Brain Transcriptome-Wide Screen for HIV-1 Nef Protein Interaction Partners Reveals Various Membrane-Associated Proteins

Abstract

HIV-1 Nef protein contributes essentially to the pathology of AIDS by a variety of protein-protein-interactions within the host cell. The versatile functionality of Nef is partially attributed to different conformational states and posttranslational modifications, such as myristoylation. Up to now, many interaction partners of Nef have been identified using classical yeast two-hybrid screens. Such screens rely on transcriptional activation of reporter genes in the nucleus to detect interactions. Thus, the identification of Nef interaction partners that are integral membrane proteins, membrane-associated proteins or other proteins that do not translocate into the nucleus is hampered. In the present study, a split-ubiquitin based yeast two-hybrid screen was used to identify novel membrane-localized interaction partners of Nef. More than 80% of the hereby identified interaction partners of Nef are transmembrane proteins. The identified hits are GPM6B, GPM6A, BAP31, TSPAN7, CYB5B, CD320/TCblR, VSIG4, PMEPA1, OCIAD1, ITGB1, CHN1, PH4, CLDN10, HSPA9, APR-3, PEBP1 and B3GNT, which are involved in diverse cellular processes like signaling, apoptosis, neurogenesis, cell adhesion and protein trafficking or quality control. For a subfraction of the hereby identified proteins we present data supporting their direct interaction with HIV-1 Nef. We discuss the results with respect to many phenotypes observed in HIV infected cells and patients. The identified Nef interaction partners may help to further elucidate the molecular basis of HIV-related diseases.

Function of ubiquitin (Ub) specific protease 15 (USP15) in HIV-1 replication and viral protein degradation

“…Nef is known to reside at the cytoplasmic membrane (Fackler et al, 1997; Kaminchik et al, 1994; Kammula et al, 2012; Yu and Felsted, 1992), while subcellular localization of USP15 is poorly understood. Since our data indicated that the stability of Nef and USP15 was affected bilaterally, we examined the possibility that Nef and USP15 co-localize to the same subcellular compartments of 293T cells transfected with pNef.GFP and pUSP15, using confocal microscopic analysis.…”

“…One of the key molecular features of Nef is that, unlike other viral proteins whose functions are phase- (or stage-) specific in the virus life cycle, Nef is the only known viral element which acts throughout the whole virus life cycle within the host cell: it is translated from the early viral messages (Cullen, 1991; Ferguson et al, 2002; Haseltine, 1991) and stays in the infected cells until the protein is packaged into virion particles (Bukovsky et al, 1997), and thus Nef has temporal advantages to determine the fate of not only viral but also cellular proteins throughout the HIV-1 infectious life cycle. Another notable feature is that subcellular localization of Nef, compared with other viral proteins which localize to specific subcellular compartments, is not confined to the cytoplasmic membrane by myristoylation (Allan et al, 1985; Franchini et al, 1986), but occurs in diverse subcellular organelles, such as endosomes (Dikeakos et al, 2012; Kueck and Neil, 2012; Singh et al, 2009), ER (Park et al, 2014), mitochondria, and even the perinuclear membrane by interacting with cellular protein partners (Kammula et al, 2012). Nef is also known to play a pivotal role in localization of Env glycoprotein and Gag in late endosomes (Sandrin and Cosset, 2006) and in regulation of various cellular proteins by inducing endocytosis (Hanna et al, 1998; Sandrin and Cosset, 2006), suggesting that Nef may have spatial advantages in governing viral and cellular protein fates.…”

VSIG4 expression on macrophages facilitates lung cancer development

“…14 VSIG4 is not only a receptor for complement C3, which mediates the clearance of pathogens, 12 but also has the capacity to inhibit CD4 þ and CD8 þ T-cell proliferation and IL-2 production by ligating an unidentified receptor on T cells. 14 VSIG4 mRNA is highly expressed in various tissues, especially the liver, lung, placenta, and possibly the central nervous system, [15][16][17] but its protein form is restricted to the surface of macrophages. 12 Recent studies have shown that VSIG4 þ cells are found in tissues from patients with rheumatoid arthritis, atherosclerosis (AS), and chronic HBV-infected livers, indicating that VSIG4 might be involved in the pathogenesis of these inflammatory diseases.…”

Immunoregulatory protein V‐set and immunoglobulin domain‐containing 4 is overexpressed in patients with endometriosis

“…V‐set and Ig domain‐containing 4 (VSIG4) is a recently identified B7 family‐related macrophage protein that plays a role in immune checkpoints with the capacity to inhibit T‐cell activation 6 . It has been reported that the expression of VSIG4 mRNA is high in various tissues such as the liver, lung, placenta and the central nervous system, 7–9 but its protein form is restrictively expressed on the surface of macrophages 10 . VSIG4 can also be a target of immune check point inhibitors for the anti‐tumor effects.…”