FUNCTIONAL AND BIOCHEMICAL RELATIONSHIPS BETWEEN TROPISM, INFECTIVITY, AND NEUTRA

将趋化因子受体确定为HIV进入的辅助受体，不仅有助于理解病毒的趋向性，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例如CCR5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。我们证明，STRL33在Jurkat细胞中的表达增加了对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以及亲本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。到目前为止，我们已经从适应的MAL病毒中克隆了一个单一的包膜基因，并表明这个包膜基因可以增强使用STRL33的能力。将获得更多的env基因，以及来自SIVmac239的适应使用STRL33的基因。我们已经确定了一种新的针对某些SIV毒株的辅助受体。我们根据HIV-1和SIV毒株的细胞嗜性预测了这种辅助受体的存在，并使用具有不同孤儿受体基因的RNase保护试验，能够识别并克隆该基因。到目前为止，这种辅助受体已经被证明在SIVagm分离株中是活跃的。我们计划扩大对其他SIV分离株和毒株以及对HIV-2的适应性研究，并寻找其他辅助受体，如APJ、CX3CR1和我们的新型辅助受体。我们的目标是获得适应的SIV，它可以使用不同的辅助受体进入。一旦这些变异被分离和鉴定，它们就可以用于体内研究，以确定哪些辅助受体用于通过不同的感染途径传播。

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.