Characterization of a herpes simplex virus type 1 mutant resistant to benzhydrazone, a selective inhibitor of herpesvirus glycosylation.

Abstract

Benzhydrazone [BH;indene-1,3(2H)-dionebis(amidinohydrazone)] significantly inhibits glycosylation of proteins, but only in cells infected with herpes simplex virus. We report on a herpes simplex virus type 1 (HSV-1) mutant resistant to BH. A syncytium-inducing mutant designated HSV-1(13)S11 was found to be biochemically resistant to BH in that [14C]glucosamine incorporation was not inhibited in infected HEp-2 cells exposed to the drug. Intertypic recombinants were obtained which showed that BH resistance is encoded in the DNA of the mutant virus and may be transferred into the genome of BH-sensitive HSV. In the recombinants the biochemical resistance marker segregated from the syncytial marker, suggesting that the two markers probably map in different loci. The BH-resistant mutant did not complement wild-type BH-sensitive HSV-1 and -2. Furthermore, resistance was apparent in HEp-2 but not in Vero cells. The paper discusses the hypothesis that inhibition of glycosylation of HSV proteins is the consequence of modification or selective transport of BH involving a HSV gene product. ing intertypic recombinants, we observed that BH resistance is encoded in HSV-1(13)S11 DNA and may be transferred from the genome of the resistant mutant into the genome of HSV-2(G).MATERIALS AND METHODS Materials. BH was synthesized as previously described (1).Viruses and Cells. The BH-resistant (BHR) mutant HSV-1(13)S11 is a Syn-mutant selected from HEp-2 cell cultures infected with stocks obtained after 5-bromodeoxyuridine mutagenesis of the non-syncytium-inducing (Syn') HSV-1(13) (4); isolation and selection were as described for HSV-1(13)B4 (5). HSV-1(HFEM)H6 was described (6). Glycoprotein and Protein Synthesis. Labeling media for glycoproteins and proteins consisted, respectively, of minimal essential medium containing '/2 the usual concentration of glucose and [14C]glucosamine (2-3 ,uCi/ml, 50 mCi/mmol, the Radiochemical Center, Amersham; 1 Ci = 37 GBq) or minimal essential medium containing V/1o the usual concentration of methionine and [35S]methionine (25 ,uCi/ml, >400 Ci/mmol, New England Nuclear). Glycoproteins were labeled between 14 and 20 hr after infection. a polypeptides were detected by exposing cells to cycloheximide (5 ,ug/ml) from 1 hr before virus adsorption to 5 hr after infection and by labeling for 20 min after cycloheximide removal. , and y polypeptides were labeled from 5 to 7 and from 15 to 18 hr after infection, respectively. Protein nomenclature was according to Morse et al. (7). Details for harvesting cells and measuring trichloroacetic acid-precipitable radioactive material were described (2). NaDodSO4 electrophoresis of glycoproteins and proteins was on 8.5% polyacrylamide gels crosslinked with NN'-diallyl tartardiamide.Intertypic Marker Transfer. Intertypic marker transfer was done essentially as described (8,9). Approximately 1 ,g of HSV-1(13)S11 DNA digested with HindIII was mixed with 0.5 ,ug of intact HSV-2(G) DNA and the mixture was used to transfect rabbit skin cells. 2440The publication co...

<i>c-fo</i><i>s</i> Proto-Oncogene Transient Transcription Is Negatively Affected in the EL&alpha;4-2 Transformed Rat Cell Line

“…In fact, in a previous paper, we showed that EGF induces the expression of a4 gene; the expression of this IE product could, itself, influence HSV-1 expression, as ICP4 has been shown to interact with both viral and cellular control sequences to either activate or repress transcription [14][15][16][17], Moreover, it has been shown that the expression of altered ICP4. with the for mation of non functional heterodimers, inhibits virus pro duction [27],…”

“…The virus strain used was HSV-l(F) [13]. Infection of cell lines was as described previously [14,15],…”

Fine Mapping and Characterization of the Syn 6 Locus in the Herpes Simplex Virus Type 1 Genome

“…The experimental results indicated that some HSV-I(13) Sll late polypeptides exhibit an altered electrophoretic mobility compared to those of the wild-type strain. Also, mutant HSV-I(13) S11, which lacks glycoprotein C (gC-) (Tognon et al, 1984), does not synthesize ICPI 5, the 105K precursor of gC; three other structural proteins, ICP23, ICP32 and ICP35, were also defective. It appeared that the kinetics of accumulation of these polypeptides were slower than those of the homologous polypeptides of the parental strain, but similar concentrations were reached late in infection (Fig.…”

“…Specifically, the BamHI S fragment (approximately 2-8 kb) contains part of the s0 gene encoding the transactivator protein ICP0 (Everett, 1987) and the sequences encoding ICP34.5, which is related to neurovirulence . We investigated the infected cell proteins (ICP) which accumulated in HEp-2 cells infected with HSV-I(13) Sll (Tognon et al, 1984), the parental strain HSV-I(13) (Cassai et al, 1972), and the prototype HSV-I(F) (Ejercito et al, 1968) and a deletion mutant derived from it, R3631 (Mavromara-Nazos et al, 1986), which lacks the ~47 gene. The last two strains were used as controls.…”

Adenosylmethionine Decarboxylase Inhibitors – Lack of Activity Against Cytopathic Effects of HIV

“…b Determined by using the AdoMetDC of baker's yeast. tiviral properties [8][9][10][11]. Because the viral disease acquired immunodeficiency syn drome is obviously the most serious medi cal problem of mankind at present, screening of AdoMetDC inhibitors and other antiviral bis(amidinohydrazones) against its etiological agent, the human immunodeficiency virus (HIV), is highly warranted.…”