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), the Zn 21 -coordinating residues of nucleocapsid (NC) perform or assist in crucial reactions necessary to complete the retrovirus life cycle. Six replication-defective mutations have been engineered in the two NC Zn 21 fingers (ZFs) of simian immunodeficiency virus [SIV(Mne)] that change or delete specific Zn 21 -interacting Cys residues and were studied by using electron microscopy, reversed-phase high-performance liquid chromatography, immunoblotting, and RNA quantification. We focused on phenotypes of produced particles, specifically morphology, Gag polyprotein processing, and genomic RNA packaging. Phenotypes were similar among viruses containing a point or deletion mutation involving the same ZF. Mutations in the proximal ZF (ZF1) resulted in near-normal Gag processing and full-length genomic RNA incorporation and were most similar to wild-type (WT) virions with electron-dense, conical cores. Mutation of the distal ZF, as well as point mutations in both ZFs, resulted 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 cores; however, the cores typically appeared less electron dense and more rod shaped than WT virions. Surprisingly, deletion of both ZFs, including the basic linker region between the ZFs, resulted in the most efficient Gag processing. However, genomic RNA packaging was ;10% of WT levels, and those particles produced were highly abnormal with respect to size and core morphology. Surprisingly, all NC mutations analyzed demonstrated a significant loss of processed NC in virus particles, suggesting that Zn 21 -coordinated NC is protected from excessive proteolytic cleavage. Together, these results indicate that Zn 21 coordination is important for correct Gag precursor processing and NC protein stability. Additionally, SIV particle morphology appears to be the result of proper and complete Gag processing and relies less on full-length genomic RNA incorporation, as dictated by the Zn 21 coordination in the ZFs of the NC protein. Retroviral nucleocapsid (NC) proteins are typically small basic proteins containing one or two conserved 14-residue peptide segments (-C-[X]2-C-[X]4-H-[X]4-C-) that bind zinc ions, forming structures referred to as zinc fingers (ZFs). NC proteins with native ZFs are required for viral replication and are apparently utilized at many stages of the replication cycle. The biological roles of retroviral NC proteins and their ZFs have been reviewed recently (18, 19) and include functions during viral assembly, maturation, and infection. Regarding particle morphology, the basic residues of NC are required for consistent and proper particle size (21), and the ZFs, when mutated or deleted, result in virions that do not mature properly (7). In fact, a number of the functions of NC are attributed to the ZFs. All human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) NC proteins contain two ZFs. It is currently believed that each ZF is required for viral replication; however, they are not necessarily equally required for each and every specific function during the replication cycle. In HIV-1, the proximal Zn 21 finger (ZF1) functions in