In vitro and in vivo functional analysis of human T cell lymphotropic virus type 1 pX open reading frames I and II

Human T lymphotropic virus type 1 (HTLV-1) is a complex retrovirus containi ng regulatory and accessory genes encoded in four open reading frames (ORF I-IV) of the pX region. It is not clear what role pX ORFs I and II-encoded proteins have in the pathogenesis of the lymphoproliferative diseases assoc iated with HTLV-1 infection. The conserved ORF I encodes for a hydrophobic 12-kDa protein, p12,(I) that contains four SW binding motifs (PXXP) that lo calizes to cellular endomembranes when overexpressed in cultured cells. Dif ferential splicing of pX ORF II results in the production of two nuclear pr oteins, p13(II) and p30(II), p13(II) also localizes to mitochondria. p30(II ) shares homology with the POU family of transcription factors. We have ide ntified functional roles of pX ORF I and ORF II in establishment and mainte nance of infection in a rabbit model. To functionally study p12(I) we have tested a proviral clone with selective ablation of ORF I (ACH.p12(I)) for i ts ability to infect quiescent peripheral blood mononuclear cells (PBMC). O ur data indicate that T cells infected with the wild-type clone of HTLV-1 ( ACH) are more efficient than ACH.p12(I) in infecting quiescent PBMC. These findings parallel our animal model data and suggest a role for p12(I) in th e activation of quiescent lymphocytes, a prerequisite for effective viral r eplication in vivo, To test the ability of p30(II) to function as a transcr iption factor we have constructed p30(II) as a Gal4-fusion protein, When tr ansfected with Gal4-driven luciferase reporter genes, the p30(II)-Gal4-fusi on protein induces transcriptional activity up to 50-fold in both 293 and H eLa-Tat cells. These systems will be useful to identify molecular mechanism s that explain the functional role of pX ORF I and ORF II-encoded proteins in HTLV-1 replication.