Genetic analysis of temperature-sensitive mutants which define the genes for the major herpes simplex virus type 2 DNA-binding protein and a new late function.

Eleven temperature-sensitive mutants of herpes simplex virus type 2 (HSV-2) exhibit overlapping patterns of complementation that define four functional groups. Recombination tests confirmed the assignment of mutants to complementation groups 1 through 4 and permitted the four groups to be ordered in an unambiguous linear array. Combined recombination and marker rescue tests (A. E. Spang, P. J. Godowski, and D. M. Knipe, J. Virol. 45:332-342, 1983) indicate that the mutations lie in a tight cluster near the center of UL to the left of the gene for DNA polymerase in the order 4-3-2-1-polymerase. The seven mutants that make up groups 1 and 2 fail to complement each other and mutants in HSV-1 complementation group 1-1, the group thought to define the structural gene for the major HSV-1 DNA-binding protein with a molecular weight of 130,000. At 38 degrees C, mutants in groups 1 and 2 synthesize little or no viral DNA, and unlike cells infected with the wild-type virus, mutant-infected cells exhibit no detectable nuclear antigen reactive with monoclonal or polypeptide-specific antibody to the major HSV-2 DNA-binding protein. The four mutants that make up groups 3 and 4 do not complement each other, nor do they complement mutants in group 2. They do, however, complement mutants in group 1 as well as representative mutants of HSV-1 complementation group 1-1. At 38 degrees C, mutants in groups 3 and 4 are phenotypically DNA+, and nuclei of mutant-infected cells contain the HSV-2 DNA-binding protein. Thus, the four functional groups appear to define two closely linked genes, one encoding an early viral function affecting both viral DNA synthesis and expression of the DNA-binding protein with a molecular weight of 130,000 (groups 1 and 2), and the other encoding a previously unidentified late viral function (groups 3 and 4). The former gene presumably represents the structural gene for the major HSV-2 DNA-binding protein.     

Degradation of intracellular DNA in KB cells infected with cyt mutants of human adenovirus type 12.

A group of mutants (cyt mutants) with much reduced oncogenicity was isolated from the highly oncogenic human adenovirus type 12 (Takemori et al., Virology 36: 575-586, 1968). These mutants induce extensive cellular destruction during lytic infection of human cells and produce low yields of virions. We report here that human KB cells infected with cyt mutants synthesized a reduced amount of viral DNA as compared with cells infected with the parental virus. Furthermore, the newly synthesized viral and cellular DNAs were extensively degraded in mutant-infected cells. Viral DNA was first synthesized as complete genome size, and most of it was degraded to subgenomic size within 6 h after synthesis. This virus-induced DNA degradation function, as well as the low yield of virions, was prevented by co-infection with the parental virus.     

Simian virus 40 maturation in cells harboring mutants deleted in the agnogene

The predominant leader region of the late 16 S mRNAs of simian virus 40 encodes a histone-like, 61-amino acid, DNA-binding protein called the agnoprotein or LP1. To test the hypothesis that this protein facilitates assembly of viral minichromosomes into virions, we have studied the synthesis of virions in cells infected with mutants deleted in this region of the SV40 genome. We found that 220 S mature virions, indistinguishable from those of wild type, were produced in cells infected with these mutants. As in wild-type-infected cells, no assembly intermediates other than 75 S chromatin were observed. However, data obtained from both steady-state and pulse-chase labeling experiments indicated that cells infected with agnogene deletion mutants produced virions more slowly than cells infected with wild-type virus. Taken together with data showing that similar levels of virion proteins were present in the wild-type- and mutant-infected cells, these findings strongly suggest that LP1 plays a role in expediting virion assembly.     

