Previous data have indicated that the development of resistance to amprenavir, an inhibitor of the human immunodeficiency virus type 1 protease, is associated with the substitution of valine for isoleucine at residue 50 (I50V) in the viral protease. We present further findings from retrospective genotypic and phenotypic analyses of plasma samples from protease inhibitor-naïve and nucleoside reverse transcriptase inhibitor (NRTI)-experienced patients who experienced virological failure while participating in a clinical trial where they had been randomized to receive either amprenavir or indinavir in combination with NRTIs. Paired baseline and on-therapy isolates from 31 of 48 (65%) amprenavir-treated patients analyzed demonstrated the selection of protease mutations. These mutations fell into four distinct categories, characterized by the presence of either I50V, I54L/I54M, I84V, or V32I؉I47V and often included accessory mutations, commonly M46I/L. The I50V and I84V genotypes displayed the greatest reductions in susceptibility to amprenavir, although each of the amprenavir-selected genotypes conferred little or no cross-resistance to other protease inhibitors. There was a significant association, for both amprenavir and indinavir, between preexisting baseline resistance to NRTIs subsequently received during the study and development of protease mutations (P ؍ 0.014 and P ؍ 0.031, respectively). Our data provide a comprehensive analysis of the mechanisms by which amprenavir resistance develops during clinical use and present evidence that resistance to concomitant agents in the treatment regimen predisposes to the development of mutations associated with protease inhibitor resistance and treatment failure.
Previous studies have suggested that the UL17 gene of herpes simplex virus type 1 (HSV-1) is essential for virus replication. In this study, viral mutants incorporating either a lacZexpression cassette in place of 1,490 bp of the 2,109-bp UL17 open reading frame [HSV-1(ΔUL17)] or a DNA oligomer containing an in-frame stop codon inserted 778 bp from the 5′ end of the UL17 open reading frame [HSV-1(UL17-stop)] were plaque purified on engineered cell lines containing the UL17 gene. A virus derived from HSV-1(UL17-stop) but containing a restored UL17 gene was also constructed and was designated HSV-1(UL17-restored). The latter virus formed plaques and cleaved genomic viral DNA in a manner indistinguishable from wild-type virus. Neither HSV-1(ΔUL17) nor HSV-1(UL17-stop) formed plaques or produced infectious progeny when propagated on noncomplementing Vero cells. Furthermore, genomic end-specific restriction fragments were not detected in DNA purified from noncomplementing cells infected with HSV-1(ΔUL17) or HSV-1(UL17-stop), whereas end-specific fragments were readily detected when the viruses were propagated on complementing cells. Electron micrographs of thin sections of cells infected with HSV-1(ΔUL17) or HSV-1(UL17-stop) illustrated that empty capsids accumulated in the nuclei of Vero cells, whereas DNA-containing capsids accumulated in the nuclei of complementing cells and enveloped virions were found in the cytoplasm and extracellular space. Additionally, protein profiles of capsids purified from cells infected with HSV-1(ΔUL17) compared to wild-type virus show no detectable differences. These data indicate that the UL17 gene is essential for virus replication and is required for cleavage and packaging of viral DNA. To characterize the UL17 gene product, an anti-UL17 rabbit polyclonal antiserum was produced. The antiserum reacted strongly with a major protein of apparent M
r 77,000 and weakly with a protein of apparent M
r 72,000 in wild-type infected cell lysates and in virions. Bands of similar sizes were also detected in electrophoretically separated tegument fractions of virions and light particles and yielded tryptic peptides of masses characteristic of the predicted UL17 protein. We therefore conclude that the UL17 gene products are associated with the virion tegument and note that they are the first tegument-associated proteins shown to be required for cleavage and packaging of viral DNA.
A panel of 10 clinical isolates of herpes simplex virus (HSV) deficient in the expression of thymidine kinase (TK) and phenotypically resistant to aciclovir was characterized. Sequence analysis revealed a variety of mutations in TK (nucleotide substitutions, insertions and deletions), most of which resulted in truncated TK polypeptides. In line with previous reports, the most common mutation was a single G insertion in the 'G-string' motif. One HSV-1 isolate and two HSV-2 isolates appeared to encode full-length polypeptides and, in each case, an amino acid substitution likely to be responsible for the phenotype was identified. Pathogenicity was determined using a zosteriform model of HSV infection in BALB/c mice. The majority of isolates appeared to show impaired growth at the inoculation site compared with wild-type virus. They also showed poor replication in the peripheral nervous system and little evidence of zosteriform spread. One exception was isolate 4, which had a double G insertion in the G-string but, nevertheless, exhibited zosteriform spread. These studies confirmed that TK-deficient viruses display a range of neurovirulence with respect to latency and zosteriform spread. These results are discussed in the light of previous experience with TK-deficient viruses.
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