PATHOGENIC DETERMINANTS OF THE SIV ENVELOPE TRANSMEMBRANE CYTOPLASMIC DOMAIN

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们之前的研究表明，干扰SIVmac239的Env胞质尾部高度保守的运输信号(GYxx)(即删除Gly-721和Tyr-721)会在恒河猴和辫子猴中产生明显减弱的表型。静脉接种后，deltaGY复制到与SIVmac239相当的高急性病毒峰，但被抑制到100拷贝/毫升，不会损失外周CD4细胞。用DeltaGY静脉感染4只恒河猴，以确定衰减的病理相关因素。两只动物在第28天进行了尸检；2只动物随后进行了高尔特和周围淋巴结的系列活组织检查。将4只动物经阴道接种，以确定deltaGY的粘膜传播能力。静脉感染动物的急性病毒高峰为1.8×10^6(2.2×10^5~6.0×10^7)，其中2只慢性感染动物的病毒水平到第16周下降到1.4×10^3和6.4×10^3。静脉注射的动物，尽管每周接种350 TCID50，但仍有1/4感染(峰值=2.6×10^7)。在解剖动物中，在肠道组织淋巴组织的生发中心和周围淋巴结处可检测到丰富的病毒。然而，与感染SIVmac239或SIVmac251的对照组相比，感染仅限于免疫诱导部位(有组织的淋巴结节)，而不在免疫效应部位(弥漫性固有层)。在慢性DeltaGY感染的动物中，肠道中的CD4+/CCR5+细胞也几乎没有耗尽(感染前的平均CD4+/CCR5+=T细胞的43.8%；感染后的33.3%)。对于身体解剖的动物，尽管血浆RNA水平很高，但中枢神经系统也没有检测到病毒。外周淋巴组织的多标记共聚焦显微镜显示T细胞中有deltaGY，但巨噬细胞中没有。因此，deltaGY复制是由免疫诱导部位的感染T细胞驱动的，而免疫效应部位的巨噬细胞和脂蛋白脂酶则不受影响。目前正在研究环境病毒转运的缺陷如何扰乱病毒的传播，以及哪些宿主免疫反应与其控制相关。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We previously showed that disrupting a highly conserved trafficking signal (GYxx¿) in the Env cytoplasmic tail of SIVmac239 (i.e. deleting Gly-721 and Tyr-721) produces a profoundly attenuated phenotype in rhesus and pigtailed macaques. Following iv inoculation, deltaGY replicates to a high acute viral peak comparable to SIVmac239, but is suppressed to 100 copies/ml with no loss of peripheral CD4 cells. To determine the pathological correlates of attenuation, 4 rhesus macaques were infected iv with deltaGY. Two animals were necropsied on day 28; 2 are being followed with serial biopsies of GALT and peripheral lymph nodes. Four animals were inoculated intravaginally to determine deltaGY's competence for mucosal transmission. For iv-infected animals, the acute viral peak was 1.8x10^6 (2.2x10^5-6.0x10^7) with levels for 2 chronically infected animals declining to 1.4x10^3 and 6.4x10^3 by week 16. For ivag-inoculated animals, 1 of 4 became infected (peak= 2.6x10^7) despite 8 weekly inoculations of 350 TCID50. For necropsied animals, abundant virus was detectable in germinal centers of organized lymphoid tissue in intestine and peripheral lymph nodes. However, in striking contrast to SIVmac239- or SIVmac251-infected controls, infection was limited to immune inductive sites (organized lymphoid nodules) and absent from immune effector sites (diffuse lamina propria). There was also little to no depletion of intestinal CD4+/CCR5+ cells in chronic, deltaGY-infected animals (average CD4+/CCR5+= 43.8% of T-cells pre-infection; 33.3% wk 22 post-infection). For necropsied animals there was also no detectable virus in CNS despite high acute levels of plasma RNA. Multilabel confocal microscopy of peripheral lymph tissue showed deltaGY in T-cells but not macrophages. Thus, deltaGY replication is driven by infected T-cells in immune inductive sites, but with sparing of macrophages and LPL in immune effector sites. How a defect in Env trafficking disrupts viral spread, and what host immune responses correlate with its control is being investigated.