Role of Gene 52 in Bacteriophage T4 DNA Synthesis

In an attempt to elucidate the mechanism of delayed DNA synthesis in phage T4, Escherichia coli B cells were infected with H17 (an amber mutant defective in gene 52 possessing a "DNA-delay" phenotype). The fate of (14)C-labeled H17 parental DNA after infection was followed: we could show that this DNA sediments more slowly in neutral sucrose than wild-type DNA 3 min postinfection. In pulse-chase experiments progeny DNA was found to undergo detachment from the membrane at 12 min postinfection. Reattachment to the membrane was found to be related to an increase in rate of DNA synthesis. A nucleolytic activity that is absent from cells infected by wild-type phage and from uninfected cells could be detected in extracts prepared from mutant-infected cells. In contrast, degradation of host DNA was found to be less extensive in am H17 compared with wild-type infected cells. Addition of chloramphenicol to mutant-infected cells 10 min postinfection inhibited the appearance of a nuclease activity on one hand and suppressed the "DNA-delay" phenotype on the other hand. We conclude that the gene 52 product controls the activity of a nuclease in infected cells whose main function may be specific strand nicking in association with DNA replication. This gene product might directly attack both E. coli and phage T4 DNA, or indirectly determine their sensitivity to degradation by another nuclease.     

The null mutant of the U(L)31 gene of herpes simplex virus 1: construction and phenotype in infected cells.

Earlier studies have shown that the U(L)31 protein is homogeneously distributed throughout the nucleus and cofractionates with nuclear matrix. We report the construction from an appropriate cosmid library a deletion mutant which replicates in rabbit skin cells carrying the U(L)31 gene under a late (gamma1) viral promoter. The mutant virus exhibits cytopathic effects and yields 0.01 to 0.1% of the yield of wild-type parent virus in noncomplementing cells but amounts of virus 10- to 1,000-fold higher than those recovered from the same cells 3 h after infection. Electron microscopic studies indicate the presence of small numbers of full capsids but a lack of enveloped virions. Viral DNA extracted from the cytoplasm of infected cells exhibits free termini indicating cleavage/packaging of viral DNA from concatemers for packaging into virions, but analyses of viral DNAs by pulsed-field electrophoresis indicate that at 16 h after infection, both the yields of viral DNA and cleavage of viral DNA for packaging are decreased. The repaired virus cannot be differentiated from the wild-type parent. These results suggest the possibility that U(L)31 protein forms a network to enable the anchorage of viral products for the synthesis and/or packaging of viral DNA into virions.     

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.     

Herpes simplex virus type 1 prereplicative sites are a heterogeneous population: only a subset are likely to be precursors to replication compartments.

When herpes simplex virus type 1 (HSV-1) DNA replication is blocked by viral polymerase inhibitors, such as phosphonoacetic acid (PAA) or acyclovir (ACV), UL29 (ICP8) localizes to numerous punctate nuclear foci which are called prereplicative sites. Since this pattern can form in cells infected with mutants which are defective in UL5, UL8, UL9, or UL52 in the presence of polymerase inhibitors (C. J. Lukonis and S. K. Weller, J. Virol. 70:1751-1758, 1996; L. M. Liptak, S. L. Uprichard, and D. M. Knipe, J. Virol. 70:1759-1767, 1996), we previously proposed that it is unlikely that these numerous UL29 foci actually represent a functional subassembly of viral replication proteins that could lead to the formation of replication compartments (C. J. Lukonis and S. K. Weller, J. Virol. 70:1751-1758, 1996). In this paper, we have investigated the requirement for formation of the prereplicative site pattern by using double mutants of HSV. From the analysis of mutants lacking both UL5 and UL9, we conclude that neither viral helicase is required for the prereplicative site pattern to form as long as a polymerase inhibitor is present. From the analysis of mutants defective in both UL30 and UL5, we suggest that the prereplicative site pattern can form under conditions in which viral and/or cellular polymerases are inhibited. Furthermore, reexamination of the UL29 staining pattern in cells infected with wild-type virus in the presence of PAA reveals that at least two different UL29 staining patterns can be detected in these cells. One population of cells contains numerous (greater than 20) punctate UL29 foci which are sites of cellular DNA synthesis. In another population of cells, fewer punctate foci (less than 15) are detected, and these structures do not colocalize with sites of cellular DNA synthesis. Instead, they colocalize with PML, a component of nuclear matrix structures known as ND10. We propose that ND10-associated UL29 sites represent domains at which replication compartments form.     

