Yoshitaka Isaka

A single amino acid change at Leu-188 in the reverse transcriptase of HIV-2 and SIV renders them sensitive to non-nucleoside reverse transcriptase inhibitors

Summary. Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are selective for human immunodeficiency virus type 1 (HIV-1) and generally not effective on HIV-2 or simian immunodeficiency virus (SIV). Only SIVagm was found to be sensitive to NNRTIs. When the amino acid differences in RT between SIVmac and SIVagm were compared with the known amino acid substitutions of NNRTI-resistance variants of HIV-1, we came to consider that the amino acid residue Leu-188 of HIV-2 and SIVmac might be related to their resistance to NNRTIs. To test this hypothesis, we substituted Leu-188 to Cys or Tyr in HIV-2 and SIVmac, and examined sensitivity of the mutant molecular clones to NNRTIs. The L188Y mutant of HIV-2 became completely sensitive to delavirdine and efavirenz, while that of SIVmac was also significantly sensitive to these NNRTIs. We further isolated NNRTI-resistant variants from these mutant viruses and determined amino acid substitutions in RT. The roles of the observed substitutions in NNRTI-resistance were further confirmed by site-directed mutagenesis. Our study reveals the crucial role of L188 in the natural resistance of HIV-2 and SIVmac to NNRTIs. Furthermore, the observed substitutions in RT of HIV-2 and SIVmac support the common mechanism of action of NNRTIs against HIV-1, HIV-2 and SIV.

Targeting of Chrolamphenicol Acetyltransferase to Human Immunodeficiency Virus Particles via Vpr and Vpx

Vpr and Vpx are the auxiliary proteins of human immunodeficiency viruses (HIVs) selectively incorporated into mature viral particles. We showed that the bacterial chloramphenicol acetyltransferase (CAT) fused to the N‐terminus of HIV‐1 Vpr, HIV‐2 Vpr, or HIV‐2 Vpx was incorporated into mature virions in a type‐selective manner. By using chimeric proteins between HIV‐1 Vpr and HIV‐2 Vpx, we found that the N‐terminal side of these proteins was mainly important for type‐selective virion incorporation. The C‐terminal arginine‐rich region of HIV‐1 Vpr was also found to transport CAT fusion proteins into virions but without any type selectivity. Furthermore, the corresponding regions of HIV‐2 Vpr and HIV‐2 Vpx had no such activity. This region of HIV‐1 Vpr may interact nonspecifically with viral genomic RNA. Collectively, Vpr and Vpx may provide a means to introduce foreign proteins and other molecules into HIV virions for therapeutic purposes.

Human sera from varicella-zoster virus (VZV) infections cross-react with human T cell leukaemia virus type 1 (HTLV-1): common epitopes in VZV gene 22 protein and HTLV-1 p19 gag protein

Twenty-nine of 100 sera from patients recently infected with varicella-zoster virus (VZV) were found to cross-react with human T cell leukaemia virus type 1 (HTLV-1) antigen in the particle agglutination (PA) assay using HTLV-1 antigen-coated gelatin particles. Anti-VZV IgM antibodies were shown to be responsible for this cross-reactivity. Western blot analysis revealed that PA-positive anti-VZV sera reacted with the HTLV-1 gag p19 protein in HTLV-1-infected cells and recombinant p19 protein produced in Escherichia coli. By using a truncated p19, the cross-reactive region was located to the C-terminal 17 amino acids of p19. One oligopeptide derived from the C terminus, PQIPPPYVEPT (amino acids 115 to 125), was capable of inhibiting PA, suggesting that this peptide carries the cross-reactive epitope. A homologous sequence was found in the VZV gene 22 protein by database analysis, and the oligopeptide TNIPPPLALLR (amino acids 1330 to 1340) had the ability to inhibit PA. These findings suggest that some IgM antibodies against the VZV gene 22 protein produced in the early phase of VZV infection are cross-reactive with the HTLV-1 gag p19 protein because they recognize an antigenic determinant containing an IPPP tetrapeptide.

Isolation and characterization of simian immunodeficiency virus variants that are resistant to nonnucleoside reverse transcriptase inhibitors

Summary. Non-nucleoside reverse transcriptase inhibitors (NNRTIs) act quite specifically on human immunodeficiency virus type 1 (HIV-1). In general, they are not effective on human immunodeficiency virus type 2 (HIV-2) or simian immunodeficiency virus (SIV). Only SIV strains from African green monkeys are sensitive to several NNRTIs. Here we isolated NNRTI- and 3TC-resistant SIVagm variants. Viruses resistant to delavirdine contained V112I and M231I substitutions, while those resistant to 3TC contained a M185I substitution. These amino acids are highly conserved in HIV-1, HIV-2, SIVmac and SIVagm, and the M184I (M185I in SIVagm) substitution was observed in 3TC-resistant HIV-1 and SIVmac. The roles of the observed mutations in NNRTI-resistance of SIVagm and HIV-1 were further confirmed by site-directed mutagenesis. The present results have provided a new insight into the common mechanism of sensitivity of HIV-1 and SIVagm to NNRTIs.

Construction of the Chimeric Reverse Transcriptase of Simian Immunodeficiency Virus Sensitive to Nonnucleoside Reverse Transcriptase Inhibitor

A number of structurally diverse compounds have been shown to be potent inhibitors of the DNA polymerase activity of human immunodeficiency virus type 1 (HIV‐1) reverse transcriptase (RT). The compounds can be grouped into two broad classes; nucleoside analogs and nonnucleoside RT inhibitors. The nonnucleoside RT inhibitors are quite specific for HIV‐1 RT but not human immunodeficiency virus type 2 (HIV‐2) and simian immunodeficiency virus (SIV) RT. We have investigated the property of SIV/HIV‐1 chimeric viruses in which portions of SIVMAC RT were exchanged with the corresponding domain of HIV‐1 RT; amino acids 176‐190, 176‐383 and 176‐495 of HIV‐1 RT. The chimeric virus, which was substituted amino acids 176‐190 of RT, had detectable RT activity, and this chimeric RT was sensitive to three nonnucleoside RT inhibitors [nevirapine, HEPT derivative (E‐EBU‐dM) and TIBO derivative (R82913)]. To further study this chimeric virus, we purified the chimeric RT enzyme expressed in Escherichia coli and determined its kinetic properties; the Km and Vmax values, and the Ki value of HEPT derivative calculated for the DNA polymerase activity. This study reveals that amino acids 176‐190 of SIVMAC RT were important for the enzymatic activity and the SIV/HIV‐1 chimeric RT, which had amino acids 176‐190 of HIV‐1, was sensitive to the nonnucleoside RT inhibitor.