Viral DNA synthesis defects in assembly-competent Rous sarcoma virus CA mutants

The major structural protein of the retroviral core (CA) contains a conserv ed sequence motif shared with the CA-like proteins of distantly related tra nsposable elements. The function of this major region of homology (MHR) has not been defined, in part due to the baffling array of phenotypes in mutan ts of several viruses and the yeast TY3. This report describes new mutation s in the CA protein of Rous sarcoma virus (RSV) that were designed to test whether these different phenotypes might indicate distinct functional subdo mains in the MHR. A comparison of 25 substitutions at 10 positions in the R SV conserved motif argues against this possibility. Most of the replacement s destroyed virus infectivity, although either of two lethal phenotypes was obtained depending on the residue introduced. At most of the positions, on e or more replacements (generally the more conservative substitutions) caus ed a severe replication defect without having any obvious effects on virus assembly, budding, Gag-Pol and genome incorporation, or protein processing. The mutant particles exhibited a defect in endogenous viral DNA synthesis and showed increased sensitivity of the core proteins to detergent, indicat ing that the mutations interfere with the formation and/or activity of the virion core. The distribution of these mutations across the MHR, with no ev idence of clustering, suggests that the entire region is important for a cr itical postbudding function. In contrast, a second class of lethal substitu tions (those that destroyed virus assembly and release) consists of alterat ions that are expected to cause severe effects on protein structure by disr uption either of the hydrophobic core of the CA carboxyl-terminal domain or of the hydrogen bond network that stabilizes the domain. We suggest that t his duality of phenotypes is consistent with a role for the MHR in the matu ration process that links the two parts of the life cycle.