Functional and biochemical relationships between tropism, infectivity, and neut

趋向性、感染性和中性之间的功能和生化关系

• 批准号: 6433512
• 负责人: KEITH PEDEN
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6433512
• 关键词: CD4 molecule; HIV envelope protein gp120; HIV envelope protein gp41; gene mutation; genetic regulatory element; genetic strain; human immunodeficiency virus 1; human immunodeficiency virus 2; tissue /cell culture; transfection; virus RNA; virus genetics; virus infection mechanism; virus replication

The identification of chemokine receptors as coreceptors for HIV entry, not only has contributed to the understanding on viral tropism but has provided an additional target for therapeutic intervention for HIV disease. Several chemokine receptors have been shown to function as coreceptors for HIV-1 entry. The main ones are CXCR4 (for T-cell line tropic viruses) and CCR5 (for macrophage-tropic viruses). Because of the capacity of HIV to adapt when selective pressures are imposed, it is likely that any drug designed to block the interaction of HIV with one coreceptor will force the virus to use additional coreceptors. Thus, the determination of the complete coreceptor repertoire is necessary. Because STRL33 is expressed in all lymphoid tissues, in collaboration with Dr J. Farber, NIAID, we tested it for coreceptor activity with HIV. STRL33 expression in Jurkat cells conferred increased permissivity to infection by the ELI1 isolate of HIV-1. Thus, STRL33 can act as an HIV-1 coreceptor in vitro. As well as testing the coreceptor activity of STRL33 with a number of HIV-1 strains of different phenotypes, we have begun studies with HIV-2 and SIV. We have shown, in an infectivity assay, that the MAL strain of HIV-1 and the mac239 isolate of SIV use STRL33 but not as well as they use CCR5. The appearance of virus only after about 30 days in culture is indicative of adaptation. To confirm this, virus emerging after about 35 days was used to infect fresh Jurkat- STRL33 cells as well as the parent Jurkat cells. In this second passage, virus production was seen after about 12 days, thus demonstrating that both SIVmac239 and HIV-1 MAL had adapted to use STRL33 more efficiently. Importantly, these passaged viruses were still unable to infect Jurkat cells. That the passaged virus had adapted to use STRL33 was demonstrated by the fact that an antibody raised to STRL33 inhibited virus infection. We have cloned 12 envelope genes from the adapted MAL virus and 6 env genes from the adapted SIVmac239. We are currently returning these genes to the infectious molecular clones to assess their biological activities. Based on the cell tropism of a number of viruses, both SIV and HIV-1, we predicted the existence of another coreceptor on SUP-T1 cells. We have cloned this coreceptor and identified it as CCR9, a chemokine receptor for TECK (CCL25). CCR9 exists in two forms, A and B, that differ by 12 amino acids at the N terminus. CCR9B has activity with several strains of SIV, including SIVagmSAB and SIVagmTAN, but as yet no HIV-1 has been found that uses this molecule as an entry cofactor. Additional HIV-1 isolates including primary isolates are being screened. Since CCR9A/B is expressed in thymocytes and in the lymphocyte subset that trafficks to the gut, and because of the obvious importance of these tissues to HIV disease and AIDS pathogenesis, we are expanding our studies to include the coreceptors expressed in these cells as well as to the infection of these cells and of dendritic cells, which secrete CCL25 and thus may recruit T cells.

趋化因子受体被确认为HIV进入的辅助受体，不仅有助于了解病毒的趋向性，而且为HIV疾病的治疗干预提供了另一个靶点。几种趋化因子受体已被证明是HIV-1进入的辅助受体。主要是CXCR4(对T细胞嗜性病毒)和CCR5(对巨噬细胞嗜性病毒)。由于艾滋病毒在施加选择性压力时的适应能力，任何旨在阻止艾滋病毒与一个辅助受体相互作用的药物都可能迫使病毒使用额外的辅助受体。因此，确定完整的辅受体谱系是必要的。由于STRL33在所有淋巴组织中都有表达，我们与NIAID的J.Farber博士合作，测试了它与HIV的共同受体活性。在Jurkat细胞中表达STRL33增加了对HIV-1ELI1分离株感染的通透性。因此，STRL33在体外可以作为HIV-1的辅助受体。除了测试STRL33与一些不同表型的HIV-1毒株的共同受体活性外，我们还开始了对HIV-2和SIV的研究。在传染性测试中，我们已经表明，HIV-1的MAL毒株和SIV的Mac239分离株使用STRL33，但没有他们使用CCR5那么好。病毒仅在培养约30天后出现，表明适应。为了证实这一点，大约35天后出现的病毒被用来感染新鲜的Jurkat-STRL33细胞和亲本Jurkat细胞。在这第二代中，大约12天后可以看到病毒的产生，从而表明SIVmac239和HIV-1 Mal都已经适应了更有效地使用STRL33。重要的是，这些传代的病毒仍然不能感染Jurkat细胞。STRL33的抗体可抑制病毒感染，证明传代病毒已适应使用STRL33。我们已经从适应株MAL中克隆了12个囊膜基因，从适应株SIVmac239中克隆了6个囊膜基因。我们目前正在将这些基因返回到具有感染性的分子克隆中，以评估它们的生物学活性。基于一些病毒的细胞嗜性，包括SIV和HIV-1，我们预测在SUP-T1细胞上存在另一个辅助受体。我们克隆了这个辅受体，并将其鉴定为CCR9，一种针对Teck的趋化因子受体(CCL25)。CCR9以A和B两种形式存在，在N末端有12个不同的氨基酸。CCR9B对几种SIV毒株具有活性，包括SIVagmSAB和SIVagmTAN，但尚未发现使用该分子作为进入辅因子的HIV-1。包括初级分离株在内的其他HIV-1分离株正在筛选中。由于CCR9A/B在胸腺细胞和运输到肠道的淋巴细胞亚群中表达，并且由于这些组织在HIV疾病和艾滋病发病机制中的明显重要性，我们正在扩大研究范围，以包括这些细胞中表达的辅助受体以及这些细胞和树突状细胞的感染，这些细胞分泌CCL25，从而可能招募T细胞。