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Early sorting of inner nuclear membrane proteins is conserved.

ABSTRACT: Spodoptera frugiperda (Sf9) importin-alpha-16 is a translocon-associated protein that participates in the early sorting pathway of baculovirus integral membrane proteins destined for the inner nuclear membrane (INM). To discern whether sorting intermediate protein complexes like those observed in insect cells are also formed with mammalian INM proteins, cross-linked complexes of importin-alpha-16 with human lamin B receptor (LBR) and nurim were examined. Both LBR and nurim cross-link with Sf9 importin-alpha-16 during cotranslational membrane integration and remain proximal with importin-alpha-16 after integration into the endoplasmic reticulum membrane and release from the translocon. Human cells encode several isoforms of importin-alpha; to determine whether any of these isoforms may recognize INM-directed proteins, they were tested for their ability to cross-link with the viral-derived INM sorting motif sequence. One cross-linked adduct was detected with a 16-kDa isoform encoded from KPNA4 (KPNA-4-16). KPNA-4-16 was easily detected in microsomal membranes prepared from KPNA4-16 recombinant virus-infected cells and was also detected in microsomes prepared from HeLa cells. Together these observations suggest that elements of the early sorting pathway of INM-directed proteins mediated by importin-alpha-16 are highly conserved, and mammalian KPNA-4-16 is a candidate partner in sorting integral membrane proteins to the INM.

SUBMITTER: Braunagel SC

PROVIDER: S-EPMC1874229 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Human immunodeficiency virus type 1 (HIV-1) infection of dividing and nondividing cells involves regulatory interactions with the nuclear pore complex (NPC), followed by translocation to the nucleus and preferential integration into genomic areas in proximity to the inner nuclear membrane (INM). To identify host proteins that may contribute to these processes, we performed an overexpression screen of known membrane-associated NE proteins. We found that the integral transmembrane proteins SUN1/UNC84A and SUN2/UNC84B are potent or modest inhibitors of HIV-1 infection, respectively, and that suppression corresponds to defects in the accumulation of viral cDNA in the nucleus. While laboratory strains (HIV-1NL4.3 and HIV-1IIIB) are sensitive to SUN1-mediated inhibition, the transmitted founder viruses RHPA and ZM247 are largely resistant. Using chimeric viruses, we identified the HIV-1 capsid (CA) protein as a major determinant of sensitivity to SUN1, and in vitro-assembled capsid-nucleocapsid (CANC) nanotubes captured SUN1 and SUN2 from cell lysates. Finally, we generated SUN1-/- and SUN2-/- cells by using CRISPR/Cas9 and found that the loss of SUN1 had no effect on HIV-1 infectivity, whereas the loss of SUN2 had a modest suppressive effect. Taken together, these observations suggest that SUN1 and SUN2 may function redundantly to modulate postentry, nuclear-associated steps of HIV-1 infection.IMPORTANCE HIV-1 causes more than 1 million deaths per year. The life cycle of HIV-1 has been studied extensively, yet important steps that occur between viral capsid release into the cytoplasm and the expression of viral genes remain elusive. We propose here that the INM components SUN1 and SUN2, two members of the linker of nucleoskeleton and cytoskeleton (LINC) complex, may interact with incoming HIV-1 replication complexes and affect key steps of infection. While overexpression of these proteins reduces HIV-1 infection, disruption of the individual SUN2 and SUN1 genes leads to a mild reduction or no effect on infectivity, respectively. We speculate that SUN1/SUN2 may function redundantly in early HIV-1 infection steps and therefore influence HIV-1 replication and pathogenesis.

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Early sorting of inner nuclear membrane proteins is conserved.

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