Quantitative Viral Outgrowth Assay to Measure the Functional SIV Reservoir in Myeloid Cells.

The role of CD4+ T cells in HIV infection and the latent reservoir, that is, latently infected cells that harbor replication competent virus, has been rigorously assessed. We have previously reported a quantitative viral outgrowth assay (QVOA) for SIV that demonstrated the frequency of latently infected CD4+ T cells is approximately 1 in a million cells, similar to that of HIV infected individuals on ART. However, the frequency of productively infected monocytes in blood and macrophages in tissues has not been similarly studied. Myeloid cells are infected during acute HIV and SIV infection; however, unlike lymphocytes, they are resistant to the cytopathic effects of the virus. Moreover, tissue-resident macrophages have the ability to self-renew and persist in the body for months to years. Thus, tissue macrophages, once infected, have the characteristics of a stable viral reservoir. A better understanding of the number of productively infected macrophages is critical to understanding the role of infected myeloid cells as a viral reservoir. In order to assess the functional latent reservoir. we have developed specific QVOAs for monocytes in blood, and macrophages in spleen, BAL and brain, which are described in detail in this chapter.

A single amino acid change and truncated TM are sufficient for simian immunodeficiency virus to enter cells using CCR5 in a CD4-independent pathway.

Entry of HIV and SIV into susceptible cells is mediated by CD4 and chemokine receptors, which act as coreceptors. To study cell entry of SIV, we constructed a cell line, xKLuSIV, derived from non-susceptible human K562 cells, that express the firefly luciferase reporter gene under control of a minimal SIV long terminal repeat (LTR). Using these susceptible cells, we studied the entry of a well-characterized molecularly cloned macrophage-tropic SIV. xKLuSIV cells that express rhesus macaque CD4 and/or the rhesus chemokine receptor CCR5 are susceptible to infection with the macrophage-tropic, neurovirulent strain SIV/17E-Fr, but only xKLuSIV cells expressing both CCR5 and CD4 were susceptible to infection by the macrophage-tropic, non-neurovirulent strain SIV/17E-Cl. CCR5-dependent, CD4-independent infection by SIV/17E-Fr was abrogated by pre-incubation of the cells with AOP-RANTES, a ligand for CCR5. In addition to viral entry occurring by a CD4-independent mechanism, neutralization of SIV/17E-Fr with rhesus mAbs from 3 different neutralization groups blocked entry into x KLuSIV cells by both CD4-dependent and -independent mechanisms. Triggering the env glycoprotein of SIV-17 EFr with soluble CD4 had no significant effect in infectivity, but triggering of the same glycoprotein of SIV/17E-Cl allowed it to enter cells in a CD4-independent fashion. Using mutant molecular clones, we studied the determinants for CD4 independence, all of which are confined to the env gene. We report here that truncation of the TM at amino acid 764 and changing a single amino acid (R751G) in the SIV envelope transmembrane protein (TM) conferred the observed CD4-independent phenotype. Our data suggest that the envelope from the neurovirulent SIV/17E-Fr interacts with CCR5 in a CD4-independent manner, and changes in the TM protein of this virus are important components that contribute to neurovirulence in SIV.