Residues within the conserved helicase motifs of UL9, the origin-binding protein of herpes simplex virus-1, are essential for helicase activity but not for dimerization or origin binding activity

UL9, an essential gene for herpes simplex virus type 1 (HSV-1) DNA replication, exhibits helicase and origin DNA binding activities. It has been hypothesized that UL9 binds and unwinds the HSV-1 origin of replication, creating a replication bubble and promoting the assembly of the viral replication machinery; however, direct confirmation of this hypothesis has not been possible. Based on the presence of conserved helicase motifs, UL9 has been classified as a superfamily II helicase. Mutations in conserved residues of the helicase motifs I-VI of UL9 have been isolated, and most of them fail to complement a UL9 null virus in vivo (Martinez R., Shao L., and Weller S. (1992) J. Virol. 66, 6735-6746). In addition, mutants in motifs I, II, and VI were found to be transdominant (Malik, A. K., and Weller, S. K. (1996) J. Virol. 70, 7859-7866). Here we present the characterization of the biochemical properties of the UL9 helicase motif mutants. We report that mutations in motifs I-IV and VI affect the ATPase activity, and all but the motif III mutation completely abolish the helicase activity. In addition, mutations in these motifs do not interfere with UL9 dimerization or the ability of UL9 to bind the HSV-1 origin of replication. Based on the similarity of the helicase motif sequences between UL9 and UvrB, another superfamily II member with helicase-like activity, we were able to map the UL9 mutations on the structure of the UvrB protein and provide an explanation for the observed phenotypes. Our results indicate that the helicase function of UL9 is indispensable for viral replication, supporting the hypothesis that UL9 is essential for unwinding the HSV-1 origin of replication in vivo. Furthermore, the data presented provide insights into the mechanism of transdominance of the UL9 helicase motif mutants.     

In Vitro Processing of Herpes Simplex Virus Type 1 DNA Replication Intermediates by the Viral Alkaline Nuclease, UL12

Herpes simplex virus type 1 (HSV-1) DNA replication intermediates exist in a complex nonlinear structure that does not migrate into a pulsed-field gel. Genetic evidence suggests that the product of the UL12 gene, termed alkaline nuclease, plays a role in processing replication intermediates (R. Martinez, R. T. Sarisky, P. C. Weber, and S. K. Weller, J. Virol. 70:2075–2085, 1996). In this study we have tested the hypothesis that alkaline nuclease acts as a structure-specific resolvase. Cruciform structures generated with oligonucleotides were treated with purified alkaline nuclease; however, instead of being resolved into linear duplexes as would be expected of a resolvase activity, the artificial cruciforms were degraded. DNA replication intermediates were isolated from the well of a pulsed-field gel (“well DNA”) and treated with purified HSV-1 alkaline nuclease. Although alkaline nuclease can degrade virion DNA to completion, digestion of well DNA results in a smaller-than-unit-length product that migrates as a heterogeneous smear; this product is resistant to further digestion by alkaline nuclease. The smaller-than-unit-length products are representative of the entire HSV genome, indicating that alkaline nuclease is not inhibited at specific sequences. To further probe the structure of replicating DNA, well DNA was treated with various known nucleases; our results indicate that replicating DNA apparently contains no accessible double-stranded ends but does contain nicks and gaps. Our data suggest that UL12 functions at nicks and gaps in replicating DNA to correctly repair or process the replicating genome into a form suitable for encapsidation.     

