) in mice. When the mice were inoculated with each virus alone in their rear footpads, no mice were killed even after infection with high doses of viruses (greater than 10(6) PFU per mouse), whereas 100% of the mice died when inoculated with 10(5) PFU of a 1:1 mixture of HSV-2 TK- mutant and nonneuroinvasive HSV-1. The 1:1 mixture exhibited even more virulence than the parental HSV-2; the mean survival time of coinfected mice was significantly shorter than that of mice inoculated with 10(5) PFU of the virulent HSV-2. We have also examined the genotypes and phenotypes of viruses isolated from the central nervous system of coinfected mice. Of 50 isolates, 7 were judged to be recombinants from their restriction endonuclease cleavage patterns, but all were nonneuroinvasive. In addition, all syn+ viruses (23 clones) tested were found to have TK- phenotypes, indicating that the majority of viruses present in the central nervous system were TK- viruses, since about 90% of viruses detected in spinal cords and brains exhibited syn+ phenotypes. These results strongly suggest that the lethal invasion of the central nervous system by HSV-2 TK- and nonneuroinvasive HSV-1 was the consequence of in vivo complementation between the two viruses.     

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.     

Second-site mutation outside of the U(S)10-12 domain of Deltagamma(1)34.5 herpes simplex virus 1 recombinant blocks the shutoff of protein synthesis induced by activated protein kinase R and partially restores neurovirulence.

Earlier studies have shown that herpes simplex virus type 1 (HSV-1) activated protein kinase R (PKR) but that the product of the product of the gamma(1)34.5 gene binds and redirects the host phosphatase 1 to dephosphorylate the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2alpha). In consequence, the gamma(1)34.5 gene product averts the threatened shutoff of protein synthesis caused by activated PKR. Serial passages of Deltagamma(1)34.5 mutants in human cells led to isolation of two classes of second-site, compensatory mutants. The first, reported earlier, resulted from the juxtaposition of the alpha promoter of the U(S)12 gene to the coding sequence of the U(S)11 gene. The mutant blocks the phosphorylation of eIF-2alpha but does not restore the virulence phenotype of the wild-type virus. We report another class of second-site, compensatory mutants that do not map to the U(S)10-12 domain of the HSV-1 genome. All mutants in this series exhibit sustained late protein synthesis, higher yields in human cells, and reduced phosphorylation of PKR that appears to be phosphatase dependent. Specific dephosphorylation of eIF-2alpha was not demonstrable. At least one mutant in this series exhibited a partial restoration of the virulence phenotype characteristic of the wild-type virus phenotype. The results suggest that the second-site mutations reflect activation of fossilized functions designed to block the interferon response pathways in cells infected with the progenitor of present HSV.     

The acidic amino-terminal region of herpes simplex virus type 1 alpha protein ICP27 is required for an essential lytic function.

The herpes simplex virus type 1 (HSV-1) alpha protein ICP27 regulates the transition between the delayed-early and late phases of the viral infection. Previous genetic analyses have suggested that the important functional domains of ICP27 map to its carboxyl-terminal half. One striking feature of the primary sequence of ICP27, however, is an extremely acidic region near its amino terminus. To determine whether this region is required for ICP27 function, we deleted the sequences in the ICP27 gene which encode it (codons 12 through 63). In transient expression assays, the deletion mutant was unable to efficiently repress the expression of a cotransfected reporter gene or to efficiently complement the growth of d27-1, an HSV-1 ICP27 null mutant. These results suggested that the acidic region of ICP27 is involved in a regulatory function required for lytic growth. To test this possibility further, we introduced the mutant allele into the HSV-1 genome by marker transfer. Two independently derived isolates of the mutant virus, designated d1-2a and d1-2b, were recovered and analyzed. Both isolates were defective for growth in Vero cells, exhibiting a 100-fold reduction in virus yield compared with the wild-type infection. Vero cells infected with the d1-2 isolates showed a three- to eightfold reduction in viral DNA replication, a moderate reduction in the expression of viral gamma genes, and a delay in the repression of beta genes. The phenotype of the d1-2 isolates differs substantially from the phenotypes of previously isolated ICP27 mutants, which show much more severe defects in viral gene expression. Our results demonstrate that the amino-terminal half of ICP27 participates in its regulatory activities in both infected and transfected cells.