The capsid lattice engages a bipartite NUP153 motif to mediate nuclear entry of HIV-1 cores.

Increasing evidence has suggested that the HIV-1 capsid enters the nucleus in a largely assembled, intact form. However, not much is known about how the cone-shaped capsid interacts with the nucleoporins (NUPs) in the nuclear pore for crossing the nuclear pore complex. Here, we elucidate how NUP153 binds HIV-1 capsid by engaging the assembled capsid protein (CA) lattice. A bipartite motif containing both canonical and noncanonical interaction modules was identified at the C-terminal tail region of NUP153. The canonical cargo-targeting phenylalanine-glycine (FG) motif engaged the CA hexamer. By contrast, a previously unidentified triple-arginine (RRR) motif in NUP153 targeted HIV-1 capsid at the CA tri-hexamer interface in the capsid. HIV-1 infection studies indicated that both FG- and RRR-motifs were important for the nuclear import of HIV-1 cores. Moreover, the presence of NUP153 stabilized tubular CA assemblies in vitro. Our results provide molecular-level mechanistic evidence that NUP153 contributes to the entry of the intact capsid into the nucleus.

HIV-1 Nef hijacks both exocytic and endocytic pathways of host intracellular trafficking through differential regulation of Rab GTPases.

BACKGROUND: HIV-1 Nef regulates several cellular functions in an infected cell which results in viral persistence and AIDS pathogenesis. The currently understood molecular mechanism(s) underlying Nef-dependent cellular function(s) are unable to explain how events are coordinately regulated in the host cell. Intracellular membranous trafficking maintains cellular homeostasis and is regulated by Rab GTPases - a member of the Ras superfamily. RESULTS: In the current study, we tried to decipher the role of Nef on the Rab GTPases-dependent complex and vesicular trafficking. Expression profiling of Rabs in Nef-expressing cells showed that Nef differentially regulates the expression of individual Rabs in a cell-specific manner. Further analysis of Rabs in HIV-1NL4-3 or ΔNef infected cells demonstrated that the Nef protein is responsible for variation in Rabs expression. Using a panel of competitive peptide inhibitors against Nef, we identified the critical domain of HIV-1 Nef involved in modulation of Rabs expression. The molecular function of Nef-mediated upregulation of Rab5 and Rab7 and downregulation of Rab11 increased the transport of SERINC5 from the cell surface to the lysosomal compartment. Moreover, the Nef-dependent increase in Rab27 expression assists exosome release. Reversal of Rabs expression using competitive inhibitors against Nef and manipulation of Rabs expression reduced viral release and infectivity of progeny virions. CONCLUSION: This study demonstrates that Nef differentially regulates the expression of Rab proteins in HIV-1 infected cells to hijack the host intracellular trafficking, which augments viral replication and HIV-1 pathogenesis. SIGNIFICANCE: Our study emphasized the indispensable role of HIV-1 protein Nef on various aspects of the intracellular trafficking regulated by Rabs GTPases, which explained how HIV-1 Nef may hijack membrane trafficking pathways in infected cells.

HIV-1 Nef-GCC185 interaction regulates assembly of cellular protein complexes at TGN targeting MHC-I downregulation.

