Establishment of a model system for studying the transcriptional regulation mechanism of HIV-1 in latently infected human monocyte cell lines

Abstract:

[Objective] With the use of anti-retroviral therapy (ART) for HIV-infected individuals, a greater control of viral replication is now possible. In 2012, however, more than 35 million people were living with HIV-1 and more than 1.6 million people died of AIDS or HIV-1 related diseases worldwide. One of the main reasons for difficulties in completely eradicating HIV-1 in vivo is the reservoir problem. Although the CD4+ T-cells and monocytes or macrophages are thought to be the reservoir of HIV-1 in vivo, the viral replication mechanism in monocytes or macrophages is yet to be studied, as compared with that of CD4+ T-cells, because of the lack of an appropriate system. Thus, to evaluate the effects of the activating or differentiating signals in monocytes on the replication of HIV-1, we attempted to establish the HIV-1 latently infected human monocyte cell lines. [Methods] We used two recombinant HIV-1 viruses, NLnGFP-Kp and NLnNanoLuc-Kp, which have a frame-shift mutation in the env region and have EGFP or NanoLuc gene, respectively, as a marker in the nef region. Two human monocyte linage cell lines, THP-1 and U937 cells, were each infected with one of the two viruses. For cloning of the infected cells, the limiting dilution method was used and the expression levels of these marker genes were determined by flow cytometry or luciferase assay. From the cell clones not expressing either marker gene, we selected latently infected cell clones after stimulating them with phorbol-12-myristate-13-acetate (PMA). [Results] We obtained four cell clones that were considered to be latently infected with HIV-1. Two of them from THP-1 had EGFP as a marker and two of them from U937 had NanoLuc as a marker. All these cell clones expressed their marker genes when stimulated with PMA but in the steady state condition of their cultures at an undetectable level. [Conclusion] We established four cell clones latently infected HIV-1 from human monocyte lineage cell lines. These cells can be a useful tool for a better understanding of the transcriptional regulation mechanisms in the HIV-1 replication.

Key words:human immunodeficiency virus (HIV), provirus, transcriptional regulation, monocyte-macrophage, cell model system, reactivation