The human cytomegalovirus UL97 protein kinase,an antiviral drug target,is required at the stage of nuclear egress

Human cytomegalovirus encodes an unusual protein kinase, UL97, that activates the established antiviral drug ganciclovir and is specifically inhibited by a new antiviral drug, maribavir. We used maribavir and a UL97 null mutant, which is severely deficient in viral replication, to determine what stage of virus infection critically requires UL97. Compared with wild-type virus, there was little or no decrease in immediate-early gene expression, viral DNA synthesis, late gene expression, or packaging of viral DNA into nuclease-resistant structures in mutant-infected or maribavir-treated cells under conditions where the virus yield was severely impaired. Electron microscopy studies revealed similar proportions of various capsid forms, including DNA-containing capsids, in the nuclei of wild-type- and mutant-infected cells. However, capsids were rare in the cytoplasm of mutant-infected or maribavir-treated cells; the magnitudes of these decreases in cytoplasmic capsids were similar to those for virus yield. Thus, genetic and pharmacological evidence indicates that UL97 is required at the stage of infection when nucleocapsids exit from the nucleus (nuclear egress), and this poorly understood stage of virus infection can be targeted by antiviral drugs. Understanding UL97 function and maribavir action should help elucidate this interesting biological process and help identify new antiviral drug targets for an important pathogen in immunocompromised patients.     

Vaccinia virus nicking-joining enzyme is encoded by K4L (VACWR035)

Vaccinia virus encodes an enzyme with DNA modifying activity that cleaves and inefficiently cross-links cruciformic DNA. This enzyme is contained within the virion, expressed at late times postinfection, and processes DNA in an energy-independent, Mg2+ ion-independent manner. Viral nuclease activity was measured in extracts from cells infected with well-defined viral mutants. Since some viral extracts lacked nuclease activity, the gene encoding the activity was postulated to be one of the open reading frames absent in the viruses lacking activity. Inducible expression of each candidate open reading frame revealed that only the gene VACWR035, or K4L, was required for nuclease activity. A recombinant virus missing only the open reading frame for K4L lacked nuclease activity. Extracts from a recombinant virus expressing K4L linked to a FLAG polypeptide were able to cleave and cross-link cruciformic DNA. There were no significant differences between the virus lacking K4L and wild-type vaccinia virus WR with respect to infectivity, growth characteristics, or processing of viral replicative intermediate DNA, including both telomeric and cross-linked forms. Purification of the K4L FLAG polypeptide expressed in bacteria yielded protein containing nicking-joining activity, implying that K4L is the only vaccinia virus protein required for the nicking-joining enzymatic activity.     

Replication of herpesvirus DNA. V. Maturation of concatemeric DNA of pseudorabies virus to genome length is related to capsid formation.

The maturation of pseudorabies virus DNA from the replicative concatemeric form to molecules of genome length was examined using nine DNA+ temperature-sensitive mutants of pseudorabies virus, each belonging to a different complementation group. At the nonpermissive temperature, cells infected with each of the mutants synthesized concatemeric DNA. Cleavage of the concatemeric DNA to genome-length viral DNA was defective in all the DNA+ ts mutants tested, indicating that several viral gene products are involved in the DNA maturation process. In none of the ts mutant-infected cells were capsids with electron-dense cores (containing DNA) formed. Empty capsids with electron-translucent cores were, however, formed in cells infected with six of the nine temperature-sensitive mutants; in cells infected with three of the mutants, no capsid assembly occurred. Because these three mutants are deficient both in maturation of DNA and in the assembly of viral capsids, we conclude that maturation of viral DNA is dependent upon the assembly of capsids. In cells infected with two of the mutants (tsN and tsIE13), normal maturation of viral DNA occurred after shiftdown of the cells to the permissive temperature in the presence of cycloheximide, indicating that the temperature-sensitive proteins involved in DNA maturation became functional after shiftdown. Furthermore, because cycloheximide reduces maturation of DNA in wild-type-infected cells but not in cells infected with these two mutants, we conclude that a protein(s) necessary for the maturation of concatemeric DNA, which is present in limiting amounts during the normal course of infection, accumulated in the mutant-infected cells at the nonpermissive temperature. Concomitant with cleavage of concatemeric DNA, full capsids with electron-dense cores appeared after shiftdown of tsN-infected cells to the permissive temperature, indicating that there may be a correlation between maturation of DNA and formation of full capsids. The number of empty and full capsids (containing electron-dense cores) present in tsN-infected cells incubated at the nonpermissive temperature, as well as after shiftdown to the permissive temperature in the presence of cycloheximide, was determined by electron microscopy and by sedimentation analysis in sucrose gradients. After shiftdown to the permissive temperature in the presence of cycloheximide, the number of empty capsids present in tsN-infected cells decreased with a concomitant accumulation of full capsids, indicating that empty capsids are precursors to full capsids.     

