Size and Composition of Marek''s Disease Virus Deoxyribonucleic Acid

Deoxyribonucleic acid (DNA) extracted from purified nucleocapsids of Marek's disease herpesvirus (MDV) was cosedimented with T4 and with herpes simplex virus (HSV) DNA in neutral sucrose density gradients and with T4 DNA in alkaline sucrose density gradients. These experiments indicated that the intact MDV DNA had a sedimentation constant of 56S corresponding to a molecular weight of 1.2 x 10(8) daltons. In the alkaline gradients, the largest and most prominent band contains a DNA sedimenting at 70S corresponding to 6.0 x 10(7) daltons in molecular weight. The DNA is therefore double-stranded and not cross-linked. Isopycnic sedimentation of the MDV DNA molecules with SPO1, Micrococcus lysodeikticus, and HSV DNA gave a density of 1.705 g/cm(3) corresponding to 46 guanine plus cytosine moles per cent. Lastly, in hybridization tests the DNA hybridized with RNA of infected cells but not with that of uninfected cells supporting the conclusion that it is viral.     

Composition and Size of Shope Fibroma Virus Deoxyribonucleic Acid

Deoxyribonucleic acid (DNA) extracted from purified virions of Shope fibroma virus (SFV) (by using DNA from Microccocus lysodeikticus as marker) had a buoyant density of 1.6996 +/- 0.0003 g/ml), hence a guanine plus cytosine (G + C) content of 40.4 +/- 0.3%, which is close to the G + C content of the DNA of susceptible rabbit cells (40.9 +/- 0.4%) and different from that of vaccinia virus DNA (35.5 +/- 0.4%). For the determination of the molecular weight of DNA, SFV and vaccinia purified virions, treated with Pronase and detergent, were cosedimented in sucrose density gradients. Results showed that SFV-DNA has a molecular weight of about 153 x 10(6) daltons. By electron microscopy, only one molecule corresponding to this value was observed (its length was 80.3 mum). The others had a median size of 49.8 mum +/- 0.9.     

Proteins Specified by Herpes Simplex Virus VIII. Characterization and Composition of Multiple Capsid Forms of Subtypes 1 and 2

Two classes of herpesvirus capsids, designated A and B, were isolated from the nuclei of human cells infected with herpes simplex virus (HSV). A and B capsids share in common four structural proteins, i.e., no. 5, 19, 23, and 24. B capsids contain 7.7 to 9.7 times more deoxyribonucleic acid than A capsids; moreover, they contain proteins no. 21 and 22a in addition. All of the proteins contained in the capsid except no. 22a are present in the enveloped nucleocapsids (virions) in approximately the same molar ratios. The capsid proteins of HSV-1 cannot be differentiated from their HSV-2 counterparts with respect to electrophoretic mobility. A third class of capsids, designated C capsids, was isolated from virions contained in the cytoplasm of infected cells by the same procedure used to obtain A and B capsids. The C capsids contain all of the proteins present in A capsids plus proteins 1 to 3 and 21.     

Deoxyribonucleic Acid Synthesis in Synchronized Mammalian KB Cells Infected with Herpes Simplex Virus

We examined the patterns of host cell and virus deoxyribonucleic acid (DNA) synthesis in synchronized cultures of KB cells infected at different stages of the cell cycle with herpes simplex virus (HSV). We found that the initiation of HSV DNA synthesis, we well as the production of new infectious virus, is independent of the S, G1, and G2 phases of the mitotic cycle of the host cell. This is in contrast to data previously found with equine abortion virus. Because HSV replicates independently of the cell cycle, we were able to establish conditions that would permit the study of rates of HSV DNA synthesized in logarithmically growing cells in the virtual absence of cellular DNA synthesis. This eliminates the need for separation of viral and cellular DNA by isopycnic centrifugation in CsCl. We found that HSV DNA synthesis was initiated between 2 to 3 hr after infection. The rate of DNA synthesis increased rapidly, reaching a maximum 4 hr after infection, and decreased to 50% of maximum by 8 hr. Evidence is also presented which suggests that HSV infection can inhibit both the ongoing synthesis of host DNA as well as the initiation of the S phase.     

