The internal N6-methyladenosine (m6A) methylation of eukaryotic nuclear RNA controls post-transcriptional gene expression, which is regulated by methyltransferases (writers), demethylases (erasers), and m6A-binding proteins (readers) in cells. The YTH domain family proteins (YTHDF1–3) bind to m6A-modified cellular RNAs and affect RNA metabolism and processing. Here, we show that YTHDF1–3 proteins recognize m6A-modified HIV-1 RNA and inhibit HIV-1 infection in cell lines and primary CD4+ T-cells. We further mapped the YTHDF1–3 binding sites in HIV-1 RNA from infected cells. We found that the overexpression of YTHDF proteins in cells inhibited HIV-1 infection mainly by decreasing HIV-1 reverse transcription, while knockdown of YTHDF1–3 in cells had the opposite effects. Moreover, silencing the m6A writers decreased HIV-1 Gag protein expression in virus-producing cells, while silencing the m6A erasers increased Gag expression. Our findings suggest an important role of m6A modification of HIV-1 RNA in viral infection and HIV-1 protein synthesis.DOI: http://dx.doi.org/10.7554/eLife.15528.001

The internal -methyladenosine (mA) modification of cellular mRNA regulates post-transcriptional gene expression. The YTH domain family proteins (YTHDF1-3 or Y1-3) bind to mA-modified cellular mRNAs and modulate their metabolism and processing, thereby affecting cellular protein translation. We previously reported that HIV-1 RNA contains the mA modification and that Y1-3 proteins inhibit HIV-1 infection by decreasing HIV-1 reverse transcription activity. Here, we investigated the mechanisms of Y1-3-mediated inhibition of HIV-1 infection in target cells and the effect of Y1-3 on viral production levels in virus-producing cells. We found that Y1-3 protein overexpression in HIV-1 target cells decreases viral genomic RNA (gRNA) levels and inhibits both early and late reverse transcription. Purified recombinant Y1-3 proteins preferentially bound to the mA-modified 5' leader sequence of gRNA compared with its unmodified RNA counterpart, consistent with the strong binding of Y1-3 proteins to HIV-1 gRNA in infected cells. HIV-1 mutants with two altered mA modification sites in the 5' leader sequence of gRNA exhibited significantly lower infectivity than WT, replication-competent HIV-1, confirming that these sites alter viral infection. HIV-1 produced from cells in which endogenous Y1, Y3, or Y1-3 proteins were knocked down singly or together had increased viral infectivity compared with HIV-1 produced in control cells. Interestingly, we found that Y1-3 proteins and HIV-1 Gag protein formed a complex with RNA in HIV-1-producing cells. Overall, these results indicate that Y1-3 proteins inhibit HIV-1 infection and provide new insights into the mechanisms by which the mA modification of HIV-1 RNA affects viral replication.

Targeting dendritic cell (DC) functions such as migration is a pivotal mechanism used by HIV-1 to disseminate within the host. The HIV-1 envelope protein is the most important of the virally encoded proteins that exploits the migratory capacity of DCs. In the present study, we elucidated the signaling machinery involved in migration of immature DCs (iDCs) in response to HIV-1 envelope protein. We observed that M-tropic HIV-1 glycoprotein 120 (gp120) induces phosphorylation of the nonreceptor tyrosine kinase, Pyk2. Inhibition of Pyk2 activity using a pharmacologic inhibitor, kinase-inactive Pyk2 mutant, and Pyk2-specific small interfering RNA blocked gp120-induced chemotaxis, confirming the role of Pyk2 in iDC migration. In addition, we also illustrated the importance of Pyk2 in iDC migration induced by virion-associated envelope protein, using aldithriol-2-inactivated M-tropic HIV-1 virus. Further analysis of the downstream signaling mechanisms involved in gp120-induced migration revealed that Pyk2 activates p38 mitogenactivated protein kinase, which in turn activates the F-actin-binding protein, leukocyte-specific protein 1, and enhances its association with actin. Taken together, our studies provide an insight into a novel gp120-mediated pathway that regulates DC chemotaxis and contributes to the dissemination of HIV-1 within an infected person. (Blood. 2009;114:3588-3600)

