Richard W. Hyman

The immediate early proteins of varicella-zoster virus

Stable, relatively high-titer varicella-zoster virus (VZV) stocks as well as a high-titer anti-VZV serum prepared in inbred guinea pigs have allowed the identification of VZV immediate early proteins using the classic inhibitor approach. VZV infection was initiated in the presence of cycloheximide. Following the removal of cycloheximide, actinomycin D and radiolabel were added. After the labeling period, extracts were immunoprecipitated with anti-VZV guinea pig serum and subjected to polyacrylamide gel electrophoresis and fluorography or autoradiography. Four immediate early proteins of mol wts 185,000, 69,000, 43,000, and 34,000 were identified. The largest three were phosphoproteins.

Specious hybridization between herpes simplex virus DNA and human cellular DNA.

Apparent hybridization between human cellular DNA and herpes simplex virus DNA was blocked by the presence of guanine-rich ribo- and deoxyribonucleic acid polymers. The data indicate that the apparent hybridization may very well be artifactual and not represent long stretches of authentic base sequence homology.

Hybridization of herpes simplex virus dna and human ribosomal DNA and RNA

A small DNA segment from the inverted repeats at the termini of the unique long sequence region of herpes simplex virus DNA was found to hybridize with human 28 S ribosomal DNA and RNA but not 18 S ribosomal DNA and RNA. The hybridization occurred under stringent conditions and was not blocked by nucleic acids high in guanine plus cytosine content. These data strongly suggest that the hybridization represented authentic base sequence homology.

Polyadenylated, cytoplasmic transcripts of varicella-zoster virus.

Cytoplasmic RNA was isolated from varicella-zoster virus (VZV)-infected cells. By oligo(dT)-cellulose chromatography, the RNA was separated into polyadenylated, poly (A)+, and nonpolyadenylated, poly (A)-, fractions. RNA blot hybridization was employed to detect and map VZV transcripts. As VZV infection cannot be coordinated, cytoplasmic RNA was isolated from VZV-infected cells when the cells showed extensive cytopathology. Therefore, while the VZV transcripts represented heterogeneous temporal classes, it may be assumed that late VZV RNA predominated. At least 41, and as many as 67 (depending on DNA probe overlap), VZV polyadenylated transcripts have been identified. Preliminary evidence for the presence of two VZV-specific nonpolyadenylated, cytoplasmic transcripts was observed.

Isomerization of the UL region of varicella-zoster virus DNA

Varicella-zoster virus (VZV) DNA exists principally as two isomers. Despite the presence of inverted repeats bounding the long sequence region, the unique long sequence, UL, is found in one (prototype) orientation in 95-98% of VZV DNA molecules and in the inverted orientation in only 2-5% of the molecules. In searching for an explanation for this disparity, we superinfected VZV-infected cells with herpes simplex virus type 1 or pseudorabies virus. Neither superinfecting virus produced a measurable change in the frequency of isomerization of the VZV DNA long sequence region.

A simple repetitive sequence common to herpes simplex virus type 1 and human ribosomal DNAs

The simple sequence GGC was tandemly repeated in herpes simplex virus type 1 DNA and human 28S rDNA and its mature 28 S rRNA transcript. The sequence homology was responsible for the observed hybridization between the two DNAs under high stringency blot hybridization conditions.

Variation in the Structure of Varicella-Zoster Virus DNA

By analyzing the fine structure of varicella-zoster virus (VZV) DNA, a naturally occurring heterogeneity was found on the right end of VZV DNA, but no evidence of a true terminal repetition was uncovered. We were unable to confirm the report of Straus and co-workers (1981) that there is a relatively high frequency of circular VZV DNA in low-passage virus. On long-term cell passage, extensive heterogeneity appeared concomitant with the accumulation of apparently defective VZV DNA.

Stability of the Cloned ‘Joint Region’ of Herpes Simplex Virus DNA

To isolate stable recombinants containing the joint region, or L-S junction, of herpes simplex virus DNA, the EcoRI restriction enzyme cleavage fragments were cloned into both coliphage lambda and plasmid vectors. The authentic joint region was found in the plasmid but not in the lambda vector. The plasmid-joint region recombinant DNAs appeared stable on limited passage. Subcloning the small BamHI L-S junction fragment into plasmid pBR322 gave rise to both stable and unstable recombinant DNAs.

Hybridization between a repeated region of herpes simplex virus type 1 DNA containing the sequence [GGC]n and heterodisperse cellular DNA and RNA

A small DNA fragment containing the simple sequence [GGC]10 from the long repeat of herpes simplex virus type 1 (HSV-1) DNA hybridized to cellular DNA and polyadenylated RNA from different mammalian species. The number and intensity of blot hybridization signals were increased in human compared with rodent and simian nucleic acids. The hybridization was blocked specifically by human 28S ribosomal DNA, which shares only the GGC repeats with the herpes simplex virus DNA. These data indicate that GGC repeats were common components of cellular DNA and were expressed in mRNA. Blot hybridization analysis of viral RNA from the HSV-1 gene regions encompassing the GGC repeats revealed abundant stable mRNAs from portions of the virus genome not previously analyzed in detail and indicated that the viral GGC sequence was not expressed in stable cytoplasmic mRNA.

Heteroduplex analysis of cloned fragments of herpes simplex virus DNAs

Abstract The heteroduplex map of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) DNAs was constructed with recombinant HSV-1 and HSV-2 DNAs so that the overall map was built up piece by piece across the virus genome. Extensive regions of good base sequence homology and some regions of partial homology were mapped. In certain cases, known virus functions were assigned to specific positions on the HSV-1-HSV-2 DNA heteroduplex map, and the sequences of HSV-1 and HSV-2 genes were compared.