Identification of a cis-acting element in human immunodeficiency virus type 2 (HIV-2) that is responsive to the HIV-1 rev and human T-cell leukemia virus types I and II rex proteins

Abstract

A simian virus 40 late replacement vector encoding human immunodeficiency virus type 1 (HIV-1) gp120 (pGP120) was used to define a region within the HIV-2 genome that could work as a rev-responsive element (RRE). Our previous work showed that gp120 expression in this system required a functional RRE in cis and required the rev protein in trans (M.-L. Hammarskjöld, J. Heimer, B. Hammarskjöld, I. Sangwan, L. Albert, and D. Rekosh, J. Virol. 63:1959-1966, 1989). Using pGP120, we first mapped an RRE to a 1,042-base-pair (bp) Sau3a fragment in the env region of HIV-2. Both HIV-1 rev (rev1) and HIV-2 rev (rev2) could work in conjunction with this fragment. Further mapping showed that a 272-bp subfragment within the 1,042-bp region was sufficient as an RRE. Surprisingly, the smaller fragment worked only with the rev1 protein and not with its homologous rev2 protein. In addition, the rev2 protein failed to function together with the RRE from HIV-1. We also utilized this system to examine the ability of the rex genes of human T-cell leukemia virus types I and II to functionally substitute for rev. These experiments showed that complementation by both the rexI and rexII proteins required the presence of an RRE. The rex proteins worked well in conjunction with either the HIV-1 or the HIV-2 RRE (the 1,042-bp as well as the 272-bp fragment).

MPER-specific antibodies induce gp120 shedding and irreversibly neutralize HIV-1

“…Detection of gp120 shedding by Western blot analysis. 293T cells were transfected in 24-well plates with CT JR-FL gp160, a cytosolic tail truncated gp160 construct known to yield higher envelope trimer expression (Binley et al, 2003), and pCMV-rev (Lewis et al, 1990) using jetPEI (Polyplus Transfection) according to the manufacturer's instruction. As mock control, empty pcDNA3.1 vector was cotransfected together with pCMV-rev under identical conditions.…”

Human T-cell leukemia virus type 2 Rex inhibits pre-mRNA splicing in vitro at an early stage of spliceosome formation

“…Investigators had previously noted Rex effects on nuclear-to-cytoplasmic transport mediated through the RxRE in the reiterated 3Ј LTR (15). The reported ability of HTLV-1/2 Rex to act as a substitute for HIV-1 Rev in Rev-deficient HIV-1 clones could conceivably be due to effects on splicing and/or transport, although we tend to favor the latter because of the distance between the HIV-1 splice donor and Rev binding sequences (18,22,28). Transport effects alone would not explain differential expression of HTLV-2 gag, env, and tax/rex mRNAs, all of which contain the reiterated 3Ј RxRE.…”

Production of Lentiviral Vectors in Protein‐free Media

“…no. 15140-122) Plasmids available from Addgene (www.addgene.org) or from the Reiser laboratory (Jakob.Reiser@fda.hhs.gov): pNL-EGFP/CMV/WPRE U3 (second-generation lentiviral transgene plasmid); Addgene plasmid 17579 pCD/NL-BH* (second-generation lentiviral packaging plasmid); Addgene plasmid 17531 pLTR-G [second-generation vesicular stomatitis virus glycoprotein (VSV-G)-encoding plasmid]; Addgene plasmid 17532 pNL(CMV)EGFP/CMV/PRE U3.1 (third-generation lentiviral transgene plasmid) pCD/NL-BH 1 (third-generation lentiviral packaging plasmid) pCMV-rev (HIV-1 Rev-encoding plasmid) (Lewis et al, 1990) pCEF-VSV-G (VSV-G glycoprotein-encoding plasmid) 5 M NaCl (…”