Cellular alterations dependent upon the polyoma virus Hr-t function: separation of mitogenic from transforming capacities.

Hr-t mutants of polyoma virus are restricted in their growth properties (host range) and defective in cell transformation and tumor induction. The present study indicates that these mutants have lost the ability to induce morphological transformation, but have retained a mitogenic function. Thus an early and dramatic difference between wild-type virus and hr-t mutant-infected cultures of rat fibroblasts is the morphological change in individual cells observed by light, fluorescence and scanning electron microscopy. Viruses containing an intact hr-t function (wild-type virus and ts-a mutants) induce a transformed phenotype consisting of stellate cell shape, loss of defined cytoplasmic actin architecture, cellular "underlapping," and increased nuclear and nucleolar sizes. These prominent alterations constitute an abortive transformation, peaking 24-48 hr post-infection, and subsequently resolving in most or all of the cells. In contrast, cells infected with hr-t mutants do not develop the above structural changes, but rather retain their preinfection appearance. Both wild-type virus and hr-t mutants induce cellular DNA synthesis in confluent monolayers of rat cells beginning 12-14 hr post-infection. Flow microfluorometric (FMF) analysis confirms the viral mediated transit of cells from the G1 to the S and G2 phases of the cell cycle, as well as an increase in the proportion of cells with an 8N (octaploid) DNA content. Approximately 50% of the clones isolated from wild-type-infected cultures are polyploid. Stable transformants are found among these polyploid clones, but the majority of the latter resemble the parental cells in their morphology and growth properties. Polyploid clones are derived from hr-t mutant-infected cultures at a much lower frequency, similar to that of mock-infected cultures. Data obtained by sequential labeling of infected cultures with 3 H-thymidine and 5-bromo-deoxyuridine, together with cell number quantitation, indicate that hr-t mutants promote only a single round of cell division, while the wild-type virus and ts-a mutants promote multiple rounds. Loss of the hr-t function in polyoma virus therefore reveals a residual viral mitogenic activity, but prevents the virus from effecting morphological transformation of cells with concomitant loss of defined actin cables, polyploidization and multiple cycles of cell division in confluent cultures.     

The Product of the Herpes Simplex Virus Type 1 UL25 Gene Is Required for Encapsidation but Not for Cleavage of Replicated Viral DNA

The herpes simplex virus type 1 (HSV-1) UL25 gene contains a 580-amino-acid open reading frame that codes for an essential protein. Previous studies have shown that the UL25 gene product is a virion component (M. A. Ali et al., Virology 216:278–283, 1996) involved in virus penetration and capsid assembly (C. Addison et al., Virology 138:246–259, 1984). In this study, we describe the isolation of a UL25 mutant (KUL25NS) that was constructed by insertion of an in-frame stop codon in the UL25 open reading frame and propagated on a complementing cell line. Although the mutant was capable of synthesis of viral DNA, it did not form plaques or produce infectious virus in noncomplementing cells. Antibodies specific for the UL25 protein were used to demonstrate that KUL25NS-infected Vero cells did not express the UL25 protein. Western immunoblotting showed that the UL25 protein was associated with purified, wild-type HSV A, B, and C capsids. Transmission electron microscopy indicated that the nucleus of Vero cells infected with KUL25NS contained large numbers of both A and B capsids but no C capsids. Analysis of infected cells by sucrose gradient sedimentation analysis confirmed that the ratio of A to B capsids was elevated in KUL25NS-infected Vero cells. Following restriction enzyme digestion, specific terminal fragments were observed in DNA isolated from KUL25NS-infected Vero cells, indicating that the UL25 gene was not required for cleavage of replicated viral DNA. The latter result was confirmed by pulsed-field gel electrophoresis (PFGE), which showed the presence of genome-size viral DNA in KUL25NS-infected Vero cells. DNase I treatment prior to PFGE demonstrated that monomeric HSV DNA was not packaged in the absence of the UL25 protein. Our results indicate that the product of the UL25 gene is required for packaging but not cleavage of replicated viral DNA.     

