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.     

Expression of herpes simplex virus type 1 major DNA-binding protein, ICP8, in transformed cell lines: complementation of deletion mutants and inhibition of wild-type virus

To minimize the contribution of residual activity associated with the temperature-sensitive (ts) form of ICP8 specified by available ts mutants, deletion mutations in this gene were constructed. Cells permissive for the generation and propagation of ICP8 deletion mutants were first obtained. Vero cells were cotransfected with pKEF-P4, which contains the gene for ICP8, and pSV2neo or a hybrid plasmid containing the G418 resistance gene linked to pKEF-P4. Of the 48 G418-resistant cell lines, 21 complemented ICP8 ts mutants in plaque assays at the nonpermissive temperature. Four of these were examined by Southern blot analysis and shown to contain 1 to 3 copies of the ICP8 gene per haploid genome equivalent. Cell line U-47 was used as the permissive host for construction of ICP8 deletion mutants. In addition to cell lines which complemented ts mutants, two lines, U-27 and U-35, significantly inhibited plaque formation by wild-type virus, contained 30 and 100 copies of the ICP8 gene per haploid genome equivalent, respectively, and expressed large amounts of ICP8 after infection with wild-type virus. At low but not high multiplicities of infection, this inhibition was accompanied by underproduction of viral polypeptides of the early, delayed-early, and late kinetic classes. For construction of deletion mutants, a 780-base-pair XhoI fragment was deleted from pSG18-SalIA, a plasmid which contains the gene for ICP8, to yield pDX. U-47 cells were then cotransfected with pDX and infectious wild-type DNA. Mutant d61, isolated from the progeny of cotransfection, was found to contain both the engineered deletion in the ICP8 gene and an oriL-associated deletion of approximately 55 base pairs. Because d61 contained two mutations, a second mutant, d21, which carried the engineered ICP8 deletion but an intact oriL, was constructed by cotransfection of U-47 cells with wild-type DNA and an SalI-KpnI fragment purified from pDX. Phenotypic analysis of d21 and d61 revealed that they were similar in all properties examined: both exhibited efficient growth in U-47 cells but not in Vero cells; both induced the synthesis of an ICP8 polypeptide which was smaller than the wild-type form of the protein and which, unlike the wild-type protein, was found in the cytoplasm and not the nucleus of infected Vero cells; and nonpermissive Vero cells infected with either mutant failed to express late viral polypeptides.     

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.     

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.     

Herpes simplex virus type 1 oriL is not required for virus replication or for the establishment and reactivation of latent infection in mice.

During the course of experiments designed to isolate deletion mutants of herpes simplex virus type 1 in the gene encoding the major DNA-binding protein, ICP8, a mutant, d61, that grew efficiently in ICP8-expressing Vero cells but not in normal Vero cells was isolated (P. K. Orberg and P. A. Schaffer, J. Virol. 61:1136-1146, 1987). d61 was derived by cotransfection of ICP8-expressing Vero cells with infectious wild-type viral DNA and a plasmid, pDX, that contains an engineered 780-base-pair (bp) deletion in the ICP8 gene, as well as a spontaneous approximately 55-bp deletion in oriL. Gel electrophoresis and Southern blot analysis indicated that d61 DNA carried both deletions present in pDX. The ability of d61 to replicate despite the deletion in oriL suggested that a functional oriL is not essential for virus replication in vitro. Because d61 harbored two mutations, a second mutant, ts+7, with a deletion in oriL-associated sequences and an intact ICP8 gene was constructed. Both d61 and ts+7 replicated efficiently in their respective permissive host cells, although their yields were slightly lower than those of control viruses with intact oriL sequences. An in vitro test of origin function of isolated oriL sequences from wild-type virus and ts+7 showed that wild-type oriL, but not ts+7 oriL, was functional upon infection with helper virus. In an effort to determine the requirement for oriL in latency, ts+7 was compared with wild-type virus for its ability to establish, maintain, and be reactivated from latent infection in a murine eye model. The mutant was reactivated as efficiently as was wild-type virus from trigeminal ganglia after cocultivation with permissive cells, and each of the seven reactivated isolates was shown to carry the approximately 150-bp deletion characteristic of ts+7. These observations demonstrate that oriL is not required for virus replication in vitro or for the establishment and reactivation of latent infection in vivo.     

