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

現地観測に基づく河川流の乱流特性に関する研究

博士（工学）神戸大

A Conserved Acidic Residue in the C-Terminal Flexible Loop of HIV-1 Nef Contributes to the Activity of SERINC5 and CD4 Downregulation

The host transmembrane protein SERINC5 is incorporated into retrovirus particles and inhibits HIV-1 infectivity. The lentiviral Nef protein counteracts SERINC5 by downregulating it from the cell surface and preventing its incorporation into virions. The ability of Nef to antagonize the host factor varies in magnitude between different HIV-1 isolates. After having identified a subtype H nef allele unable to promote HIV-1 infectivity in the presence of SERINC5, we investigated the molecular determinants responsible for the defective counteraction of the host factor. Chimeric molecules with a subtype C Nef highly active against SERINC5 were constructed to locate Nef residues crucial for the activity against SERINC5. An Asn at the base of the C-terminal loop of the defective nef allele was found in place of a highly conserved acidic residue (D/E 150). The conversion of Asn to Asp restored the ability of the defective Nef to downregulate SERINC5 and promote HIV-1 infectivity. The substitution was also found to be crucial for the ability of Nef to downregulate CD4, but not for Nef activities that do not rely on the internalization of receptors from the cell surface, suggesting a general implication in promoting clathrin-mediated endocytosis. Accordingly, bimolecular fluorescence complementation revealed that the conserved acidic residue contributes to the recruitment of AP2 by Nef. Altogether, our results confirm that Nef downregulates SERINC5 and CD4 by engaging a similar machinery and indicates that, in addition to the di-leucine motif, other residues in the C-terminal flexible loop are important for the ability of the protein to sustain clathrin-mediated endocytosis

The decrease in T cell transduction efficiency by SIV_MAC is not explained by differences in reporter gene expression.

<p>(A) CRFK cells (left panel) and Jurkat T cells (right panel) were transduced with VSV G-pseudotyped, single-cycle, two-part HIV-1_NL4-3GFP or SIV_MAC239-GFP vectors, as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005050#ppat.1005050.g001" target="_blank">Fig 1</a>_. Virus stocks were normalized by reverse transcriptase activity prior to transduction. 48 hrs after transduction, cells were visualized by phase contrast and fluorescence microscopy. Shown are representative fields for each condition at 100x magnification. Fluorescence intensity of individual T cells transduced with SIV_MAC239-GFP is at least as strong as that in cells transduced with HIV-1_NL4-3GFP. (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 Jurkat T cells. 48 hrs post vector challenge, the percentage 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).</p

As₂O₃ specifically increases SIV_MAC infectivity in human blood cells.

<p>TE671 cells (A), Jurkat T cells (B), human PBMC (C), or human CD4⁺ T cells (D) were transduced with two-part, VSV G-pseudotyped HIV-1_<b>NL4-3</b>-GFP or SIV_<b>MAC</b>GFP vectors using a predetermined quantity of virus such that 1% of cells were infected. As_<b>2</b>O_<b>3</b> was added 1 hr prior to vector challenge and maintained for 12 hrs post-infection, at the concentrations indicated on the X axis. 48 hrs post-challenge the percentage of GFP-expressing cells was determined. The Y axis shows the fold increase relative to infection without As_<b>2</b>O_<b>3</b>.</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

The capsid of SIV_MAC, HIV-2, or SIV_SM is sufficient to decrease HIV-1 transduction efficiency in a T cell-specific manner.

<p>(A) Chimeric vectors were generated in which the coding sequence for HIV-1 CA amino acid residues 1 to 202 of the two-part HIV-1_NL4-3GFP vector (white squares) was replaced with sequence encoding the corresponding amino acid residues from HIV-2_ROD (grey triangles) or SIV_MAC239 (black circles). VSV G-pseudotyped vector was generated for each by transfection of 293T cells. Stocks were normalized by RT and used to challenge CRFK cells (B) or HeLa cells (C). Stocks were then normalized for CRFK transduction activity and used to challenge Jurkat T cells (D). 48 hrs post-challenge, the percentage of GFP-expressing cells was determined by FACS. Data is plotted as percent GFP⁺ (infected) cells (Y axis) versus RT activity (B and C), or versus CRFK infectious units (IU) x 1000 (X axis). (E) Chimeric vectors were generated in which the coding sequence for HIV-1 CA amino acid residues 1 to 202 of the HIV-1 <i>gag-pol</i> expression vector (white) was replaced with sequence encoding the corresponding amino acid residues from various HIV-2 isolates (grey) or SIV_SME041 (black). Three-part, VSV G-pseudotyped, SFFV-GFP bearing vectors were generated for each CA chimera by transfection of 293T cells. Stocks were then normalized for CRFK transduction activity and used to challenge Jurkat T cells. 48 hrs post-challenge, the percentage of GFP-expressing cells was determined by FACS. Data is plotted as CRFK normalized transduction of Jurkat cells, relative to the parental HIV-1 vector (F). Accession numbers for the different CA coding sequences are as follows: HIV-2(AB), 731744; HIV-2(A), GH123; HIV-2(D), L33083; HIV-2(E), L33087; HIV-2(F), U75441; HIV-2(H), AY5308; SIV_SME041, HM059825.</p