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Development of quantitative real time PCR to assess human brain microvascular endothelial cell susceptibility to HIV -1 infection

  • Author(s): Chao, Ying Sheng
  • et al.
Abstract

Neuroimaging studies identified blood brain barrier disruption as a common feature in HIV associated dementia (HAD) and suggested that human brain microvascular endothelial cells (HBMECs) are involved in neuropathogenesis. Direct infection of HBMECs by HIV-1 has been reported by in vivo and in vitro studies, yet data interpretation has been compounded by technical limitations and productive infection has not been reproducible. Variability between these studies is very likely the result of different HIV-1 strains employed and unidentified host factors affecting the eventual outcome. The main goal of the present study is to address the possibility of intracellular restriction to HIV-1 replication in HBMECs. Toward this goal, a VSV-G pseudotyped HIV-1 NL4-3 virus was constructed which could mediate cytoplasmic delivery in the apparent absence of appropriate host cell receptors and a series of qRT-PCR assays were developed to monitor HIV-1 lifecycle progression in HBMECs. Overall, the qRT-PCR assays were demonstrated to be specific, sensitive, efficient, cover a dynamic linage range, and reproducible. Utilizing these qRT-PCR assays, it was shown that HBMECs exhibit comparable reverse transcription, provirus integration, yet a diminished early regulatory gene expression exemplified by rev transcription. Interestingly, late gene expression and whole genome replication, indicated by the detection of comparable number of gag transcripts, were not affected. However, it was shown that HBMECs might promote a modification, or degradation of HIV-1 p24 capsid protein

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