DEFECTIVE HIV-1 PROVIRUS ENCODING A MULTITARGET-RIBOZYME INHIBITS ACCUMULATION OF SPLICED AND UNSPLICED HIV-1 MESSENGER-RNAS, REDUCES INFECTIVITY OF VIRAL PROGENY, AND PROTECTS THE CELLS FROM PATHOGENESIS

A HeLa T4 cell line containing a defective human immunodeficiency viru s type 1 (HIV-1) DNA (HD4) was isolated. After transactivation with Ta t, the HD4 DNA was transcribed into a single 3.7-kb mRNA that encodes a chimeric CD4/Env protein and a multitarget-ribozyme directed against multiple sites within the gp120 coding region of HIV-1 RNA (Chen et a l., 1992). Early steps in HIV infection such as entry, reverse transcr iption, and proviral DNA formation were not affected in HD4 cells, and HD4 was efficiently transactivated after either HIV-1 or HIV-2 infect ions. HIV-2, which lacks all of the HIV-1-specific ribozyme target sit es, replicated to high levels in HD4 cells whereas HIV-1 replication w as selectively inhibited. Despite a reduced accumulation of all HIV-1 transcripts, transactivation of HD4 was efficient. Surprisingly, the m ost abundant, multiply spliced mRNAs were reduced even though they lac k all of the ribozyme target sites. These results strongly suggest tha t the ribozyme co-localizes with unspliced HIV-1 pre-mRNA and/or genom ic HIV-1 RNA in the nucleus. Cleavage of these precursor RNAs explains the reduction of all spliced and unspliced HIV-1 RNAs. Cleavage of ge nomic RNA probably contributed to the three-fold reduction in the infe ctivity of viral progeny. Thus, the HD4 ribozyme RNA functioned as a r ibozyme in the nucleus and as a mRNA for a chimeric CD4/Env protein in the cytoplasm. Its unusual large size for a ribozyme (3.7 kb) indicat es that, in the future, other antiviral proteins, like negative transd ominant mutant HIV-1 proteins, may also be encoded to increase its ant iviral potential in a gene therapy approach.