Viral DNA Synthesis Defects in Assembly-Competent Rous Sarcoma Virus CA Mutants

Abstract

The major structural protein of the retroviral core (CA) contains a conserved sequence motif shared with the CA-like proteins of distantly related transposable elements. The function of this major region of homology (MHR) has not been defined, in part due to the baffling array of phenotypes in mutants of several viruses and the yeast TY3. This report describes new mutations in the CA protein of Rous sarcoma virus (RSV) that were designed to test whether these different phenotypes might indicate distinct functional subdomains in the MHR. A comparison of 25 substitutions at 10 positions in the RSV conserved motif argues against this possibility. Most of the replacements destroyed virus infectivity, although either of two lethal phenotypes was obtained depending on the residue introduced. At most of the positions, one or more replacements (generally the more conservative substitutions) caused 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, indicating that the mutations interfere with the formation and/or activity of the virion core. The distribution of these mutations across the MHR, with no evidence of clustering, suggests that the entire region is important for a critical postbudding function. In contrast, a second class of lethal substitutions (those that destroyed virus assembly and release) consists of alterations that are expected to cause severe effects on protein structure by disruption either of the hydrophobic core of the CA carboxyl-terminal domain or of the hydrogen bond network that stabilizes the domain. We suggest that this duality of phenotypes is consistent with a role for the MHR in the maturation process that links the two parts of the life cycle.

Element-specific localization of Drosophila retrotransposon Gag proteins occurs in both nucleus and cytoplasm

“…Studies of mutations in the sequence implicate it in many activities in viral assembly as well as infection of new hosts (22)(23)(24)(25). This motif also has been found in the Saccharomyces cerevisiae retrotransposon, Ty3.…”

Formation of a Human Immunodeficiency Virus Type 1 Core of Optimal Stability Is Crucial for Viral Replication

“…Similar studies have recently been performed on the CA protein of RSV (3,7). Several mutations in the major homology region of RSV resulted in efficient assembly of virions that were blocked for reverse transcription in target cells and exhibited core stability defects (7).…”

“…Several mutations in the major homology region of RSV resulted in efficient assembly of virions that were blocked for reverse transcription in target cells and exhibited core stability defects (7). Subsequent analysis of CA mutant pseudorevertants revealed restoration of wild-type replication kinetics (3).…”

“…The endogenous reaction has been used in studies of retroviral minus-and plus-strand DNA synthesis and to identify defects in Vif-defective virions (19) and an MA point mutant (25,26). Furthermore, this assay has been used to reveal defects in the reverse transcription machinery of Rous sarcoma virus (RSV) CA mutants (7).…”

Second-Site Suppressors of Rous Sarcoma Virus CA Mutations: Evidence for Interdomain Interactions

“…The most well studied of these mutants have substitutions in a highly conserved part of CA named the major homology region (MHR) (7, 25, 32, 34a). For RSV, biochemical analyses of these mutants have shown that the released particles are normal in size and density and contain normal amounts of the envelope glycoproteins, genomic RNA, and tRNA primer but exhibit a defect in reverse transcription (4,7). These data suggest that CA has an important postassembly function that is needed for the establishment of infection once the particle enters a new cell.…”