Sensitivity of Herpes Simplex Virus, Vaccinia Virus, and Adenoviruses to Deoxyribonucleic Acid Inhibitors and Thiosemicarbazones in a Plaque Suppression Test

Herpes simplex and vaccinia viruses and adenovirus types 1, 2, 5, and 7 were tested by plaque suppression methods for sensitivity to halogenated deoxyuridines (5-iodo-, 5-bromo-, 5-chloro-, and 5-fluoro-), cytosine arabinoside, isatin-beta-thiosemicarbazone, and N-methylisatin-beta-thiosemicarbazone. After incubation for 12 days in HeLa cell cultures, vaccinia virus plaques were still readily suppressed by deoxyribonucleic acid (DNA) inhibitors and thiosemicarbazones. Herpes simplex virus plaques were likewise suppressed by at least three DNA inhibitors. Adenovirus plaques were not suppressed by DNA inhibitors or thiosemicarbazones. 5-Fluoro-2'-deoxyuridine could not be shown to have any antiviral activity, but it did produce a substantial lethal action on the cells.     

Herpes Simplex Virus Types 1 and 2 Completely Help Adenovirus-Associated Virus Replication

In addition to adenoviruses, which are capable of completely helping adenovirus-associated virus (AAV) multiplication, only herpesviruses are known to provide any AAV helper activity, but this activity has been thought to be partial (i.e., AAV DNA, RNA, and protein syntheses are induced, but infectious particles are not assembled). In this study, however, we show that herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) are in fact complete AAV helpers and that AAV type 2 (AAV2) infectivity yields can approach those obtained when coinfections are carried out with a helper adenovirus. AAV helper activity was demonstrated in KB cells with two HSV-1 strains (11124 and 17MP) and an HSV-2 strain (HG52). Each herpesvirus supported AAV2 multiplication with comparable efficiency. AAV2 multiplication was similarly efficient in HSV-1 coinfections of HeLa cells, whereas lower yields were obtained in HEp-2 and primary human embryonic kidney cells. HSV-1 also supported AAV1 multiplication in HeLa cells but, at corresponding multiplicities of infection, AAV1 grew less efficiently than AAV2. Comparisons of the time courses of AAV2 DNA, RNA, and protein syntheses after coinfection with either adenovirus type 5 or HSV-1 revealed that, in each case, the onset of synthesis and attainment of maximal synthesis rate occurred earlier in coinfections with HSV-1. These findings demonstrate the linkage of AAV macromolecular synthesis to an event(s) in the helper virus cycle. Aside from this temporal association, helper-related differences in AAV macromolecular synthesis were not apparent.     

Transport Media for Herpes Simplex Virus Types 1 and 2

An evaluation was made of the recovery rate of herpes simplex virus (HSV) type 1 or 2 from 197 clinical specimens obtained in two or three charcoal transport media: Leibovitz viral transport medium, a modified Leibovitz-Emory medium (LEM), in which agarose was used instead of agar, and Amies bacterial transport medium. The specimens were stored and shipped for 1 to 19 days in these media at ambient temperature or in Hanks buffered-salt solution in dry ice. The results indicate that the LEM was most effective, particularly in the recovery of HSV type 2 from clinical specimens held at ambient temperature. In vitro and in vivo studies in genitally infected mice corroborated the observations obtained with human clinical specimens. The availability of transport media which can be used for shipment at ambient temperature offers clinicians easier accessibility to laboratory confirmation and antigenic typing of HSV from suspect herpetic infections.     

Suppression of Herpes Simplex Virus Infection by Phosphonoacetic Acid

Disodium phosphonoacetate when administered orally or topically to mice experimentally infected with herpes simplex virus was able to significantly reduce the mortality associated with the agent. In addition, this compound was able to reduce herpesvirus lesions on the corneas of infected rabbits.     

Herpes Simplex Virus: Genome Size and Redundancy Studied by Renaturation Kinetics

Herpes simplex virus subtype 1 deoxyribonucleic acid (DNA) was sheared in a French press to uniform fragments, denatured by heating, then allowed to reassociate. The renaturation reaction followed second-order kinetics with a single rate constant indicating that at least 95% of the genome was unique and that repetitive sequences, if present, were not detectable by this technique. The kinetic complexity of the herpes simplex genome was determined by DNA renaturation kinetics to be (95 ± 1) × 10⁶ daltons. Since this value is in excellent agreement with the molecular weight of viral DNA [(99 ± 5) × 10⁶ daltons] obtained from velocity sedimentation studies, it is concluded that virions contain only one species of double-stranded DNA molecules 95 × 10⁶ to 99 × 10⁶ daltons in molecular weight.     

