ROLE OF MASON-PFIZER MONKEY VIRUS (MPMV) CONSTITUTIVE TRANSPORT ELEMENT (CTE) IN THE PROPAGATION OF MPMV VECTORS BY GENETIC COMPLEMENTATIONUSING HOMOLOGOUS HETEROLOGOUS ENV GENES/

To study Mason-Pfizer monkey virus (MPMV) replication over a single ro und, virus particles were generated that contain a replication-defecti ve vector encoding a dominant selectable marker, the hygromycin B phos photransferase (hyg') gene. Genetic complementation with a homologous MPMV envelope glycoprotein (Env-gp) or pseudotyping by several heterol ogous Env-gps from a variety of viruses resulted in infectious MPMV pa rticles containing the replication-defective RNA. Recently, it has bee n shown that human immunodeficiency virus type 1 (HIV-1) and simian im munodeficiency virus (SIV) Rev and Rev-responsive element (RRE) functi ons can be substituted in vitro by a cis-acting sequence, the constitu tive transport element (CTE), from simian type D retroviruses like MPM V and simian retrovirus type 1 (SRV-1). To determine whether CTE of MP MV is necessary for MPMV nucleic acid propagation, an MPMV vector that lacked the terminally located CTE was generated. Propagation of this vector was completely abrogated in the absence of CTE, showing the imp ortance of CTE in MPMV replication. Insertion of CTE back into the MPM V genome in the sense orientation rescued replication to wild-type lev els. Slot-blot analysis of nuclear versus cytoplasmic RNA fractions re vealed that most of the messages were sequestered in the nucleus of ce lls transfected with the CTE(-) vectors and very little was transporte d to the cytoplasm. To test whether HIV-1 or SIV RREs could complement CTE function, the HIV-1 or SIV RREs were inserted in the CTE(-) vecto rs. trans complementation of CTE(-)RRE(+) vectors with Env- and Rev- e xpression plasmids rescued propagation of the CTE(-) vectors. Computer analysis predicted an RNA secondary structure in MPMV CTE analogous t o the HIV-1 and SIV RREs that could form three stable stem loops, the first of which contains a site similar to the Rev-binding domain in th e HIV-1 RRE. The presence of a higher-order CTE structure was analyzed by mutational analysis. We conclude that CTE is important in the repl ication of MPMV and affects the nucleocytoplasmic transport and/or sta bility of viral messages similar to the Rev/RRE regulatory system of H IV-1 and SIV. (C) 1996 Academic Press, Inc.