Detection of human immunodeficiency virus type 1 after infection of unstimulated peripheral blood mononuclear cells.

Abstract

Application of a highly sensitive PCR-based reverse transcriptase (RT) assay to the analysis of the infection of CD4 M cell lines with human immunodeficiency virus type 1 (HIV-1) demonstrated that virus production can be detected as early as 24 h after infection. Most of the signal at 24 h was due to virus production, as it could be substantially reduced by prior treatment with the RT inhibitor zidovudine. Virus production at 24 and 48 h was unaffected by the protease inhibitor indinavir. Infection of unstimulated peripheral blood mononuclear cells (PBMC) with a macrophage-tropic HIV-1 isolate yielded increasing virus production for 2-3 weeks, while infection with a T-cell line-tropic isolate yielded only low and sporadic virus production. Productive infection of unstimulated PBMC by the macrophage-tropic virus required functional Gag matrix and Vpr proteins ; therefore, the monocyte-derived macrophage is probably the virus-producing cell in these cultures.Studies on human immunodeficiency virus type 1 (HIV-1) replication in vitro most commonly use a p24 antigen ELISA or reverse transcriptase (RT) assay to quantify virus released into the culture supernatant from infected cells. The p24 assay is capable of detecting HIV-1 between the pg and ng range, while conventional RT assays are about 10 to 100 times less sensitive. Several highly sensitive RT assays have been described that are approximately 10'-fold more sensitive than conventional RT assays (Heneine et al., 1995 ;Pyra et al., 1994 ;Silver et al., 1993). These assays rely on the ability of a retroviral RT to copy an exogenous RNA template of known sequence into a complementary DNA (cDNA). This cDNA is then amplified by PCR, and the PCR product can be detected and quantified. These assays have the potential to detect a single retroviral virion (Heneine et al

Roles for CXC Chemokine Ligands 10 and 11 in Recruiting CD4+ T Cells to HIV-1-Infected Monocyte-Derived Macrophages, Dendritic Cells, and Lymph Nodes

“…Pseudotyped virus was concentrated, and infections were performed as described above. The viable mutants of AD (Vif, MA.Vpr, Vpu, and Nef) have been described previously (32,35).…”

“…The study protocol was reviewed and approved by the institutional review board of the National Institute of Allergy and Infectious Diseases. Hybridizations were performed on sections from paraffin-embedded tissue using 35 S-labeled antisense and sense riboprobes. The probes for CXCL10 and CXCL11 were made as previously described (36).…”