Most of the compounds currently used for treatment of HIV-1 infection are reverse transcriptase inhibitors and protease inhibitors. Several early steps in the HIV-1 life cycle such as virus attachment to host cell and cell-virus fusion are potential targets for drugs. Since most of the target molecules involved in this infection step are cellular, it is expected that the drug resistant mutations occur less frequently than those against viral enzymes. In this review, new inhibitors of entry and fusion are summarized that are promising.
We previously described chimeric HIV-1, EcoHIV, which can infect mouse cells in culture and cause spreading infection in conventional immunocompetant mice. We have now applied this system as a model for preclinical evaluation of anti-retroviral drugs.We used chimeric virus EcoHIV/NDK constructed on the backbone of subtype D NDK. EcoHIV/NDK expression in mice was characterized 5-10 days after infection by testing viral DNA, RNA, and protein burdens in spleen and macrophages by real-time PCR (QPCR), RT-PCR, and p24 ELISA. For antiviral evaluation, groups of 5-7 mice were pretreated with 2',3'-dideoxycytidine (ddC), abacavir, or vehicle; mice were then infected with EcoHIV/NDK, treatment maintained for additional 48 h, and tested for viral DNA and RNA burdens in spleens and macrophages by QPCR.EcoHIV/NDK infected mice reproducibly showed viral burdens of up to 1.4 x 10 viral DNA copies and 200 pg p24 per 10 spleen cells and expressed spliced Vif RNA and mature p24 in macrophages 5-10 days after infection. Treatment of mice with 60 or 300 mg ddC/kg/day blocked EcoHIV/NDK infection in a dose-dependent manner with significantly lower viral DNA and RNA burdens at both drug doses (P < 0.001) in the spleens of infected mice. Abacavir tested at 100 mg/kg/day caused 96% inhibition of viral DNA synthesis in spleen and it almost completely abolished viral spliced RNA synthesis in spleens and macrophages.The system of chimeric HIV-1 infection of mice permits rapid, statistically powerful, and inexpensive evaluation of antiretroviral drugs in vivo.
Molecular epidemiology of HIV-1 in Zambia was investigated by direct sequencing of PCR products from samples collected from antenatal attendees in Lusaka, Zambia. One hundred and forty samples were initially screened for HIV, using antibody assays. Thirty-three (23.6%) samples were HIV-1 positive. Sequences of the HIV-1 env gp120 region were obtained from 28 of 33 (85%) HIV-1-positive samples. Twenty-six of the 28 sequences were HIV-1 env subtype C-like as previously reported. However, one HIV-1 env subtype D-like virus and one HIV-1 env subtype G-like virus were identified. This is the first time that these two HIV-1 env subtype viruses have been identified in Zambia, suggesting that more subtypes could be in existence.
A low molecular weight nonpeptide compound, KRH-1636, efficiently blocked replication of various T cell line-tropic (X4) HIV type 1 (HIV-1) in MT-4 cells and peripheral blood mononuclear cells through the inhibition of viral entry and membrane fusion via the CXC chemokine receptor (CXCR)4 coreceptor but not via CC chemokine receptor 5. It also inhibited binding of the CXC chemokine, stromal cell-derived factor 1α, to CXCR4 specifically and subsequent signal transduction. KRH-1636 prevented monoclonal antibodies from binding to CXCR4 without down-modulation of the coreceptor. The inhibitory effect against X4 viral replication by KRH-1636 was clearly reproduced in the human peripheral blood lymphocyte/severe combined immunodeficiency mouse system. Furthermore, this compound was absorbed into the blood after intraduodenal administration as judged by anti-HIV-1 activity and liquid chromatography MS in the plasma. Thus, KRH-1636 seems to be a promising agent for the treatment of HIV-1 infection.
To study how HIV-1 viral infectivity factor (Vif) mediates proteasome-dependent depletion of host factor APOBEC3G, functional and nonfunctional Vif-APOBEC3G interactions were correlated with subcellular localization. APOBEC3G localized throughout the cytoplasm and co-localized with gamma-tubulin, 20 S proteasome subunit, and ubiquitin at punctate cytoplasmic bodies that can be used to monitor the Vif-APOBEC3G interaction in the cell. Through immunostaining and live imaging, we showed that a substantial fraction of Vif localized to the nucleus, and this localization was impaired by deletion of amino acids 12-23. When co-expressed, Vif exhibited more pronounced localization to the cytoplasm and reduced the total cellular levels of APOBEC3G but rarely co-localized with APOBEC3G at cytoplasmic bodies. On the contrary, Vif(C114S), which is inactive but continues to interact with APOBEC3G, stably associated with APOBEC3G in the cytoplasm, resulting in complete co-localization at cytoplasmic bodies and a dose-dependent exclusion of Vif(C114S) from the nucleus. Following proteasome inhibition, cytoplasmic APOBEC3G levels increased, and both proteins co-accumulated nonspecifically into a vimentin-encaged aggresome. Furthermore in the presence or absence of APOBEC3G, Vif localization was significantly altered by proteasome inhibition, suggesting that aberrant localization may also contribute to the loss of Vif function. Finally mutations at Vif Ile(9) disrupted the ability of Vif or Vif(C114S) to coimmunoprecipitate and to co-localize with APOBEC3G, suggesting that the N terminus of Vif mediates interactions with APOBEC3G. Taken together, these results demonstrate that cytoplasmic Vif-APOBEC3G interactions are required but are not sufficient for Vif to modulate APOBEC3G and can be monitored by co-localization in vivo.
