Deletion of a Cys–His motif from the Alpharetrovirus nucleocapsid domain reveals late domain mutant-like budding defects

The Rous sarcoma virus (RSV) Gag polyprotein is the only protein required for virus assembly and release. We previously found that deletion of either one of the two Cys–His (CH) motifs in the RSV nucleocapsid (NC) protein did not abrogate Gag–Gag interactions, RNA binding, or packaging but greatly reduced virus production (E-G. Lee, A. Alidina et al., J. Virol. 77: 2010–2020, 2003). In this report, we have further investigated the effects of mutations in the CH motifs on virus assembly and release. Precise deletion of either CH motif, without affecting surrounding basic residues, reduced virus production by approximately 10-fold, similar to levels seen for late (L) domain mutants. Strikingly, transmission electron microscopy revealed that virions of both ΔCH1 and ΔCH2 mutants were assembled normally at the plasma membrane but were arrested in budding. Virus particles remained tethered to the membrane or to each other, reminiscent of L domain mutants, although the release defect appears to be independent of the L domain functions. Therefore, two CH motifs are likely to be required for budding independent of a requirement for either Gag–Gag interactions or RNA packaging.