Hamida Qavi

Nucleotide sequence of the herpes simplex virus type 2 (HSV-2) thymidine kinase gene and predicted amino acid sequence of thymidine kinase polypeptide and its comparison with the HSV-1 thymidine kinase gene

To analyze the boundaries of the functional coding region of the HSV-2(333) thymidine kinase gene (TK gene), deletion mutants of hybrid plasmid pMAR401 H2G, which contains the 17.5 kbp BglII-G fragment of HSV-2 DNA, were prepared and tested for capacity to transform LM(TK-) cells to the thymidine kinase-positive phenotype. These studies showed that hybrid plasmids containing 2.2-2.4 kbp subfragments of HSV-2 BglII-G DNA transformed LM(TK-) cells to the thymidine kinase-positive phenotype and suggested that the region critical for transformation might be less than 2 kbp. That the activity expressed in the transformants was HSV-2 thymidine kinase was shown by experiments with type-specific enzyme-inhibiting rabbit antisera and by disc-polyacrylamide gel electrophoresis analyses. DNA fragments of the HSV-2 TK gene were subcloned in phage M13mp9 and M13mp8. A sequence of 1656 bp containing the entire coding region of the TK gene and the flanking sequences was determined by the dideoxynucleotide chain termination method. Comparisons with the HSV-1(Cl 101) TK gene revealed that PstI, PvuII, and EcoRI cleavage sites had homologous locations as did promoter, translational start and stop, and polyadenylation signals. Extensive homology was observed in the nucleotide sequence preceding the ATG translational start signal and in portions of the coding region of the genes. Comparisons of the predicted amino acid sequences of the HSV-1 and HSV-2 thymidine kinase polypeptides revealed that both were enriched in alanine, arginine, glycine, leucine, and proline residues and that clear, but interrupted homology existed within several regions of the polypeptide chains. Stretches of 15-30 amino acid residues were identical in conserved regions. The possibility is suggested that domains containing some of the conserved amino acid sequences might have a role in substrate binding and as major antigenic determinants.

Demonstration of HIV-1 and HHV-6 in AIDS-associated retinitis

Using an immunohistochemical technique and monoclonal antisera, HIV-1 and CMV antigens were demonstrated in lesioned areas of retinal tissues from selected AIDS patients. Polymerase chain reaction (PCR) was utilized to detect HIV-1 and HHV-6 DNA sequences in total retinal tissues from these patients. In this study of six eyes from four patients, two of the retinas contained three different viruses, HIV-1, HHV-6 and CMV. To determine whether HIV-1 and HHV-6 DNA sequences were restricted to the intraretinal lesions, normal and lesioned areas were dissected from the retina, DNA was extracted and subjected to amplification using PCR. The results showed that HIV-1 and HHV-6 DNA were restricted to the lesioned areas. All four lesions (from two different patients) utilized in this study showed the presence of CMV antigens immunohistochemically. The combination of viruses present in each lesion was either HIV-1 and CMV or HHV-6 and CMV. Two of four lesions contained HIV-1 and CMV; a third lesion showed the presence of HHV-6 and CMV. The fourth lesion contained only CMV antigens.

Thymidine kinase (TK) induction after infection of TK-deficient rabbit cell mutants with bovine herpesvirus type 1 (BHV-1): Isolation of TK− BHV-1 mutants☆

Cytosol thymidine kinase (TK) activity is enhanced at 6 hr after bovine embryo tracheal (EBTr) and rabbit skin fibroblast (RAB-9) cells are infected with the Los Angeles and Cooper strains of bovine herpesvirus type 1 (BHV-1). To learn whether this enhancement resulted from the induction of a virus-specific TK activity, biochemical and genetic studies were carried out. The biochemical experiments demonstrated that: (i) the BHV-1-induced TK activity had a relative disc PAGE mobility (Rm) characteristic of other herpesvirus-encoded TKs and distinctly different from the Rm value of the cytosol TK of host cells; and (ii) the BHV-1-induced TK was significantly more sensitive to competitive inhibition by arabinosylthymine (araT) than the cytosol TKs of EBTr and RAB-9 cells. The genetic experiments entailed the isolation of a bromodeoxyuridine (BrdUrd)-resistant rabbit cell line [RAB(BU)] deficient in cytosol TK activity and of BrdUrd- and araT-resistant BHV-1 mutants. RAB(BU) cells acquired TK activity after they were infected by wild-type, TK+ BHV-1, but not drug-resistant BHV-1 mutants. The experiments strongly suggest that wild-type BHV-1 induces a virus-specific TK activity.

