REV RRE-INDEPENDENT MASON-PFIZER MONKEY VIRUS CONSTITUTIVE TRANSPORT ELEMENT-DEPENDENT PROPAGATION OF SIVMAC239 VECTORS USING A SINGLE ROUND OF REPLICATION ASSAY/

In a step toward creating live-attenuated or DNA subunit vaccines for AIDS, the replication of simian immunodeficiency virus (SIV) was studi ed independently of the Rev and RRE (Rev-responsive element) regulator y system, over a single round. To accomplish this, the env gene of an SIV vector was made defective by the insertion of a SV40 promoter/enha ncer hygromycin B phosphotransferase gene cassette. Using this vector as the backbone, molecular clones of SIV were generated that contained a mutated Rev, Rev(-), a deleted RRE, RRE(-), or both, Rev(-)RRE(-). It has been shown recently that human immunodeficiency virus type 1 (H IV-I) Rev and RRE functions can be replaced in vitro by a cis-acting s equence, constitutive transport element (CTE), from simian type D retr oviruses. To determine whether such a cis-acting element from Mason-Pf izer monkey virus (MPMV) would substitute for SIV Rev and RRE function s, the MPMV CTE was inserted either into the Nef ORF or at the junctio n of vpx and vpr of our Rev(-), RRE(-), and Rev(-)RRE(-) SIV molecular clones. Cell-free viral stocks harvested from Cos cells following tra nsfections of these molecular clones revealed that these stocks were i nfectious over a single round of replication; however, their replicati on was attenuated 18-fold compared to that of wild-type virus. In addi tion, our experiments revealed that CTE functions in a position-depend ent manner such that its insertion at the junction of vpx and vpr atte nuated SIV replication 8- to 12-fold compared to the attenuation obser ved when it was inserted in the nef region. Our results demonstrate th at MPMV CTE is capable of substituting for SIV Rev and RRE functions, resulting in an attenuated ability to produce infectious virus. (C) 19 96 Academic Press, Inc.