Biomedical subjects
Publications and source records attributed to C E Dabrowski.
Cinnamyl derivatives of thieno[2,3-d]oxazinones are mechanism-based inhibitors of the HSV-2, VZV and CMV herpes proteases which demonstrate nanomolar potency. Compounds 5 and 28 inhibit protease processing in HSV-2 infected cells with a selectivity index of at least 30.
Enedione derivatives of thieno[2,3-d]oxazinones are nanomolar inhibitors of CMV protease which act through a novel dual acylation of the catalytic serine and alkylation of the protease cysteine 161 via a Michael addition to the enedione moiety of the inhibitor.
We have recently identified a novel 53-kDa herpes simplex virus type 1 (HSV-1) protein encoded by, and in frame with, the 3' half of the UL9 open reading frame, designated OBPC (K. Baradaran, C. Dabrowski and P. A. Schaffer, J. Virol. 68:4251-4261, 1994). Here we show that OBPC is a nuclear protein synthesized at both early and late times postinfection. In gel-shift assays in vitro-synthesized OBPC bound to oriS site I DNA to form a complex identical in mobility to complex A, generated with infected cell extracts and site I DNA. OBPC inhibited both plaque formation and viral DNA replication in transient assays, consistent with its ability to bind to site I DNA and its limited ability to interact with other essential DNA replication proteins. These properties suggest that OBPC may play a role in the initiation, elongation, or packaging of viral DNA.
The region of the UL component of the herpes simplex virus type 1 genome between nucleotides 17,793 and 25,150 includes three open reading frames that code for the protein products of the UL8, UL9, and UL10 genes (D.J. McGeogh, M.A. Dalrymple, A.J. Davison, A. Dolan, M.C. Frame, D. McNab, L.J. Perry, J.E. Scott, and P. Taylor, J. Gen. Virol. 69:1531-1574, 1988). We have mapped and characterized the overlapping transcripts in this region and have found that, in addition to the low-abundance UL8 and UL9 transcripts and the abundant UL10 transcript, at least two additional transcription units, designated UL8.5 and UL9.5, are specified by this region of the genome. The 5' ends of the UL8, UL8.5, and UL9 transcripts were mapped to nucleotides 20,682, 22,351, and 23,381, respectively. The 5' terminus of the UL9.5 transcript has not yet been mapped. The 3' ends of the UL8, UL8.5, UL9, and UL9.5 transcripts are coterminal at nucleotide 18,197. The 5' end of the UL10 mRNA, which is transcribed from the strand opposite that specifying the UL8, UL8.5, UL9, and UL9.5 transcripts, lies within the UL9 open reading frame at nucleotide 22,944, while the 3' terminus was mapped to nucleotide 24,666. Time course studies demonstrated that the UL8 and UL9 transcripts are members of the early kinetic class, the UL8.5 mRNA is a delayed-early transcript, and the UL9.5 and UL10 transcripts belong to the true-late kinetic class. Examination of the nucleotide sequence of the UL8.5 transcript revealed a potential open reading frame that overlaps and is in frame with the C-terminal half of the open reading frame encoding the origin-binding protein (OBP), the product of the UL9 gene. In vitro translation of the UL8.5 transcript demonstrated that it encodes a protein with an apparent molecular mass of 53 kDa. This protein was recognized by antibody directed against the C-terminal region of OBP and has thus been designated OBPC. A protein with an identical apparent molecular mass was also recognized by this antibody in infected-cell lysates, indicating that OBPC is synthesized during viral infection.
The herpes simplex virus type 1 (HSV-1) origin of DNA replication, oriS, contains an AT-rich region and three highly homologous sequences, sites I, II, and III, identified as binding sites for the HSV-1 origin-binding protein (OBP). In the present study, interactions between specific oriS DNA sequences and proteins in uninfected cell extracts were characterized. The formation of one predominant protein-DNA complex, M, was demonstrated in gel shift assays following incubation of uninfected cell extracts with site I DNA. The cellular protein(s) that comprises complex M has been designated origin factor I (OF-I). The OF-I binding site was shown to partially overlap the OBP binding site within site I. Complexes with mobilities indistinguishable from that of complex M also formed with site II and III DNAs in gel shift assays. oriS-containing plasmid DNA mutated in the OF-I binding site exhibited reduced replication efficiency in transient assays, demonstrating a role for this site in oriS function. The OF-I binding site is highly homologous to binding sites for the cellular CCAAT DNA-binding proteins. The binding site for the CCAAT protein CP2 was found to compete for OF-I binding to site I DNA. These studies support a model involving the participation of cellular proteins in the initiation of HSV-1 DNA synthesis at oriS.
The herpes simplex virus type 1 (HSV-1) origin of replication, oriS, contains three highly homologous sequences, sites I, II, and III. The HSV-1 origin-binding protein (OBP), the product of the UL9 gene, has been shown to bind specifically to sites I and II. In this study, gel shift analysis was used to characterize interactions between site I DNA and proteins in infected and uninfected cell extracts. The formation of two protein-DNA complexes, bands A and B, was demonstrated with infected cell extracts, and one predominant protein-DNA complex, band M, was identified with mock-infected extracts. Protein interactions with the highly homologous site II and III DNAs were also characterized. Incubation of infected cell extracts with the lower-affinity site II DNA as a probe resulted in the appearance of two protein-DNA complexes with mobilities identical to those of the A and B complexes, while incubation with site III DNA resulted in the formation of a single complex with the mobility of band B; no A-like band was observed. Incubation of high concentrations of partially purified OBP with site I DNA resulted in the formation of two novel complexes, bands 9-1 and 9-2. Addition of uninfected or HSV-1-infected cell extracts to the purified OBP-site I DNA mix significantly enhanced the formation of complex 9-1. The enhanced formation of complex 9-1 by uninfected cell extracts implicates a cellular factor or factors in the formation or stabilization of the OBP-site I DNA complex.