DEVELOPMENT OF AN IN-VITRO MESSENGER-RNA DEGRADATION ASSAY UTILIZING EXTRACTS FROM HIV-1-INFECTED AND SIV-INFECTED CELLS

We previously demonstrated that cellular mRNAs are degraded in CD4 pos itive lymphocytes infected by the human immunodeficiency virus, HIV-1, but not in cells infected by the simian lentivirus, SIV. To begin to define the molecular mechanisms underlying this RNA degradation, we ha ve established an in vitro RNA degradation assay utilizing extracts fr om both infected and uninfected cells. We found that in vitro transcri bed, P-32-radiolabeled actin RNA was degraded in extracts prepared fro m CEM, CEMx174, and C8166 cells which were infected with HIV-1. Minima l actin RNA degradation was observed in extracts prepared from uninfec ted cells. Similarly little degradation was observed in cell-free extr acts prepared from SIV-infected cells. To determine if viral RNA seque nces could impart enhanced stability to cellular RNAs in our in vitro assay, we prepared radiolabeled RNAs that contained selected viral RNA determinants. One such RNA contained the HIV-1-specific TAR (transact ivating region) sequence (nucleotides 1-111) appended to a reporter CA T RNA. Like the cellular actin RNA, these TARCAT RNAs were degraded in HIV-1-infected cell extracts, but not in extracts from uninfected cel ls or extracts prepared from SIV-infected cells. In contrast, an RNA c ontaining only authentic HIV-1 sequences comprising TAR and gag sequen ces was more stable than actin RNA in HIV-1-infected extracts, These r esults, taken together, suggest that the in vitro assay reproduces eve nts that occur in vivo and provide a starting point for identifying th e factors responsible for cellular RNA degradation in HIV-1-infected c ells. (C) 1996 Academic Press, Inc.