Effect of Cocaine and LTR Polymorphism on HIV-1 Pathogenesis

描述（由申请人提供）：该实验室以前的研究表明，与感染HIV-1的非吸毒者相比，非法吸毒者的病毒遗传多样性明显更高，对蛋白酶抑制剂（PI）的原发性耐药频率更高。为了探索药物使用与病毒复制、多样性和抗逆转录病毒耐药性之间的潜在联系，我们建议利用以下发现：1)可卡因滥用刺激高水平的AP-1转录因子相关的即时早期基因；2)只有约40%的进化支B感染个体携带含有AP-1结合位点的HIV-1 LTR长度多态性（MFNLP）。通过在范德比尔特临床护理中心对大量使用可卡因的HIV-1感染受试者进行随访，我们将提出这样的假设：与不携带LTR多态性的可卡因使用者或非可卡因使用者相比，携带带有AP-1结合位点的Clade B HIV-1 LTR多态性的可卡因使用者具有更高的病毒载量、更大的遗传多样性和更大的抗逆转录病毒初级耐药性。为了探索这一假设，我们将1)在一组HIV-1阳性、未接受抗逆转录病毒治疗的个体中，识别出LTR内携带MFNLP病毒的可卡因使用者和非可卡因使用者。因此，我们将确定四组进行进一步分析：携带或不携带MFNLP病毒的HIV-1感染可卡因使用者和携带或不携带MFNLP病毒的非可卡因使用者。2)四组内定量测定AP-1的直接早期基因组分c-fos和JunB在外周血CD4 T淋巴细胞和单核细胞中的表达水平。同样，使用凝胶移位法或新的高通量试剂盒，我们将量化四组细胞群中AP-1的水平。3)利用454高通量测序技术对蛋白酶区进行测序，以确定宿主遗传多样性和对PI的初级抗性突变。使用多样性、对PI的抗性突变和病毒载量作为结果，我们将使用多变量分析、可卡因使用的影响、即时早期基因表达水平、AP-1数量和MFNLP的存在进行检查。公共卫生相关性：艾滋病毒-1的某些变体携带遗传变异，可能使它们在暴露于可卡因的细胞中更快地复制。这项研究将评估可卡因使用者中这些变异的存在是否会改变疾病进展的各种参数。

DESCRIPTION (provided by applicant): Previous studies from this laboratory have demonstrated significantly higher viral genetic diversity and a higher frequency of primary resistance to protease inhibitors (PI) among illicit drug users compared to HIV-1 infected non-drug users. To explore the potential link between drug use and greater viral replication, diversity and antiretroviral resistance, we propose to exploit the findings that 1) cocaine abuse stimulates high levels of the AP-1 transcription factor associated immediate early genes and 2) only about 40% of Clade B infected individuals carry an HIV-1 LTR length polymorphism (MFNLP) containing an AP-1 binding site. Using a large cohort of cocaine using HIV-1 infected subjects followed at the Vanderbilt Clinical Care Center, we will address the hypothesis that cocaine users infected with HIV-1 carrying the Clade B HIV-1 LTR polymorphism with an AP-1 binding site will have higher viral loads, greater genetic diversity, and greater primary resistance to antiretrovirals than either cocaine users whose virus does not carry the LTR polymorphism or non-cocaine users. To explore this hypothesis we will 1) Identify among a cohort of HIV-1 positive, antiretroviral therapy naive individuals those cocaine-using and non-cocaine using individuals with virus carrying the MFNLP within the LTR. We will thereby have identified four groups for additional analyses: HIV-1 infected cocaine users with and without the MFNLP and non-cocaine users with and without the MFNLP. 2) Within the four groups quantify the expression levels within peripheral blood CD4 T lymphocytes and monocytes of c-fos and JunB, as representative immediate early gene components of AP-1. Similarly, using gel-shift assays or a new high throughput kit, we will quantify levels of AP-1 within those cell populations from the four groups. 3) Using 454 high throughput sequencing technology we will sequence the protease region to define within host genetic diversity and primary resistance mutations to PI. Using diversity, resistance mutations to PI and viral load as outcomes, we will examine using a multivariate analysis, the effect of cocaine use, immediate early gene expression levels, AP-1 quantities, and the presence of the MFNLP. PUBLIC HEALTH RELEVANCE: Certain variants of HIV-1 carry genetic alterations that might enable them to replicate more rapidly in cells exposed to cocaine. This study will evaluate whether the presence of those variants in cocaine users alters various parameters of disease progression.