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Posted: 7 Jul 2011
30 years in 30 weeks, 1990

Categories: careers, comment, opinion piece

From the very first report of AIDS, which identified the deficiency of the CD4+ subset of T cells in patients, HIV research relied on the latest findings and technological advances in human immunology. The information flow, however, occurred in both directions, with HIV research uncovering many previously unknown facts about the immune system. In 1990, Zack et al. described a defect in HIV replication, occurring in quiescent T cells. This paper initiated a line of extensive studies that eventually transformed the simplistic dichotomy between quiescent and activated immune cells into a complex picture of multiple levels of activation, characterized by distinctive cellular properties, which influence, among other things, the susceptibility of the cell to infection by HIV.

HIV-1 Entry into Quiescent Primary Lymphocytes: Molecular Analysis Reveals a Labile, Latent Viral Structure
Cell, Vol. 61. 213-222, April 20, 1990, Copyright 0 1990 by Cell Press
Jerome A. Zack, Salvatore J. Arrigo, Stacy R. Weitsman, Alan S. Go, Allyson Haislip, and Irvin S. Y. Chen

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180
Commentary by Drs. Zack and Chen

The major finding of our paper was that quiescent T cells could be infected by HIV-1, but the infection was very limited due to a defect in the reverse transcription process. The partially reverse transcribed form could be activated to express virus to some extent, but this ability waned with time. Thus, the latent intermediate in these quiescent cells was labile. This observation was important for the field for several reasons. First, on a scientific basis it showed that the virus could establish a latent infection in primary T cells and explained why activation of the target cell was important for HIV replication.  The work was also of interest to the field on a technical level. To assess the progress of reverse transcription, we designed PCR probes specific for various regions of the viral genome that allowed us to measure the amounts of the reverse transcription intermediates.  To make data from these probes interpretable, we adapted the PCR technology such that it was quantitative. This was one of the first reports in the field using quantitative PCR, and one of the first reports using PCR for other reasons than mere determination of the presence or absence of DNA. This quantitative technique became quite useful for assessing the amount of HIV infection. At the time we did our study, PCR was done manually (we actually hired an undergraduate to move samples back and forth between waterbaths set at different temperatures). To the relief of many researchers across the world, the process is now automated.

Perhaps the most challenging aspect of the work, aside from the technical challenges in creating quantitative PCR, was that we had very little appreciation of how difficult it was to prevent PCR contamination. We initially submitted our manuscript claiming that full length reverse transcription occurred in these cells. While the manuscript was being reviewed, we realized that we were detecting contamination arising from amplified PCR product that had found its way into our samples. It took us over a year to resolve this issue by establishing a room exclusively for extraction of DNA for low copy number analysis. We bleached all the surfaces in the laboratory to remove any contaminating DNA, obtained all new reagents and established Standard Operating Procedures (SOPs) to modify how we processed samples for PCR in the lab.  These rules of behavior apply in our labs today.

The results of our study have been validated by many others since that time, allowing for some added nuances in interpretation. Investigators have identified additional blocks to HIV infection in quiescent T cells. The term “quiescence”, however, is not absolute, so interpretation depends on the criteria being used.  Quiescent T cells in the blood are in the G0 stage of the cell cycle, and, to a degree, are transcriptionally inactive. However, T cells in tissues may be exposed to certain cytokines that activate them to move out of G0, but not to the extent that they replicate their DNA and proliferate. These partially activated cells are still considered “quiescent”, but they can be infected by HIV. This also holds true for macrophages, which are generally non-dividing cells albeit with an activation level sufficient to allow successful HIV infection.
It still remains unclear exactly what mechanisms are preventing efficient HIV infection in truly quiescent cell types. However many laboratories, including ours, continue to use old and new techniques to investigate this long-standing mystery.

About the authors: Dr. Zack is a Professor of Medicine and of Microbiology, Immunology and Molecular Genetics at the David Geffen School of Medicine at UCLA. He is also the director of the UCLA Center for AIDS Research (CFAR).
Dr. Chen is a Professor of Microbiology, Immunology and Molecular Genetics and Medicine at the David Geffen School of Medicine at UCLA. He is also Director of the UCLA AIDS Institute.


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