Sequential genital infections by herpes simplex viruses types 1 and 2: Restriction nuclease analyses of viruses from recurrent infections

Virus isolated from a woman presenting with the first symptomatic episode of genital herpes was identified as herpes simplex virus type 1 (HSV-1) by restriction nuclease fingerprinting. Testing for IgM antibody to HSV indicated that the patient had recently contracted a new HSV infection. Virus microneutralization and the micro-solid phase radioimmunometric test for IgG, however, showed that the patient had had prior infection with herpes simplex virus type 2 (HSV-2); thus the HSV-1 infection was acquired despite the presence of antibody to HSV-2. Genital herpes recurred about four, seven, and nine months after the HSV-1 infection. Isolates from the latter three episodes all were of an identical strain of HSV-2 and were not recombinants or a mixture of the viruses. The data show that two distinctly different herpes simplex viruses can initiate genital infections in one individual and suggest that HSV-2 is more likely to recur than HSV-1.

Functional expression of the Herpes simplex virus thymidine kinase gene in Escherichia coli K-12.

The recombinant plasmid pAGO contains the Herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene and consists of a 2-kb PvuII fragment of HSV-1 DNA inserted into the PvuII site of pBR322. A deletion mutant of pAGO, designated pMH110, has been isolated which removes the normal HSV-1 TK gene promoter but places the promoter of the pBR322 tetracycline-resistance (tetr) gene only about 400 bp from the translational start codon of the HSV-1 TK polypeptide. In contrast to pAGO, which transforms mouse LM(TK-) cells to TK+ but is only weakly expressed in TK- bacteria, pMH110 not only efficiently transforms LM(TK-) cells to TK+ but also enables TK- Escherichia coli K-12 cells to form colonies on selective plates containing 5-fluorodeoxyuridine (FdUrd) plus thymidine (dThd) and to exhibit fully restored ability to incorporate [3H]dThd into DNA. The levels of TK activity expressed by bacteria harboring pMH110 were about as high as those expressed by bacteria harboring plasmid pTK3, which contains the wild-type E. coli TK gene. The TK activity expressed in bacteria harboring pMH110 was partially purified and shown to be HSV-1-specific by serological and disc PAGE analyses and by experiments demonstrating that this enzyme phosphorylated [125I]deoxycytidine.

The incidence of HIV-1 and HHV-6 in corneal buttons

Twenty pairs of corneas from asymptomatic carriers of HIV-1 and seven pairs from AIDS patients were analyzed for the presence of HIV-1 and HHV-6 antigens, viral transcripts, DNA sequences, and intact and infectious particles. Although serum from all donors was positive for both HIV-1 and HHV-6 antibody by Western blot analysis, only one cornea from an asymptomatic carrier of HIV-1 was positive for HIV-1 and HHV-6. The cornea was positive when tested by tissue culture, PCR, in situ hybridization, and electron microscopy. There was no tear film contamination. These results suggest that HIV-1 and HHV-6 may be capable of invading corneal tissue.

Biochemical transformation of LM(TK−) cells by hybrid plasmids containing the coding region of the herpes simplex virus type 1 thymidine kinase gene

Abstract Recombinant plasmid pAGO codes for herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) and consists of a 2-kbp HSV-1 DNA fragment inserted at the unique Pvu II cleavage site of plasmid pBR322. A hybrid plasmid, designated pMH110, has been derived from plasmid pAGO by deleting the 1689-bp pBR322 nucleotide sequence of pAGO, which extends from the Bam HI to the Pvu II cleavage site, and the 250-bp HSV-1 nucleotide sequence of pAGO, which extends from the Pvu II to the Bgl II cleavage site. Plasmid pMH110 biochemically transformed LM(TK − )cells to the TK + phenotype. The biochemically transformed cell lines had the following properties: (i) they were resistant to the growth-inhibiting effects of 1 m M thymidine; and (ii) they expressed an HSV-1-specific TK activity. This HSV-1 TK activity was purified after labeling biochemically transformed cell lines [LM(TK − )/TF pMH110 E2 and LM(TK − )/TF pMH110 Hc2] with [ 35 S]methionine. Immunoprecipitation experiments revealed that the TK polypeptides made in the biochemically transformed cells had molecular weights of about 39,000 to 40,000, which are about the same as the molecular weights of the TK polypeptides previously purified from HSV-1-infected LM(TK − ) cells and other biochemically transformed cell lines. The experiments support the hypothesis that the functional coding region of the HSV-1 TK gene is 3′ to the Bgl II cleavage site, and they also suggest that the HSV-1 TK messenger RNA may have been initiated in cells transformed by Hinc II- and Eco RI-cleaved pMH110 DNA at a site in cellular (or plasmid) DNA upstream from the HSV-1 DNA Bgl II cleavage site.

