SUN2 Silencing Impairs CD4 T Cell Proliferation and Alters Sensitivity to HIV-1 Infection Independently of Cyclophilin A

Abstract

Linker of nucleoskeleton and cytoskeleton (LINC) complexes connect the nucleus to the cytoskeleton in eukaryotic cells. We previously reported that the overexpression of SUN2, an inner nuclear membrane protein and LINC complex component, inhibits HIV infection between the steps of reverse transcription and nuclear import in a capsid-specific manner. We also reported that SUN2 silencing does not modulate HIV infection in several cell lines. Silencing of SUN2 was recently reported to decrease HIV infection of CD4 T cells, an effect which was suggested to result from modulation of cyclophilin A (CypA)-dependent steps of HIV infection. We confirm here that HIV infection of primary CD4 T cells is compromised in the absence of endogenous SUN2, and we extend these findings to additional viral strains. However, we find that CypA is not required for the decreased infection observed in SUN2-silenced cells and, conversely, that endogenous SUN2 is not required for the well-documented positive modulation of HIV infection by CypA. In contrast, CD4 T cells lacking SUN2 exhibit a considerable defect in proliferative capacity and display reduced levels of activation markers and decreased viability. Additionally, SUN2-silenced CD4 T cells that become infected support reduced levels of viral protein expression. Our results demonstrate that SUN2 is required for the optimal activation and proliferation of primary CD4 T cells and suggest that the disruption of these processes explains the contribution of endogenous SUN2 to HIV infection in primary lymphocytes.IMPORTANCE Linker of nucleoskeleton and cytoskeleton (LINC) complexes connect the nucleus to the cytoskeleton. We previously reported that the overexpression of the LINC complex protein SUN2 inhibits HIV infection by targeting the viral capsid and blocking infection before the virus enters the nucleus. A recent report showed that the depletion of endogenous SUN2 in primary CD4 T cells results in decreased HIV infection and that this involves cyclophilin A (CypA), a host protein that interacts with the capsid of HIV to promote infection. We confirm that HIV infection is reduced in CD4 T cells lacking SUN2, but we find no role for CypA. Instead, SUN2 silencing results in CD4 T cells with decreased viability and much lower proliferation rates. Our results show that SUN2 is required for optimal CD4 T cell activation and proliferation and explain the reduced level of HIV infection in the absence of SUN2.KEYWORDS CD4 T cell, SUN2, human immunodeficiency virus L inker of nucleoskeleton and cytoskeleton (LINC) complexes connect the nucleus to the cytoskeleton and are evolutionarily conserved in eukaryotes from budding and fission yeasts and Caenorhabditis elegans to D. melanogaster, plants, and mammals (1). The LINC complex is defined by the interaction of KASH domain proteins and SUN

Dynamics and regulation of nuclear import and nuclear movements of HIV-1 complexes

“…CsA treatment inhibits HIV-1 replication in some T cell lines and primary CD4 + T cells [ 29 – 31 , 35 ], suggesting that CypA binding evolved to facilitate HIV-1 replication in its natural target cells of infection. Our observation that disrupting the CA-CypA interaction in HeLa cells leads to faster NE docking and nuclear import indicates that one function of CypA binding is to slow down nuclear import.…”

“…The CA-CypA interaction can be disrupted by treatment with cyclosporine A (CsA), which binds to CypA, or by introducing mutations into the CA loop that is involved in binding to CypA (reviewed in [ 7 ]). Disruption of the CA-CypA interaction results in a reduction in viral titers in primary CD4 + T cells, monocyte derived macrophages, and in some T cell lines, but has minimal effects on the viral infectivity in other cell lines such as HeLa cells [ 28 – 31 ]. It is also known that disruption of the CA-CypA interaction influences nuclear import and the requirement for some nuclear pore proteins [ 32 – 35 ].…”

Are Evolution and the Intracellular Innate Immune System Key Determinants in HIV Transmission?

“…SUN2 is an integral membrane protein that spans the inner nuclear membrane and forms part of a multiprotein complex (LINC) that physically bridges the nucleoskeleton and cytoplasm ( 166 ). Several recent studies published in quick succession have suggested that manipulation of SUN2 can either inhibit or promote HIV-1 infection, depending on the level of expression ( 167 – 169 ).…”

“…This was again proposed to be dependent on CypA recruitment to the capsid, as CypA inhibitors had no additive effects with SUN2 silencing ( 168 ). However, the defect in primary T cells has since been convincingly attributed to defects in T cell proliferation, activation status and viability resulting from SUN2 silencing ( 169 ). Discrepancies in cell viability between the two studies could be explained by depletion efficiencies and the duration of silencing experiments.…”

SUN1 Regulates HIV-1 Nuclear Import in a Manner Dependent on the Interaction between the Viral Capsid and Cellular Cyclophilin A

“…This activity is exerted mainly via the nucleoplasmic domain of SUN2 (22). Downregulation of endogenous SUN2 did not affect HIV-1 infection efficiency in HeLa cells but impaired the infection efficiency in primary CD4 ϩ T cells, possibly due to the reduced activation and proliferation of these cells upon SUN2 downregulation (22)(23)(24).…”