The herpes simplex virus type 1 regulatory protein ICP27 is required for the prevention of apoptosis in infected human cells

The herpes simplex virus type 1 (HSV-1) ICP27 protein is an immediate-early or alpha protein which is essential for the optimal expression of late genes as well as the synthesis of viral DNA in cultures of Vero cells. Our specific goal was to characterize the replication of a virus incapable of synthesizing ICP27 in cultured human cells. We found that infection with an HSV-1 ICP27 deletion virus of at least three separate strains of human cells did not produce immediate-early or late proteins at the levels observed following wild-type virus infections. Cell morphology, chromatin condensation, and genomic DNA fragmentation measurements demonstrated that the human cells died by apoptosis after infection with the ICP27 deletion virus. These features of the apoptosis were identical to those which occur during wild-type infections of human cells when total protein synthesis has been inhibited. Vero cells infected with the ICP27 deletion virus did not exhibit any of the features of apoptosis. Based on these results, we conclude that while HSV-1 infection likely induced apoptosis in all cells, viral evasion of the response differed among the cells tested in this study.     

Intragenic complementation of herpes simplex virus ICP8 DNA-binding protein mutants.

The major DNA-binding protein, or infected-cell protein 8 (ICP8), of herpes simplex virus is required for viral DNA synthesis and normal regulation of viral gene expression. Previous genetic analysis has indicated that the carboxyl-terminal 28 residues are the only portion of ICP8 capable of acting independently as a nuclear localization signal. In this study, we constructed a mutant virus (n11SV) in which the carboxyl-terminal 28 residues of ICP8 were replaced by the simian virus 40 large-T-antigen nuclear localization signal. The n11SV ICP8 localized into the nucleus and bound to single-stranded DNA in vitro as tightly as wild-type ICP8 did but was defective for viral DNA synthesis and viral growth in Vero cells. Two mutant ICP8 proteins (TL4 and TL5) containing amino-terminal alterations could complement the n11SV mutant but not ICP8 gene deletion mutants. Cell lines expressing TL4 and TL5 ICP8 were isolated, and in these cells, complementation of n11SV was observed at the levels of both viral DNA replication and viral growth. Therefore, complementation between n11SV ICP8 and TL4 or TL5 ICP8 reconstituted wild-type ICP8 functions. Our results demonstrate that (i) the carboxyl-terminal 28 residues of ICP8 are required for a function(s) involved in viral DNA replication, (ii) this function can be supplied in trans by another mutant ICP8, and (iii) ICP8 has multiple domains possessing different functions, and at least some of these functions can complement in trans.     

Genetic evidence for two distinct transactivation functions of the herpes simplex virus alpha protein ICP27.

Infected-cell protein 27 (ICP27) is a herpes simplex virus type 1 alpha, or immediate-early, protein involved in the regulation of viral gene expression. To better understand the function(s) of ICP27 in infected cells, we have isolated and characterized viral recombinants containing defined alterations in the ICP27 gene. The mutant virus d27-1 contains a 1.6-kilobase deletion which removes the ICP27 gene promoter and most of the coding sequences, while n59R, n263R, n406R, and n504R are mutants containing nonsense mutations which encode ICP27 molecules truncated at their carboxyl termini. All five mutants were defective for lytic replication in Vero cells. Analysis of the mutant phenotypes suggests that ICP27 has the following regulatory effects during the viral infection: (i) stimulation of expression of gamma-1 genes, (ii) induction of expression of gamma-2 genes, (iii) down regulation of expression of alpha and beta genes late in infection, and (iv) stimulation of viral DNA replication. Cells infected with the mutant n504R expressed wild-type levels of gamma-1 proteins but appeared to be unable to efficiently express gamma-2 mRNAs or proteins. This result suggests that ICP27 mediates two distinct transactivation functions, one which stimulates gamma-1 gene expression and a second one required for gamma-2 gene induction. Analysis of the mutant n406R suggested that a truncated ICP27 polypeptide can interfere with the expression of many viral beta genes. Our results demonstrate that ICP27 has a variety of positive and negative effects on the expression of viral genes during infection.     

Amino acid substitution mutations in the herpes simplex virus ICP27 protein define an essential gene regulation function.

