HIV-1 Nef-mediated T-cell activation and chemotaxis are decoupled using a HIV-1/SIVpbj1.9. chimeric nef variant

Department of Cell Biology and Anatomy, Graduate School of Biomedical Sciences, University of North Texas Health Science Center, CBH-316, 3500 Camp Bowie Blvd, Fort Worth, TX, 76107, USA, .
Archives of Virology (Impact Factor: 2.39). 12/2012; 158(4). DOI: 10.1007/s00705-012-1560-z
Source: PubMed


HIV-1 Nef is known to activate CD4+ T cells but inhibit their migration toward SDF-1α. However, it is not clear how this protein orchestrates these two seemingly concomitant events. In this study, by comparing these two activities of HIV-1 Nef with those of its primate counterpart, SIVpbj1.9, we found that HIV-1 Nef activated T cells only in the presence of CD3/ CD28 stimulation, while SIVpbj1.9 Nef did even without CD3/CD28. We also observed that HIV-1 Nef inhibited T-cell chemotaxis toward SDF-1α, while SIVpbj1.9 Nef did not. A hybrid between HIV-1 and SIVpbj1.9 Nef completely abrogated the chemotaxis blockade by HIV-1 Nef while failing to activate T cells without CD3/CD28 co-stimulation. Mutations in the myristoylation and SH3-binding site, but not the basic-rich domain, in Nef were unresponsive to CD3/CD28 stimulation but reversed the inhibition of migration. These findings indicate that the signals for T-cell activation by Nef do not necessarily parallel those for T-cell migration.

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    ABSTRACT: A central feature of HIV-1 infection is the inability of entering virus to integrate into chromosomes of resting T lymphocytes unless they are mitogenically activated. In contrast, SIVpbj1.9 replicates in initially resting T lymphocytes by activating infected cells. Previous reports have shown that a difference in Nef-mediated T cell activation between HIV-1 and SIVpbj1.9 plays a critical role in the differing abilities of these viruses to replicate in resting lymphocytes. However, the molecular details of these differences are still unclear. Here, we show that infection with a chimeric virus, HSIVnef, which harbors the 5′ 308 nucleotides of SIVpbj1.9 nef in place of the 5′ 221 nucleotides of HIV-1 nef in the HIV-1 proviral backbone, resulted in integration of the provirus into host chromosomes without mitogenic activation and thereby replication in resting human PBMCs (hPBMCs). These results indicate that Nef is an essential viral determinant for the integration of provirus into host chromosomes in resting T cells. Using the yeast two-hybrid system, we identified integrase interactor-1 (INI1/SMARCB1) as a cellular factor that is involved in the integration process via interaction with Nef. Although INI1 interacted with both SIVpbj1.9 and HIV-1 Nefs, SIVpbj1.9 Nef, but not HIV-1 Nef, enhanced proviral integration into host DNA. Furthermore, mutational analysis revealed that the basic-amino-acid-rich amino-terminal domain in SIVpbj1.9 Nef is crucial for interaction with INI1 and virus replication in resting hPBMCs. Taken together, these data indicate that Nef is a critical viral protein for incorporating nascent proviral DNA into host chromosomes in resting PBMCs and that this occurs through interaction with INI1. This elucidates the basis for replication of the integrated provirus when the host cell is in a resting state.
    Archives of Virology 01/2015; 160(3). DOI:10.1007/s00705-014-2315-9 · 2.39 Impact Factor
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    ABSTRACT: HIV-1 Nef is an important pathogenic factor for HIV/AIDS pathogenesis. Several recent studies including ours have demonstrated that Nef can be transferred to neighboring cells and alters the function of these cells. However, how the intercellular Nef transfer occurs is in dispute. In the current study, we attempted to address this important issue using several complementary strategies, a panel of exosomal markers, and human CD4+ T lymphocyte cell line Jurkat and a commonly used cell line 293T. First, we showed that Nef was transferred from Nef-expressing or HIV-infected Jurkat to naïve Jurkat and other non-Jurkat cells and that the transfer required the membrane targeting function of Nef and was cell densitydependent. Then, we showed that Nef transfer was cell-cell contact-dependent, as exposure to culture supernatants or exosomes from HIV-infected Jurkat or Nef-expressing Jurkat and 293T led to little Nef detection in the target cells Jurkat. Thirdly, we demonstrated that Nef was only detected to be associated with HIV virions but not with acetylcholinesterase (AChE+) exosomes from HIV-infected Jurkat and not in the exosomes from Nef-expressing Jurkat. In comparison, when it was over-expressed in 293T, Nef was detected in detergent-insoluble AChE+/CD81low/TSG101low exosomes, but not in detergent-soluble AChE-/CD81high/TSG101high exosomes. Lastly, microscopic imaging showed no significant Nef detection in exosomal vesicle-like structures in and out 293T. Taken together, these results show that exosomes are unlikely involved in intercellular Nef transfer. In addition, this study reveals existence of two types of exosomes: AChE+/CD81low/TSG101low exosomes and AChE-/CD81high/TSG101high exosomes.
    PLoS ONE 04/2015; 10(4):e0124436. DOI:10.1371/journal.pone.0124436 · 3.23 Impact Factor