Spina C A, Guatelli J C, Richman D D
Department of Pathology, University of California at San Diego, La Jolla 92093-0679, USA.
J Virol. 1995 May;69(5):2977-88. doi: 10.1128/JVI.69.5.2977-2988.1995.
Human immunodeficiency virus type 1 (HIV-1) possesses the ability to establish a complete infection in nondividing host cells. The capacity of HIV-1 to infect nondividing cells probably contributes significantly to its pathology in vivo, as reflected by infection of peripheral T lymphocytes, tissue macrophages, and microglial cells. However, the in vitro demonstration of the establishment of stable HIV-1 infection in quiescent T cells remains controversial. We have developed a primary T-cell model of acute HIV-1 infection of quiescent CD4 lymphocytes that demonstrates the development of a complete, reverse-transcribed form of virus that is stable for over 10 days in culture. To ensure that our primary cell culture was representative of a quiescent population, the CD4 lymphocyte targets were monitored for membrane expression of activation antigens and for shifts in cell cycle from G0/G1 to S/G2 phase. The presence of viral DNA fragments reflecting progressive reverse transcription was determined by PCR analysis. HIV entered primary CD4 cells rapidly, but viral DNA accumulated slowly in the resting cell cultures. DNA species containing regions of full-length reverse transcription were not detected until 3 to 5 days after infection. In parallel with the appearance of complete viral DNA, spliced RNA transcripts, predominantly of the nef species, were detected by reverse transcriptase PCR amplification. When infected CD4 cells were sorted on the basis of cell cycle analysis of DNA content, the accumulation of a complete viral DNA form was found to occur in both the purified G0/G1-phase cell subset and the cell fraction enriched for the minor S-phase subset. In contrast, spliced viral RNA products could be detected only in the enriched S-phase cell fraction. These results demonstrate that HIV-1 can infect and establish a complete, stable form of viral DNA in primary CD4 lymphocytes in vitro but is blocked from transcription in the absence of cell activation. The findings are consistent with in vivo data from HIV-infected individuals that show the existence of viral DNA predominantly as a stable, extrachromosomal form in T cells of the peripheral circulation.
1型人类免疫缺陷病毒（HIV-1）具有在非分裂宿主细胞中建立完整感染的能力。HIV-1感染非分裂细胞的能力可能在很大程度上导致了其体内病理变化，外周T淋巴细胞、组织巨噬细胞和小胶质细胞的感染就反映了这一点。然而，在体外静止T细胞中建立稳定HIV-1感染的证据仍存在争议。我们建立了一个静止CD4淋巴细胞急性HIV-1感染的原代T细胞模型，该模型显示出完整的、经逆转录的病毒形式的形成，这种病毒形式在培养中可稳定存在超过10天。为确保我们的原代细胞培养代表静止细胞群体，对CD4淋巴细胞靶标监测了活化抗原的膜表达以及细胞周期从G0/G1期到S/G2期的转变。通过PCR分析确定反映进行性逆转录的病毒DNA片段的存在。HIV迅速进入原代CD4细胞，但病毒DNA在静止细胞培养物中积累缓慢。直到感染后3至5天，才检测到含有全长逆转录区域的DNA种类。与完整病毒DNA的出现同时，通过逆转录酶PCR扩增检测到主要为nef种类的剪接RNA转录本。当根据DNA含量的细胞周期分析对感染的CD4细胞进行分选时，发现完整病毒DNA形式的积累在纯化的G0/G1期细胞亚群和富含少量S期亚群的细胞部分中均会发生。相比之下，仅在富含S期的细胞部分中可检测到剪接的病毒RNA产物。这些结果表明，HIV-1能够在体外原代CD4淋巴细胞中感染并建立完整、稳定的病毒DNA形式，但在缺乏细胞活化的情况下转录受阻。这些发现与来自HIV感染个体的体内数据一致，这些数据表明病毒DNA主要以外染色体稳定形式存在于外周循环T细胞中。