Cloning of the marmoset herpesvirus thymidine kinase gene and analyses of the boundaries of the coding region.

Abstract In order to delimit the approximate boundaries of the marmoset herpesvirus (MarHV) thymidine kinase (TK) gene, Hind III and Bam HI digests of MarHV DNA were cloned in plasmid pBR322. Several recombinant plasmids which transformed E. coli K12 strain RR1 to ampicillin resistance were isolated. The MarHV DNA inserts in these plasmids accounted for about half of the MarHV genome. One of the plasmids, pMAR4, contained a 9.1-kbp fragment of MarHV DNA ( Hind III-G), transformed LM(TK − ) cells to TK + , and hybridized to the Bam HI-I fragment of MarHV DNA, which had previously been shown to have TK-transforming activity. pMAR4 DNA had little or no homology to the 2-kbp Puv II fragment of HSV-1 DNA, which contains the HSV-1 TK gene. Cleavage with Pvu II, Sac I, Sma I, and Kpn I inactivated the TK-transforming activity of pMAR4, but cleavage with Hind III, Pst I, Eco RI, Xho I, Xba I, and Bam HI did not. Deletion mutants pMAR401 and pMAR420, which lacked the 2.6-kbp Kpn I and the 2.75-kbp Eco RI fragments, respectively, of pMAR4, lost transforming activity, whereas pMAR410, which lacked a 2.9kbp Xho I fragment of pMAR4 did not. Recombinant plasmid pMAR430, which contained a 3-kbp Pst I fragment of pMAR4, also transformed LM(TK − ) cells to TK + . The results strongly suggest that the coding region of the MarHV TK gene was within a 2.4-kbp pMAR4 sequence extending from the Pst I (0.33 kbp) to the Eco RI (2.7 kbp) cleavage sites.

Biochemical transformation of mouse cells by a purified fragment of marmoset herpesvirus DNA.

Although the size of marmoset herpesvirus (MarHV) DNA, estimated by velocity sedimentation in sucrose gradients, was similar to that of herpes simplex virus type 1 (HSV-1) DNA, the restriction endonuclease sites of MarHV and HSV-1 DNAs were quite different. A specific BamHI restriction fragment (6.2 x 10(6) daltons) of MarHV DNA biochemically transformed LM(TK-) mouse fibroblasts to the thymidine kinase(TK)-positive phenotype. Rabbit antisera, prepared against MarHV TK, inhibited MarHV-induced TK, but not HSV-1, HSV-2, or cellular TKs. Disc PAGE analyses and enzyme neutralization experiments with the anti-MarHV TK sera demonstrated that the TK expressed in MarHV transformants was MarHV-specific.

Integration site(s) of herpes simplex virus type 1 thymidine kinase gene and regional assignment of the gene for aminoacylase-1 in human chromosomes

To investigate the chromosomal sites of integration of the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene in HSV-1-transformed human HeLa(BU25)/KOS 8-1 cells, the biochemically transformed cells were fused with TK-negative mouse LM(TK-) cells, and human-mouse somatic cell hybrid lines (LH81) were isolated using a HATG-ouabain selection system. The presence of HSV-1 TK activity in the hybrid lines was verified by disc polyacrylamide gel electrophoresis (PAGE) and by enzyme neutralization with type-specific rabbit anti-HSV-1 TK immunoglobulin. Karyotype analyses of several somatic cell hybrid clones using G-banding, Hoechst 33258 staining, and combined G-banding and Hoechst staining demonstrated that they retained only a few human chromosomes. A marker chromosome, M7, consisting of a chromosome 17 translocated to the short arm of 3, occurred in 25 of the 28 metaphases examined. Also chromosomes 8 and X were found in a minority of metaphases. Isozyme analyses showed that all 19 hybrid clones analyzed expressed human aminoacylase-1 (ACY1) and esterase D (ESD), markers for 3 and 13, respectively. Back-selection of somatic cell hybrid clones with 5-bromodeoxyuridine resulted in the isolation of several subclones lacking HSV-1 TK activity, human ACY1, human ESD, and the human chromosomes. These experiments suggest that the HSV-1 TK gene is associated with either M7 or a segment of 13, or both, in biochemically transformed HeLa(BU25)/KOS 8-1 cells. These experiments also permit localization of the ACY1 structural gene to the pter leads to p12 region of 3.