HIV-1 Nef Induces Proliferation and Anchorage-Independent Growth in Podocytes

Abstract

ABSTRACT. HIV-associated nephropathy (HIVAN) is now the third leading cause of end-stage renal disease in the African American population. HIV-1 infects renal tubular and glomerular epithelial cells or podocytes, cells that are a critical part of the filtration barrier. HIV-1 infection induces the loss of podocyte differentiation markers and increases podocyte proliferation. It has been previously shown that HIV-infection induces loss of contact inhibition. Here, the HIV-1 gene responsible for proliferative changes is identified by using cultured podocytesin vitro. The HIV-1 proviral construct, pNL4-3 was rendered noninfectious by replacing the HIV-1gag/polsequences with an EGFP reporter gene (pNL4-3: ΔG/P-EGFP). This construct was then pseudotyped with VSV.G envelope to infect podocytes that were conditionally immortalized with SV-40 T antigen. In addition, mutated constructs were engineered with premature stop codons in the HIV-1env,vif,vpr,vpu,nef, orrevgenes. The parental construct and all the other mutated constructs, with the exception ofnef, induced proliferation under nonpermissive conditions and anchorage-independent growth (colony formation in soft agar) under permissive conditions. In contrast, deletion ofnefmarkedly reduced proliferation and colony formation. Althoughtatalone, ortatplusrevinduced marginal levels of anchorage-independent growth, coexpression withnefsignificantly increased colony formation. Finally, stable expression of Nef in a retroviral vector, pBabe-puro, was sufficient to induce increased proliferation and colony formation. Moreover,nefinduced saturation density and loss of contact inhibition. These data indicate that Nef induces multiple proliferative effects in podocytes in culture and thatnefmay therefore be an important gene in the pathogenesis of HIVANin vivo.