Glycopeptides of the Type-Common Glycoprotein gD of Herpes Simplex Virus Types 1 and 2

We have carried out detailed structural studies of the glycopeptides of glycoprotein gD of herpes simplex virus types 1 and 2. We first examined and compared the number of N-asparagine-linked oligosaccharides present in each glycoprotein. We found that treatment of either pgD-1 or pgD-2 with endo-β-N-acetylglucosaminidase H (Endo H) generated three polypeptides which migrated more rapidly than pgD on gradient sodium dodecyl sulfate-polyacrylamide gels. Two of the faster-migrating polypeptides were labeled with [³H]mannose, suggesting that both pgD-1 and pgD-2 contained three N-asparagine-linked oligosaccharides. Second, we characterized the [³H]mannose-labeled tryptic peptides of pgD-1 and pgD-2. We found that both glycoproteins contained three tryptic glycopeptides, termed glycopeptides 1, 2, and 3. Gel filtration studies indicated that the molecular weights of these three peptides were approximately 10,000, 3,900, and 1,800, respectively, for both pgD-1 and pgD-2. Three methods were employed to determine the size of the attached oligosaccharides. First, the [³H]mannose-labeled glycopeptides were treated with Endo H, and the released oligosaccharide was chromatographed on Bio-Gel P6. The size of this molecule was estimated to be approximately 1,200 daltons. Second, Endo H treatment of [³⁵S]methionine-labeled glycopeptide 2 reduced the molecular size of this peptide from approximately 3,900 to approximately 2,400 daltons. Third, glycopeptide 2 isolated from the gD-like molecule formed in the presence of tunicamycin was approximately 2,200 daltons. From these experiments, the size of each N-asparagine-linked oligosaccharide was estimated to be approximately 1,400 to 1,600 daltons. Our experiments indicated that glycopeptides 2 and 3 each contained one N-asparagine-linked oligosaccharide chain. Although glycopeptide 1 was large enough to accommodate more than one oligosaccharide chain, the experiments with Endo H treatment of the glycoprotein indicated that there were only three N-asparagine-linked oligosaccharides present in pgD-1 and pgD-2. Further studies of the tryptic glycopeptides by reverse-phase high-performance liquid chromatography indicated that all of the glycopeptides were hydrophobic in nature. In the case of glycopeptide 2, we observed that when the carbohydrate was not present, the hydrophobicity of the peptide increased. The properties of the tryptic glycopeptides of pgD-1 were compared with the properties predicted from the deduced amino acid sequence of gD-1. The size and amino acid composition compared favorably for glycopeptides 1 and 2. Glycopeptide 3 appeared to be somewhat smaller than would be predicted from the deduced sequence of gD-1. It appears that all three potential glycosylation sites predicted by the amino acid sequence are utilized in gD-1 and that a similar number of glycosylation sites are present in gD-2.     

Isolation and Characterization of the Herpes Simplex Virus 1 Terminase Complex

During herpes simplex virus 1 (HSV-1) infection, empty procapsids are assembled and subsequently filled with the viral genome by means of a protein complex called the terminase, which is comprised of the HSV-1 UL15, UL28, and UL33 proteins. Biochemical studies of the terminase proteins have been hampered by the inability to purify the intact terminase complex. In this study, terminase complexes were isolated by tandem-affinity purification (TAP) using recombinant viruses expressing either a full-length NTAP-UL28 fusion protein (vFH476) or a C-terminally truncated NTAP-UL28 fusion protein (vFH499). TAP of the UL28 protein from vFH476-infected cells, followed by silver staining, Western blotting, and mass spectrometry, identified the UL15, UL28, and UL33 subunits, while TAP of vFH499-infected cells confirmed previous findings that the C terminus of UL28 is required for UL28 interaction with UL33 and UL15. Analysis of the oligomeric state of the purified complexes by sucrose density gradient ultracentrifugation revealed that the three proteins formed a complex with a molecular mass that is consistent with the formation of a UL15-UL28-UL33 heterotrimer. In order to assess the importance of conserved regions of the UL15 and UL28 proteins, recombinant NTAP-UL28 viruses with mutations of the putative UL28 metal-binding domain or within the UL15 nuclease domain were generated. TAP of UL28 complexes from cells infected with each domain mutant demonstrated that the conserved cysteine residues of the putative UL28 metal-binding domain and conserved amino acids within the UL15 nuclease domain are required for the cleavage and packaging functions of the viral terminase, but not for terminase complex assembly.     