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Edited by Charles E. Samuel N 6 -methyladenosine (m 6 A) modification of HIV-1 RNA regulates viral replication and protein expression. The m 6 A modification is regulated by two groups of cellular proteins named writers and erasers that add or remove m 6 A, respectively. HIV-1 infection of CD4 ؉ T-cells increases m 6 A levels of cellular mRNA, but the underlying mechanism is unknown. Here, we show that HIV-1 infection of CD4 ؉ primary T-cells or Jurkat cells significantly increases m 6 A levels of cellular RNA independently of viral replication. Compared with HIV-1-infected CD4 ؉ T-cells, similar m 6 A up-regulation was detected in total RNA from HIV-1-infected cells treated with a reverse-transcriptase inhibitor or with heat-inactivated HIV-1. Compared with mock controls, significantly increased m 6 A levels were detected in total RNA from Jurkat cells infected by single-cycle HIV-1 pseudotyped with an HIV-1 envelope (Env) glycoprotein, but not with vesicular stomatitis virus glycoprotein G (VSV-G).Overexpression of HIV-1 Env in HEK293T cells did not affect m 6 A levels of cellular RNA, suggesting that de novo synthesis of Env is not required for m 6 A up-regulation. Interestingly, treatment of Jurkat cells with recombinant gp120 of HIV-1 Env significantly increased m 6 A levels of cellular RNA, which was reduced by a gp120-neutralizing antibody. Preincubation of Jurkat cells with a CD4 receptor-neutralizing antibody blocked HIV-1-induced up-regulation of m 6 A levels in cellular RNA. Moreover, HIV-1 infection or gp120 treatment did not alter the protein expression of m 6 A writers and erasers in cells. Our findings suggest that HIV-1 gp120 binding to the CD4 receptor is required for m 6 A up-regulation in cells. . 2 The abbreviations used are: m 6 A, N 6 -methyladenosine; MOI, multiplicity of infection; hpi, hours post-infection; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NVP, nevirapine; Env, HIV-1 envelope proteins; METTL, methyltransferase-like protein; FTO, fat mass and obesity-associated protein; AlkBH5, AlkB family member 5; MB, methylene blue; sncRNA, small noncoding RNA; sCD4, soluble CD4; NARP, National Institutes of Health AIDS Reagent Program; SSC, saline-sodium citrate.

1 Very young and adult Wistar rats were given As5+, 5 mg arsenic kg-1 body weight day-1 (sodium arsenate). 2 Operant learning was tested in a Skinner box at the end of exposure and, in the case of developing animals, also after a recovery period. 3 Acetylcholine esterase (AChE) activity was estimated in discrete brain regions of these animals. 4 The animals exposed to arsenic took longer to acquire the learned behaviour and to extinguish the operant. AChE activity was inhibited in some regions of the brain.

BackgroundSlit2 is a ~ 200 kDa secreted glycoprotein that has been recently shown to regulate immune functions. However, not much is known about its role in HIV (human immunodeficiency virus)-1 pathogenesis.ResultsIn the present study, we have shown that the N-terminal fragment of Slit2 (Slit2N) (~120 kDa) inhibits replication of both CXCR4 and CCR5-tropic HIV-1 viruses in T-cell lines and peripheral blood T-cells. Furthermore, we demonstrated inhibition of HIV-1 infection in resting CD4+ T-cells. In addition, we showed that Slit2N blocks cell-to-cell transmission of HIV-1. We have shown that Slit2N inhibits HIV-1 infection by blocking viral entry into T-cells. We also ruled out Slit2N-mediated inhibition of various other steps in the life cycle including binding, integration and viral transcription. Elucidation of the molecular mechanism revealed that Slit2N mediates its functional effects by binding to Robo1 receptor. Furthermore, we found that Slit2N inhibited Gp120-induced Robo1-actin association suggesting that Slit2N may inhibit cytoskeletal rearrangements facilitating HIV-1 entry. Studies into the mechanism of inhibition of HIV-1 revealed that Slit2N abrogated HIV-1 envelope-induced actin cytoskeletal dynamics in both T-cell lines and primary T-cells. We further showed that Slit2N specifically attenuated the HIV-1 envelope-induced signaling pathway consisting of Rac1, LIMK and cofilin that regulates actin polymerization.ConclusionsTaken together, our results show that Slit2N inhibits HIV-1 replication through novel mechanisms involving modulation of cytoskeletal dynamics. Our study, thus, provides insights into the role of Slit2N in HIV-1 infection and underscores its potential in limiting viral replication in T-cells.

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