Lv4 Is a Capsid-Specific Antiviral Activity in Human Blood Cells That Restricts Viruses of the SIVMAC/SIVSM/HIV-2 Lineage Prior to Integration

HIV-2 and SIVMAC are AIDS-causing, zoonotic lentiviruses that jumped to humans and rhesus macaques, respectively, from SIVSM-bearing sooty mangabey monkeys. Cross-species transmission events such as these sometimes necessitate virus adaptation to species-specific, host restriction factors such as TRIM5. Here, a new human restriction activity is described that blocks viruses of the SIVSM/SIVMAC/HIV-2 lineage. Human T, B, and myeloid cell lines, peripheral blood mononuclear cells and dendritic cells were 4 to \u3e 100-fold less transducible by VSV G-pseudotyped SIVMAC, HIV-2, or SIVSM than by HIV-1. In contrast, transduction of six epithelial cell lines was equivalent to that by HIV-1. Substitution of HIV-1 CA with the SIVMAC or HIV-2 CA was sufficient to reduce HIV-1 transduction to the level of the respective vectors. Among such CA chimeras there was a general trend such that CAs from epidemic HIV-2 Group A and B isolates were the most infectious on human T cells, CA from a 1 degrees sooty mangabey isolate was the least infectious, and non-epidemic HIV-2 Group D, E, F, and G CAs were in the middle. The CA-specific decrease in infectivity was observed with either HIV-1, HIV-2, ecotropic MLV, or ALV Env pseudotypes, indicating that it was independent of the virus entry pathway. As2O3, a drug that suppresses TRIM5-mediated restriction, increased human blood cell transduction by SIVMAC but not by HIV-1. Nonetheless, elimination of TRIM5 restriction activity did not rescue SIVMAC transduction. Also, in contrast to TRIM5-mediated restriction, the SIVMAC CA-specific block occurred after completion of reverse transcription and the formation of 2-LTR circles, but before establishment of the provirus. Transduction efficiency in heterokaryons generated by fusing epithelial cells with T cells resembled that in the T cells, indicative of a dominant-acting SIVMAC restriction activity in the latter. These results suggest that the nucleus of human blood cells possesses a restriction factor specific for the CA of HIV-2/SIVMAC/SIVSM and that cross-species transmission of SIVSM to human T cells necessitated adaptation of HIV-2 to this putative restriction factor

Potent inhibition of HIV-1 by TRIM5-cyclophilin fusion proteins engineered from human components

New World monkeys of the genus Aotus synthesize a fusion protein (AoT5Cyp) containing tripartite motif-containing 5 (TRIM5) and cyclophilin A (CypA) that potently blocks HIV-1 infection. We attempted to generate a human HIV-1 inhibitor modeled after AoT5Cyp, by fusing human CypA to human TRIM5 (hT5Cyp). Of 13 constructs, 3 showed substantial HIV-1–inhibitory activity when expressed in human cell lines. This activity required capsid binding by CypA and correlated with CypA linkage to the TRIM5a capsid-specificity determinant and the ability to form cytoplasmic bodies. CXCR4- and CCR5-tropic HIV-1 clones and primary isolates were inhibited from infecting multiple human macrophage and T cell lines and primary cells by hT5Cyp, as were HIV-2ROD, SIVAGMtan, FIVPET, and a circulating HIV-1 isolate previously reported to be AoT5Cyp resistant. The anti–HIV-1 activity of hT5Cyp was surprisingly more effective than that of the well-characterized rhesus TRIM5α, especially in T cells. hT5Cyp also blocked HIV-1 infection of primary CD4+ T cells and macrophages and conferred a survival advantage to these cells without disrupting their function. Extensive attempts to elicit HIV-1 resistance to hT5Cyp were unsuccessful. Finally, Rag2–/–γc–/– mice were engrafted with human CD4+ T cells that had been transduced by optimized lentiviral vectors bearing hT5Cyp. Upon challenge with HIV-1, these mice showed decreased viremia and productive infection in lymphoid organs and preserved numbers of human CD4+ T cells. We conclude that hT5Cyp is an extraordinarily robust inhibitor of HIV-1 replication and a promising anti–HIV-1 gene therapy candidate

Evidence for a dominant-acting, capsid-specific, restriction activity in Jurkat T cells.