AIM: HIV-1 Nef downregulates surface MHC-I to protect the infected cells from CTLs-mediated killing. Although MHC-I downregulation has been extensively studied, the Nef-dependent assembly of the multi-protein complex and subsequent pathways activation has not yet been well explored. The present study is aimed for the identification of Nef-mediated sequential recruitment of cellular proteins that constitute the functional multi-protein complex, required for the downregulation of MHC-I. MAIN METHODS: Different Cellular protein complexes were identified by co-immunoprecipitation in Nef or NefE4A mutant-expressing Jurkat T, and THP-1 cells followed by exposure to Nef-specific peptides 24 h post infection. The MHC-I downregulation was analyzed by confocal microscopy and flow cytometry. KEY FINDINGS: We found the association of Nef with PACS-2, GCC185, PI3K, AP-1, SFK, and MHC-I proteins that probably constitute a functional multi-protein complex. Furthermore, the immunoprecipitations with PACS-2 and GCC185 in the presence or absence of Nef, Nef E4A mutant and Nef with CP-inhibitor divide the functional complex of Nef into Nef-dependent (AP-1 and PI3K) and GCC185-dependent complex (MHC-I and SFK). The molecular mechanisms for activation of cellular pathways have been deciphered on the basis of these interactions that are brought in close proximity through Nef-GCC185 interaction. Knockdown of GCC185 using siRNA in Jurkat T cells showed a direct relationship between the assembly of functional multi-protein complex and MHC-I accumulation at GCC185. SIGNIFICANCE: Overall, our study elucidates that GCC185 is a focal point for the assembly of the Nef-mediated multi-protein complex at TGN.

HIV-1 Nef physically associate with CAMKIIδ - ASK-1 complex to inhibit p38MAPK signalling and apoptosis in infected cells.

Human immunodeficiency type 1 virus accessory protein Nef is a key modulator of AIDS pathogenesis. With no enzymatic activity, Nef regulated functions in host cells largely depends on its ability to form multi-protein complex with the cellular proteins. Here, we identified Calcium (Ca2+)/Calmodulin dependent protein kinase II subunit delta (CAMKIIδ) as novel Nef interacting host protein. Further, we confirmed that Nef mediated [Ca2+]I promote formation of Nef-CAMKIIδ - apoptosis signal-regulating kinase (ASK-1) heterotrimeric complex. The assembly of Nef with CAMKIIδ - ASK-1 inhibits the downstream p38MAPK phosphorylation resulting in abrogation of apoptosis. Further, using competitive peptide inhibitors against Nef binding domains to CAMKIIδ, identified in the present study and ASK-1, individually blocked physical interaction of Nef with CAMKIIδ-ASK-1 complex and restored p38MAPK phosphorylation and apoptosis. Altogether, our study indicates that HIV-Nef modulates cytosolic [Ca2+]I and blocks CAMKIIδ - ASK-1 kinase activity to inhibit apoptosis of infected cells.

HIV-1 Nef-POTEE; A novel interaction modulates macrophage dissemination via mTORC2 signaling pathway.

AIMS: Human immunodeficiency virus -1 [HIV-1] Nef, localizes in different cellular compartments and modulates several cellular pathways. Nef promotes virus pathogenicity through alteration in cell surface receptor expression, apoptosis, protein trafficking etc. Nef regulates viral pathogenesis through interaction with different host proteins. Thus, molecular mechanisms of pathogenesis could be deciphered by identifying novel Nef interacting proteins. MAIN METHODS: HIV-1 Nef interacting proteins were identified by pull down assay and MALDI-TOF analysis. The interaction was further validated through mammalian two hybrid assay. Functional role of this interaction was identified by immunoprecipitation assay, cell invasion and cell migration studies. Fold Change in mRNA levels of CD163, CD206, CCL17 and CCL18 was analyzed using qPCR. KEY FINDINGS: In current study, C. elegans protein ACT4C and its human homolog POTEE was identified to be interacting with Nef. This interaction activates mTORC2 complex, which in-turn activates AKT and PKC-α. The activation of mTORC2 complex was found to be initiated by the interaction of Nef, mTORC2, Rictor to POTEE. The cellular phenotype and functions affected by Nef-POTEE interaction resulted in significant increase in cell invasion and migration of macrophages (MΦ). SIGNIFICANCE: MΦ is primary target of HIV-1 infection where HIV-1 replicates and polarizes immunosuppressive M2 phenotype. Combine effect of M2 phenotype and Viral-host protein interactions compromise the MΦ associated physiological functions. Infected MΦ dissemination into other system also leads to HIV-1 induced malignancies. Therefore, targeting POTEE-Nef interaction can lead to formulating better therapeutic strategy against HIV-1.