Alteration of zinc-binding residues of simian immunodeficiency virus p8(NC) results in subtle differences in gag processing and virion maturation associated with degradative loss of mutant NC

In all retroviruses analyzed to date (except for the spumaretroviruses), th e Zn2+-coordinating residues of nucleocapsid (NC) perform or assist in cruc ial reactions necessary to complete the retrovirus life cycle. Six replicat ion-defective mutations have been engineered in the two NC Zn2+ fingers (ZF s) of simian immunodeficiency virus [SIV(Mne)] that change or delete specif ic Zn2+-interacting Cys residues and were studied by using electron microsc opy, reversed-phase high-performance liquid chromatography, immunoblotting, and RNA quantification. We focused on phenotypes of produced particles, sp ecifically morphology, Gag polyprotein processing, and genomic RNA packagin g. Phenotypes were similar among viruses containing a point or deletion mut ation involving the same ZF. Mutations in the proximal ZF (ZF1) resulted in near-normal Gag processing and full-length genomic RNA incorporation and w ere most similar to wild-type (WT) virions with electron-dense, conical cor es. Mutation of the distal ZF, as well as point mutations in both ZFs, resu lted in more unprocessed Gag proteins than a deletion or point mutation in ZF1, with an approximate 30% reduction in levels of full-length genomic RNA in virions. These mutant virions contained condensed teres; however, the c ores typically appeared less electron dense and more rod shaped than WT vir ions. Surprisingly, deletion of both ZFs, including the basic linker region between the ZFs, resulted in the most efficient Gag processing. However, g enomic RNA packaging was similar to 10% of WT levels, and those particles p roduced were highly abnormal with respect to size and core morphology. Surp risingly, all NC mutations analyzed demonstrated a significant loss of proc essed NC in virus particles, suggesting that Zn2+-coordinated NC is protect ed from excessive proteolytic cleavage. Together, these results indicate th at Zn2+ coordination is important for correct Gag precursor processing and NC protein stability. Additionally, SIV particle morphology appears to be t he result of proper and complete Gag processing and relies less on full-len gth genomic RNA incorporation, as dictated by the Zn2+ coordination in the ZFs of the NC protein.