Role of the constitutive splicing factors U2AF65 and SAP49 in suboptimal RNA splicing of novel retroviral mutants

Retroviruses display a unique form of alternative splicing in which both spliced and unspliced RNAs accumulate in the cytoplasm, Simple retroviruses, such as avian sarcoma virus, do not encode regulatory proteins that affect splicing; this process is controlled solely through interactions between the viral RNA and the host cell splicing machinery. Previously, we described the selection and characterization of novel avian sarcoma virus mutants. These viruses were separated into two classes based upon analysis of splicing intermediates produced in infected cells and in a cell-free system. One class, which included mutants with altered polypyrimidine tract or branch point sequences, showed significant accumulation of intermediates, suggesting that splicing was regulated in step 2, The other class, which included mutants with deletions of exonic enhancer sequences, did not accumulate splicing intermediates, suggesting that splicing was regulated before step 1 of the splicing reaction. In this report, we show that a mutant blocked at step 1 fails to form a stable spliceosomal complex, whereas one blocked at step 2 shows a defect in its ability to transit through the last spliceosomal complex. Using UV cross-linking methods, we show that regulation at each step is associated with specific changes in the binding of cellular splicing factors. Regulation at step 1 is correlated with decreased crosslinking of the factor U2AF(65), whereas regulation at step 2 is correlated with enhanced cross-linking of the factor SAP49, Because these mutations were isolated by selection for replication-competent viruses, we conclude that retroviral splicing may be regulated in vivo through altered binding of constitutive splicing factors.