Molecular characterization of human immunodeficiency virus type 1 cloned directly from uncultured human brain tissue: identification of replication-competent and -defective viral genomes

Abstract

All presently available replication-competent proviral clones of human immunodeficiency virus type 1 (HIV-1) are derived from cell culture-amplified virus. Since tissue culture is highly selective for viral strains with an in vitro growth advantage, such clones may not be representative of the biologically relevant virus present in vivo. In this study, we report the molecular cloning and genotypic characterization of 10 HIV-1 genomes directly from uncultured brain tissue of a patient with AIDS dementia complex. Targeting unintegrated circular HIV-1 molecules for recombinant lambda phage cloning, we obtained four full-length genomes with one or two long terminal repeats (LTRs), three defective genomes with internal deletions, two rearranged genomes with inverted LTR sequences, and one integrated proviral half with flanking cellular sequences. Nucleotide sequence analysis of these clones demonstrated chromosomal integration, circle formation, genomic inversion, and LTR-mediated autointegration of HIV-1 genomes in vivo. Comparison of a 510-bp hypervariable envelope region among 8 lambda phage-derived and 12 polymerase chain reactionderived clones from the same brain specimen identified a predominant viral form as well as genetically divergent variants. Variability among 19 of 20 clones ranged between 0.2 and 1.2%. One clone exhibited 8.2% nucleotide sequence differences consisting almost exclusively of G-to-A changes. Transfection of the four fulllength HIV-1 genomes identified one clone (YU-2) as replication competent and exhibiting growth characteristics similar to those of tissue culture-derived macrophage tropic strains of HIV-1. These results demonstrate, for the first time, that replication-competent HIV-1 genomes, complex mixtures of defective viral forms, and chromosomally integrated provirus persist in vivo. In addition, the brain-derived viral clones are expected to prove valuable for future studies of macrophage and neurotropism as well as for the analysis of other viral properties that are subject to in vitro selection pressures.

Infection of Human Immunodeficiency Virus and Intracellular Viral Tat Protein Exert a Pro-survival Effect in a Human Microglial Cell Line

“…23 First, we attempted to validate the CHME5 cell line as a target cell type of M-tropic HIV-1 by examining whether CHME5 cells can support the replication of an M-tropic HIV-1 strain, YU-2, which was cloned from HIV-1 infected human brain tissue. 37 As shown in Figure 1, the CHME5 cells were able to produce and amplify M-tropic virus during serial viral passages, and the efficiency of the HIV-1 production in this cell line was similar with that in primary human peripheral blood mononuclear cells (PBMCs). However, as expected, Figure 1.…”

Macropinocytosis-Like HIV-1 Internalization in Macrophages Is CCR5 Dependent and Leads to Efficient but Delayed Degradation in Endosomal Compartments

“…Molecular constructs. The X4-tropic pNL4-3 (27) and the R5-utilizing pYU-2 (28,29) full-length molecular HIV-1 clones were obtained from the NIH AIDS Research and Reference Reagent program. The pNL4-3Balenv R5-tropic molecular clone was a kind gift of R. Pomerantz (Thomas Jefferson University, Philadelphia, PA) (30).…”

GPI-anchored single chain Fv - an effective way to capture transiently-exposed neutralization epitopes on HIV-1 envelope spike

“…HIV-1 envelope Q168 is derived from a R5 tropic clade A virus [63]. HIV-1 envelopes AD8, ADA, JR-FL, Yu2 are derived from R5 tropic clade B viruses [64][65][66][67]. HIV-1 envelope HxBc2 is derived from a X4 tropic clade B virus [68].…”