Functional and biochemical relationships between tropism

总结：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的嗜性，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作为HIV-1进入的辅助受体。 主要的是CXCR 4（针对T细胞系嗜性病毒）和CCR 5（针对巨噬细胞嗜性病毒）。 由于艾滋病毒在选择性压力下的适应能力，任何旨在阻断艾滋病毒与一种辅助受体相互作用的药物都可能迫使病毒使用其他辅助受体。 因此，确定完整的辅助受体库是必要的。 由于CXCR 6（STRL 33）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的共受体活性。 Jurkat细胞中CXCR 6的表达增加了HIV-1的ELI 1分离株的感染概率。 因此，CXCR 6在体外可作为HIV-1辅助受体。 除了用不同表型的HIV-1毒株测试CXCR 6的辅助受体活性外，我们还开始了HIV-2和SIV的研究。 我们已经在感染性试验中表明，HIV-1的MAL株和SIV的mac 239分离株使用CXCR 6，但不如它们使用CCR 5。 仅在培养约30天后才出现病毒，这表明存在适应。 为了证实这一点，使用约35天后出现的病毒感染新鲜Jurkat-CXCR 6细胞以及亲本Jurkat细胞。 在第二次传代中，在约12天后观察到病毒产生，从而证明SIVmac 239和HIV-1 MAL均已适应更有效地使用CXCR 6。 重要的是，这些传代病毒仍然不能感染Jurkat细胞。 传代的病毒已经适应使用CXCR 6，这一点通过针对CXCR 6产生的抗体抑制病毒感染的事实得到证明。 我们已经从CXCR 6适应的MAL病毒中克隆了12个包膜基因，从CXCR 6适应的SIVmac 239中克隆了6个env基因。 我们已经表征了来自MAL的7个功能性env基因和来自SIVmac 239的4个功能性env基因。 所有适应的克隆在单循环测定和生产性感染测定中使用CXCR 6的能力都比CCR 5增强。 MAL env的测序表明，虽然在gp 120和gp 41的几个区域中发现了变化，但gp 120的V3区域中的变化足以赋予适应的表型。 已经评估了MAL中单个突变的活性，V3和gp 41的变化可以赋予使用CXCR 6的能力，而V2和V5的变化不能。 CXCR 6适应的MAL的继续传代产生了可以感染亲本Jurkat细胞的病毒，这强烈表明适应是对CXCR 4的使用。 我们已经证明，由HIV-1的R5株而不是X4株感染单核细胞衍生的巨噬细胞（MDM）和单核细胞衍生的树突状细胞（MDDC）导致诱导IP-10和I-TAC（CXCR 3的趋化因子），但未观察到对CCR 5、CXCR 4或CCR 5配体的诱导。 这对于HIV疾病是有意义的，因为所有表达CCR 5的记忆性CD 4细胞也表达CXCR 3，因此感染的组织巨噬细胞或DC将分泌IP-10和I-TAC，其然后将募集记忆性CD 4 T细胞，其可以被感染并因此传播感染。 募集的CD 8 T细胞也可以发挥作用，但在这种情况下，在宿主防御中。 IP-10和I-TAC的诱导依赖于病毒复制，因为该作用被RT抑制剂ZDV阻断，并且病毒与细胞受体的结合是不充分的。 目前的工作是针对理解的诱导机制。

Summary: 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 CXCR6 (STRL33) is expressed in all lymphoid tissues, in collaboration with Dr J. Farber, NIAID, we tested it for coreceptor activity with HIV. CXCR6 expression in Jurkat cells conferred increased permissivity to infection by the ELI1 isolate of HIV-1. Thus, CXCR6 can act as an HIV-1 coreceptor in vitro. As well as testing the coreceptor activity of CXCR6 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 CXCR6 but not as well as they use CCR5. The appearance of virus only after about 30 days in culture was indicative of adaptation. To confirm this, virus emerging after about 35 days was used to infect fresh Jurkat-CXCR6 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 CXCR6 more efficiently. Importantly, these passaged viruses were still unable to infect Jurkat cells. That the passaged virus had adapted to use CXCR6 was demonstrated by the fact that an antibody raised to CXCR6 inhibited virus infection. We have cloned 12 envelope genes from the CXCR6-adapted MAL virus and 6 env genes from the CXCR6-adapted SIVmac239. We have characterized 7 functional env genes from MAL and four from SIVmac239. All of the adapted clones had enhanced capacity to use CXCR6 over CCR5 in both a single-cycle assay and a productive infection assay. Sequncing of the MAL envs demonstrated that, while changes were found in several regions of gp120 and gp41, changes in the V3 region of gp120 were sufficient to confer the adapted phenotype. The activity of individual mutations in MAL has been assessed and changes in V3 and gp41 can confer the ability to use CXCR6, while changes in V2 and V5 cannot. Continued passage of the CXCR6-adapted MAL has resulted in virus that can infect the parent Jurkat cells, strongly suggesting that adaptation has been to CXCR4 use. We have demonstrated that infection of monocyte-derived macrophages (MDM) and monocyte-derived dendritic cells (MDDC) by R5 strains, but not X4 strains, of HIV-1 results in the induction of IP-10 and I-TAC, chemokines for CXCR3, but no induction was seen for CCR5, CXCR4, or CCR5 ligands. This is of interest for HIV disease, since all CCR5-expressing memory CD4 cells also express CXCR3, and thus infected tissue macrophages or DC would secrete IP-10 and I-TAC, which would then recruit memory CD4 T cells, which could be infected and thus disseminate infection. Recruited CD8 T cells could also play a role but in this case that in host defence. Induction of IP-10 and I-TAC depends on viral replication, since the effect is blocked by the RT inhibitor ZDV, and association of virus with the cellular receptors is not sufficient. Current work is directed towards understanding the mechanism of the induction.