ICP27 is an essential herpes simplex virus type 1 (HSV-1) alpha protein that is required for the transition from the beta to the gamma phase of infection. To identify functional regions of ICP27, we constructed 16 plasmids that contain nucleotide substitution mutations in the ICP27 gene. The mutations created XhoI restriction sites, altered one or two codons, and were spaced at semiregular intervals throughout the coding region. Three mutations completely inactivated an essential function of ICP27, as demonstrated by the inability of the transfected plasmids to complement the growth of an HSV-1 ICP27 deletion mutant. These mutations, M11, M15, and M16, mapped in the carboxyl-terminal one-third of ICP27 at residues 340 and 341, 465 and 466, and 488, respectively. In cotransfection assays, all three defective-plasmid mutants retained the transrepression function of ICP27 but were defective at transactivation. To define the lytic functions that are mediated by the transactivation activity of ICP27, we engineered HSV-1 recombinants containing the M11, M15, or M16 mutation. All three viral mutants failed to grow in Vero cells and possessed similar phenotypes. The viral mutants replicated their DNA similarly to the wild-type virus but showed several defects in viral gene expression. These were a failure to down-regulate alpha and beta genes at late times after infection and an inability to induce certain gamma-2 genes. Our results demonstrate that the transactivation function of ICP27 (as it is defined in cotransfection assays) mediates an essential gene regulation function during the HSV-1 infection. This activity is not required for ICP27-dependent enhancement of viral DNA replication. Our work supports and extends previous studies which suggest that ICP27 carries out two distinct regulatory activities during the HSV-1 infection.     

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.     

Phenotype of the herpes simplex virus type 1 protease substrate ICP35 mutant virus.

The herpes simplex virus type 1 ICP35 assembly protein is involved in the formation of viral capsids. ICP35 is encoded by the UL26.5 gene and is specifically processed by the herpes simplex virus type 1 protease encoded by the UL26 gene. To better understand the functions of ICP35 in infected cells, we have isolated and characterized an ICP35 mutant virus, delta ICP35. The mutant virus was propagated in complementing 35J cells, which express wild-type ICP35. Phenotypic analysis of delta ICP35 shows that (i) mutant virus growth in Vero cells was severely restricted, although small amounts of progeny virus was produced; (ii) full-length ICP35 protein was not produced, although autoproteolysis of the protease still occurred in mutant-infected nonpermissive cells; (iii) viral DNA replication of the mutant proceeded at wild-type levels, but only a very small portion of the replicated DNA was processed to unit length and encapsidated; (iv) capsid structures were observed in delta ICP35-infected Vero cells by electron microscopy and by sucrose sedimentation analysis; (v) assembly of VP5 into hexons of the capsids was conformationally altered; and (vi) ICP35 has a novel function which is involved in the nuclear transport of VP5.     

Simian virus 40 host range/helper function mutations cause multiple defects in viral late gene expression.

Simian virus 40 (SV40) deletion mutants dlA2459 and dlA2475 express T antigens that lack the normal carboxy terminus. These mutants are called host range/helper function (hr/hf) mutants because they form plaques at 37 degrees C on BSC-1 and Vero monkey kidney cell lines but not on CV-1p monkey kidney cells. Wild-type SV40 can provide a helper function to permit growth of human adenoviruses in monkey kidney cells; the hr/hf mutants cannot. Progeny yields of hr/hf mutants are also cold sensitive in all cell lines tested. Patterns of viral macromolecular synthesis in three cell lines (Vero, BSC-1, and CV-1) at three temperatures (40, 37, and 32 degrees C) were examined to determine the nature of the growth defect of hr/hf mutants. Mutant viral DNA replication was similar to that of the wild type in all three cell lines, indicating that the mutations affect late events in the viral lytic cycle. In mutant-infected Vero cells, in which viral yields were highest, late mRNA levels were similar to those observed during wild-type infection. Levels of viral late mRNA from mutant-infected CV-1 and BSC-1 cells at 32 and 37 degrees C were reduced relative to those of wild-type-infected cells. The steady-state level of the major viral capsid protein, VP1, in mutant-infected CV-1 cells was reduced to the same extent as was late mRNA. The synthesis of agnoprotein could not be detected in mutant-infected CV-1 cells but was readily detected in CV-1 cells infected by wild-type SV40. Primer extension analyses indicated that most late mRNAs from mutant-infected CV-1 cells utilize start sites downstream from the major wild-type cap site (nucleotide 325) and the agnoprotein initiation codon (nucleotide 335). These results indicate that deletion of the carboxyl-terminal domain of T antigen affects viral late mRNA production, both quantitatively and qualitatively. The agnoprotein is detected late in the wild-type SV40 lytic cycle and is thought to play a role in the assembly or maturation of virions. Reduced hr/hf progeny yields could result from decreased capsid protein synthesis and, in the absence of detectable levels of agnoprotein, from inefficient use of available capsid proteins.