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.     

The U(L)15 gene of herpes simplex virus type 1 contains within its second exon a novel open reading frame that is translated in frame with the U(L)15 gene product.

The U(L)15 gene of herpes simplex virus type 1 is composed of two exons. A mutation previously shown to preclude viral DNA cleavage and packaging at the nonpermissive temperature was identified as a change from a highly conserved serine to proline at codon 653. Separate viral mutants that contained stop codons inserted into exon I of U(L)15 (designated S648) or an insertion of the Escherichia coli lacZ gene into a truncated U(L)15 exon II [designated HSV-1(delta U(L)15ExII)] were constructed. Recombinant viruses derived from S648 and HSV-1(delta U(L)15ExII) and containing restored U(L)15 genes were constructed and designated S648R and HSV-1(delta U(L)15ExIIR), respectively. Unlike HSV-1(delta U(L)15ExIIR) and S648R, the viruses containing mutant U(L)15 genes failed to cleave and package viral DNA when propagated on noncomplementing cells. As revealed by electron microscopy, large numbers of enveloped capsids lacking viral DNA accumulated within the cytoplasm of cells infected with either S648 or HSV-1(delta U(L)15ExII) but not in cells infected with HSV-1(delta U(L)15ExIIR) or S648R. Thus, one function of the U(L)15 gene is to effectively prevent immature particles lacking DNA from exiting the nucleus by envelopment at the inner lamella of the nuclear membrane. Cells infected with HSV-1(delta U(L)15ExII) did not express the 75,000- or 35,000-apparent-Mr proteins previously shown to be products of the U(L)15 open reading frame, whereas the 35,000-apparent-Mr protein was readily detectable in cells infected with S648. We conclude that at least the 75,000-Mr protein is required for viral DNA cleavage and packaging and hypothesize that the 35,000-Mr protein is derived from translation of a novel mRNA located partially or completely within the second exon of U(L)15.     

Fragments from both termini of the herpes simplex virus type 1 genome contain signals required for the encapsidation of viral DNA.

A 535 base pair DNA fragment which maps entirely within the IRS/TRS regions of the herpes simplex virus type 1 (HSV-1) genome and contains all the cis-acting signals necessary for it to function as an origin of viral DNA replication has previously been identified (N.D. Stow and E.C. McMonagle, Virology, in press). When BHK cells were transfected with circular plasmid molecules containing cloned copies of this DNA fragment, and superinfected with wt HSV-1 as helper, amplification of the input plasmid was detected. Two observations indicated that the amplified DNA was not packaged into virus particles. Firstly, when the transfected cells were disrupted the amplified DNA was susceptible to digestion by added DNase, and secondly, it was not possible to further propagate the DNA when virus from the cells was passaged. Fragments from the joint region and from both termini of the viral genome were inserted into origin-containing plasmids and the resulting constructs analysed. In all cases the inserted fragment allowed the amplified DNA to be further passaged, and a proportion to become resistant to digestion with DNase. These observations suggest that signals required for the encapsidation of HSV-1 DNA are located within DNA sequences shared by the inserted fragments and therefore lie within the reiterated 'a' sequence of the viral genome.