<p>(A) Jurkat and HeLa cells stably expressing the ALV-A receptor (TvA) or TagRFP-657, as indicated, were fused by treatment with PEG and transduced with ALV-A Env-pseudotyped HIV-1_NL4-3-GFP, or with isogenic vector bearing the SIV_MAC239 CA^1-202. Shown are flow cytometry dot plots obtained 48 hrs post-transduction. HeLa-TagRFP-657 cells are only permissive to infection with ALV-A Env-pseudotyped vectors after fusion with Jurkat-TvA. Infected heterokaryons were visualized as GFP and TagRFP-657 double-positive cells. As a positive transduction control, TagRFP-657 and TvA were also co-expressed in HeLa cells, as indicated. The percentage of transduced cells are indicated. (B) Bar graph showing the infectivity of the SIV_MAC239 CA^1-202-bearing vector relative to the isogenic vector bearing HIV-1 CA, for the HeLa, Jurkat and heterokaryons. Data from the flow cytometry data shown in A, and two repeat experiments, is shown with the standard deviation.</p

The block to SIV_MAC infection of Jurkat T cells occurs after formation of 2-LTR circles.

<p>CRFK and Jurkat (A), or Hela and Jurkat (B), or PBMCs (C) were infected with VSV G-pseudotyped HIV-1_NL4-3-GFP, or with isogenic vector bearing the SIV_MAC239 CA residues 1 to 202. 24 hrs post-infection, DNA was collected from the cells and subjected to qPCR using primers specific for full-length linear viral cDNA, 2-LTR circles, or proviral DNA, as indicated. Shown is the abundance of signal from vector bearing the SIV_MAC239 CA^1-202, relative to the amount of signal from HIV-1_NL4-3-GFP. In each case, infection was performed in the presence of an RT inhibitor to control for background levels of signal.</p

The transduction defect associated with SIV_MAC CA is independent of the virus entry pathway.

<p>A two-part, <i>env</i>-minus HIV-1 vector with GFP in place of nef (black squares), or an isogenic vector in which CA^1-202 coding sequences were replaced with those from SIV_MAC239 (white circles), were produced by 293T transfection. Each vector was pseudotyped with Env glycoprotein from either HIV-1_HXB2(A), HIV-2_MCN (B), ecotropic MLV (C), or ALV-A (D) and transduction efficiency was measured on HeLa cells bearing human CD4 (A and B), the mCAT1 ecotropic receptor (C), or the avian TVA receptor (D), and then used to challenge Jurkat cells bearing the same receptors. 48 hrs post-challenge, the percentage of GFP-expressing cells was determined by FACS.</p

SIV_MAC transduction of human peripheral blood mononuclear cells or of monocyte derived dendritic cells is less efficient than by HIV-1.

<p>(A) VSV G-pseudotyped HIV-1_NL4-3GFP (black squares) and SIV_MAC239GFP (white circles) two-part vectors were generated by plasmid transfection of 293T cells. Vector stocks were normalized by titer on CRFK cells, and then used to challenge human peripheral blood mononuclear cells. (B) VSV G-pseudotyped, HIV-1_NL4-3 (black squares) and SIV_MAC239 (white circles) three-part vectors were generated by plasmid transfection of 293T cells. In each case, the viral genomic RNA was designed to transduce an identical SFFV-GFP reporter gene. Vector stocks were normalized by titer on CRFK cells, and then used to challenge monocyte derived dendritic cells (DCs). 2 days post-challenge, the percentage of GFP-expressing cells was determined by FACS. Data is plotted as percent GFP⁺ (infected) cells (Y axis) versus CRFK infectious units (IU) x 1,000 (X axis). Shown are representative data with cells from 4 independent blood donors.</p