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 studied independently of the Rev and RRE (Rev-responsive element) regulatory system, over a single round. To accomplish this, theenvgene of an SIV vector was made defective by the insertion of a SV40 promoter/enhancerhygromycin B phosphotransferasegene 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 (HIV-1) Rev and RRE functions can be replacedin vitroby acis-acting sequence, constitutive transport element (CTE), from simian type D retroviruses. To determine whether such acis-acting element from Mason–Pfizer monkey virus (MPMV) would substitute for SIV Rev and RRE functions, the MPMV CTE was inserted either into the Nef ORF or at the junction ofvpxandvprof our Rev(−), RRE(−), and Rev(−)RRE(−) SIV molecular clones. Cell-free viral stocks harvested from Cos cells following transfections of these molecular clones revealed that these stocks were infectious over a single round of replication; however, their replication was attenuated 16-fold compared to that of wild-type virus. In addition, our experiments revealed that CTE functions in a position-dependent manner such that its insertion at the junction ofvpxandvprattenuated SIV replication 8- to 12-fold compared to the attenuation observed when it was inserted in thenefregion. Our results demonstrate that MPMV CTE is capable of substituting for SIV Rev and RRE functions, resulting in an attenuated ability to produce infectious virus.