FUNCTIONAL AND BIOCHEMICAL RELATIONSHIPS BETWEEN TROPISM, INFECTIVITY, AND NEUTRA

向性、感染性和中性之间的功能和生物化学关系

• 批准号: 6293728
• 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/6293728
• 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, in addition to CD4, 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, say, CCR5 will force the virus to use additional coreceptors. Thus, the determination of the complete coreceptor repertoire will be necessary. Because STRL33 is expressed in all lymphoid tissues, in collaboration with Dr J. Farber, NIAID, we tested it for coreceptor activity with HIV. We demonstrated that the expression of STRL33 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 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. So far, we have cloned a single envelope gene from the adapted MAL virus and shown that this Env can confer an increased capacity to use STRL33. Additional env genes will be obtained, as well as ones from SIVmac239 adapted to use STRL33. We have identified a novel coreceptor for certain SIV strains. We predicted the existence of this coreceptor from the cellular tropism of HIV-1 and SIV strains, and, using an RNase protection assay with different orphan receptor genes, were able to identify the gene and clone it. So far this coreceptor has been shown to be active with SIVagm isolates. We plan to expand the study on adaptation to additional SIV isolates and strains and to HIV-2, as well as looking at addtional coreceptors such as APJ, CX3CR1, and our novel coreceptor. Our aim is to obtain adapted SIVs that can use different coreceptors for entry. Once these variants have been isolated and characterized, they could be used for in vivo studies to determine which coreceptors are used for transmission via different routes of infection.

除了 CD4 之外，趋化因子受体作为 HIV 进入的辅助受体的鉴定不仅有助于理解病毒趋向性，而且为 HIV 疾病的治疗干预提供了额外的靶点。 多种趋化因子受体已被证明可作为 HIV-1 进入的辅助受体。 主要是CXCR4（针对T细胞系嗜性病毒）和CCR5（针对巨噬细胞嗜性病毒）。 由于 HIV 在施加选择性压力时具有适应能力，因此任何旨在阻止 HIV 与 CCR5 等相互作用的药物都可能会迫使病毒使用额外的辅助受体。 因此，有必要确定完整的辅助受体库。 由于 STRL33 在所有淋巴组织中表达，因此我们与 NIAID 的 J. Farber 博士合作，测试了它与 HIV 的辅助受体活性。 我们证明 Jurkat 细胞中 STRL33 的表达增加了对 HIV-1 ELI1 分离株感染的耐受性。 因此，STRL33 在体外可以充当 HIV-1 辅助受体。 除了用许多不同表型的 HIV-1 毒株测试 STRL33 的辅助受体活性外，我们还开始了 HIV-2 和 SIV 的研究。 我们在感染性测定中表明，HIV-1 的 MAL 毒株和 SIV 的 mac239 分离株使用 STRL33，但不如使用 CCR5。 仅在培养约 30 天后病毒出现才表明已适应。 为了证实这一点，使用约 35 天后出现的病毒感染新鲜的 Jurkat-STRL33 以及亲本 Jurkat 细胞。 在第二次传代中，大约 12 天后观察到病毒产生，从而证明 SIVmac239 和 HIV-1 MAL 都已适应更有效地使用 STRL33。 重要的是，这些传代的病毒仍然无法感染 Jurkat 细胞。 针对 STRL33 产生的抗体抑制病毒感染的事实证明了传代的病毒已适应使用 STRL33。 到目前为止，我们已经从适应的 MAL 病毒中克隆了一个包膜基因，并表明该包膜基因可以增强使用 STRL33 的能力。 将获得额外的 env 基因，以及来自适合使用 STRL33 的 SIVmac239 的基因。 我们已经为某些 SIV 毒株鉴定出一种新的辅助受体。 我们根据 HIV-1 和 SIV 毒株的细胞向性预测了这种辅助受体的存在，并且使用不同孤儿受体基因的 RNase 保护测定，能够识别该基因并克隆它。 到目前为止，该辅助受体已被证明对 SIVagm 分离株具有活性。 我们计划扩大对其他 SIV 分离株和毒株以及 HIV-2 的适应研究，并研究其他辅助受体，如 APJ、CX3CR1 和我们的新型辅助受体。 我们的目标是获得可以使用不同的辅助受体进入的适应性 SIV。 一旦这些变体被分离和表征，它们就可以用于体内研究，以确定哪些共同受体用于通过不同的感染途径进行传播。