Solution structure and dynamics of the bioactive retroviral M domain from Rous sarcoma virus.

A biologically active construct of the retroviral M domain from the avian Rous sarcoma virus is defined and its solution structure described. This M domain is fully active in budding and infectivity without myristylation. In spite of a sequence homology level that suggests no relationship among M domains and the family of matrix proteins in mammalian retroviruses, the conserved structural elements of a central core allow an M domain sequence motif to be described for all retroviruses. The surface of the M domain has a highly clustered positive patch comprised of sequentially distant residues. An analysis of the backbone dynamics, incorporating rotational anisotropy, is used to estimate the thermodynamics of proposed domain oligomerization.

A large region within the Rous sarcoma virus matrix protein is dispensable for budding and infectivity.

All retroviruses have a layer of matrix protein (MA) situated directly beneath the lipid of their envelope. This protein is initially expressed as the amino-terminal sequence of the Gag polyprotein, where it plays an important role in binding Gag to the plasma membrane during the early steps of the budding process. Others have suggested that MA may provide additional functions during virion assembly, including the selective incorporation of viral glycoproteins and the RNA genome into the emerging virion. To further study the role of the Rous sarcoma virus MA sequence in the viral replication cycle, we have pursued an extensive deletion analysis. Surprisingly, the entire second half of MA (residues 87 to 155) and part of the neighboring p2 sequence were found to be dispensable not only for budding but also for infectivity in avian cells. Thus, all of the functions associated with the Rous sarcoma virus MA sequence must be contained within its first half.

Functional chimeras of the Rous sarcoma virus and human immunodeficiency virus gag proteins.

The Gag protein encoded by Rous sarcoma virus (RSV) is the only viral product required for the process of budding whereby virus particles are formed at the plasma membrane. Deletion analysis of this Gag molecule has revealed several regions (assembly domains) that are important for budding. One of these domains is located at the amino terminus and is needed for membrane binding. Another is located within the carboxy-terminal third of the protein. Though there is little sequence homology among the Gag proteins of unrelated retroviruses, it seemed possible that their assembly domains might be functionally conserved, and to explore this idea, numerous Gag chimeras were made. The results indicate that the first 10 amino acids of the human immunodeficiency virus (HIV) Gag protein can suppress the block to budding caused by deletions in the RSV MA sequence, much as described previously for the first 10 residues from the Src oncoprotein (J.W. Wills, R.C. Craven, R. A. Weldon, Jr., T. D. Nelle, and C.R. Erdie, J. Virol. 65:3804-3812, 1991). In addition, the carboxy-terminal half of the HIV Gag protein was fused to a truncated RSV Gag molecule, mutant Bg-Bs, which is unable to direct core assembly. This chimera was able to produce particles at a rate identical to that of RSV and of a density similar to that of authentic virions. Deletion analysis of the carboxy-terminal chimera revealed two small regions within the HIV NC protein that were sufficient for endowing mutant Bg-Bs with these properties. Chimeras lacking both regions produced particles of a low density, suggesting that these sequences may be involved in the tight packing of Gag molecules during assembly. In a related set of experiments, replacement of the RSV protease with that of HIV resulted in premature processing within the RSV sequence and a block to budding. Particle assembly was restored when the HIV PR activity was inactivated by mutagenesis. Collectively, the data presented here illustrate the functional similarities of Gag proteins from unrelated retroviruses.

The membrane-binding domain of the Rous sarcoma virus Gag protein.

The Gag protein of Rous sarcoma virus (RSV) can direct particle assembly and budding at the plasma membrane independently of the other virus-encoded products. A previous deletion analysis has suggested that the first 86 amino acids of RSV Gag constitute a large membrane-binding domain that is absolutely required for these processes. To test this hypothesis, we inserted these residues in place of the N-terminal membrane-binding domain of the pp60v-src, a transforming protein whose biological activity requires plasma membrane localization. The ability of the Src chimera to induce cellular transformation suggests that the RSV sequence indeed contains an independent, functional domain.

The major site of phosphorylation within the Rous sarcoma virus MA protein is not required for replication.

About one-third of the MA protein in Rous sarcoma virus (RSV) is phosphorylated. Previous analyses of this fraction have suggested that serine residues 68 and 106 are the major sites of phosphorylation. As a follow-up to that study, we have characterized mutants which have these putative phosphorylation sites changed to alanine, either separately or together. None of the substitutions (S68A, S106A, or S68/106A) had an effect on the budding efficiency or infectivity of the virus. Upon examination of the 32P-labeled viral proteins, we found that the S68A substitution did not affect phosphorylation in vivo at all. In contrast, the S106A substitution prevented all detectable phosphorylation of MA, suggesting that there is only one major site of phosphorylation in MA. We also found that the RSV MA protein is phosphorylated on tyrosine, but the amount was low and detectable only with large numbers of virions and an antibody specific for phosphotyrosine.

Suppression of retroviral MA deletions by the amino-terminal membrane-binding domain of p60src.

The molecular mechanism by which retroviral Gag proteins are directed to the plasma membrane for the formation of particles (budding) is unknown, but it is widely believed that the MA domain, located at the amino terminus, plays a critical role. Consistent with this idea, we found that small deletions in this segment of the Rous sarcoma virus Gag protein completely blocked particle formation. The mutant proteins appear to have suffered only localized structural damage since they could be rescued (i.e., packaged into particles) when coexpressed with Gag proteins that are competent for particle formation. To our surprise, the effects of the MA deletions could be completely suppressed by fusing as few as seven residues of the myristylated amino terminus of the oncoprotein p60src to the beginning of the mutant Gag proteins. Particles produced by the chimeras were of the same density as the wild type. Two myristylated peptides having sequences distinct from that of p60src were entirely unable to suppress MA deletions, indicating that myristate alone is not a sufficient membrane targeting signal. We hypothesize that the amino terminus of p60src suppresses the effects of MA deletions by diverting the Rous sarcoma virus Gag protein from its normal site of assembly to the Src receptor for particle formation.

Positionally independent and exchangeable late budding functions of the Rous sarcoma virus and human immunodeficiency virus Gag proteins.

The Gag proteins of Rous sarcoma virus and human immunodeficiency virus (HIV) each contain a function involved in a late step in budding, defects in which result in the accumulation of these molecules at the plasma membrane. In the Rous sarcoma virus Gag protein (Pr76gag), this assembly domain is associated with a PPPY motif, which is located at an internal position between the MA and CA sequences. This motif is not contained anywhere within the HIV Gag protein (Pr55gag), and the MA sequence is linked directly to CA. Instead, a late assembly function of HIV has been associated with the p6 sequence situated at the C terminus of Gag. Here we demonstrate the remarkable finding that the late assembly domains from these two unrelated Gag proteins are exchangeable between retroviruses and can function in a positionally independent manner.