Biological Characteristics of Cloned Populations of Herpes Simplex Virus Types 1 and 2

By using cloned types 1 and 2 herpes simplex virus, obtained by selecting large and small plaques produced by material from human lesions, studies were performed to compare properties between preparations of each type. Regarding the rate of inactivation by ultraviolet light, no differences were found between the two antigenic types and none between the preparations obtained from either type. In contrast, type 1 preparations were found to be more readily inactivated at 45 C than type 2. Plaque size of cloned preparations changed by passage in cell culture. A broader range of plaque sizes was obtained, and average plaque size was larger. After 20 passages, preparations obtained from different types gave rise to one of three kinds of cytopathic effect. The cytopathic effect produced by type 1 preparations remained as before 20 passages and consisted of round cells in a compact central mass. For type 2, two kinds of cytopathic effect were seen in cloned preparations. This consisted of aggregates of round cells (seen in preparations before 20 passages) or of large, loose aggregates of round cells of various sizes. Results from neutralization studies using virus before and after 20 passages in cell culture versus antisera prepared against live or ultraviolet-inactivated virus showed no differences between cloned preparations obtained from a given type.     

PKR-Dependent Xenophagic Degradation of Herpes Simplex Virus Type 1

The lysosomal pathway of autophagy is the major catabolic mechanism for degrading long-lived cellular proteins and cytoplasmic organelles. Recent studies have also shown that autophagy (xenophagy) may be used to degrade bacterial pathogens that invade intracellularly. However, it is not yet known whether xenophagy is a mechanism for degrading viruses. Previously, we showed that autophagy induction requires the antiviral eIF2alpha kinase signaling pathway (including PKR and eIF2alpha) and that this function ofeIF2alpha kinase signaling is antagonized by the herpes simplex virus (HSV-1) neurovirulence gene product, ICP34.5. Here, we show quantitative morphologic evidence of PKR-dependent xenophagic degradation of herpes simplex virions and biochemical evidence of PKR and eIF2alpha-dependent degradation of HSV-1 proteins, both of which are blocked by ICP34.5. Together, these findings indicate that xenophagy degrades HSV-1 and that this cellular function is antagonized by the HSV-1 neurovirulence gene product, ICP34.5. Thus, autophagy-related pathways are involved in degrading not only cellular constituents and intracellular bacteria, but also viruses.     

Genome Sequence of Herpes Simplex Virus 1 Strain McKrae

The herpes simplex virus 1 (HSV-1) strain McKrae is highly virulent compared to other wild-type strains of HSV-1. To help us better understand the genetic determinants that lead to differences in the pathogenicity of McKrae and other HSV-1 strains, we sequenced its genome. Comparing the sequence of McKrae's genome to that of strain 17 revealed that the genomes differ by at least 752 single nucleotide polymorphisms (SNPs) and 86 insertion/deletion events (indels). Although the majority of these polymorphisms reside in noncoding regions, 241 SNPs and 10 indels alter the protein-coding sequences of 58 open reading frames. Some of these variations are expected to contribute to the pathogenic phenotype of McKrae.     

Preparation of Type 2 Herpes Simplex Virus Complement-Fixing Antigen

Comparable complement-fixing antigens of type 1 and type 2 herpes simplex virus were produced by extraction of infected African green monkey cells with 0.85% NaCl which was buffered at pH 9.0 with 0.05 m glycine-NaOH. The optimal antigen dilutions were higher in titrations against hyperimmune animal sera than in titrations against human sera. Complement-fixing antibody to type 2 herpes antigen was detected in 5 of 17 sera from healthy humans.