Dataset Information

In vivo selection of Rous sarcoma virus mutants with randomized sequences in the packaging signal.

ABSTRACT: Retrovirus genomes contain a sequence at the 5' end which directs their packaging into virions. In Rous sarcoma virus, previous studies have identified important segments of the packaging signal, Psi, and support elements of a secondary-structure prediction. To further characterize this sequence, we used an in vivo selection strategy to test large collections of mutants. We generated pools of full-length viral DNA molecules with short stretches of random sequence in Psi and transfected each pool into avian cells. Resulting infectious virus was allowed to spread by multiple passages, so that sequences could compete and the best could be selected. This method provides information on the kinds of sequences allowed, as well as those that are most fit. Several predicted stem-loop structures in Psi were tested. A stem at the base of element O3 was highly favored; only sequences which maintained base pairing were selected. Two other stems, at the base and in the middle of element L3, were not conserved: neither base pairing nor sequence was maintained. A single mutation, G213U, was seen upstream of the randomized region in all selected L3 stem mutants; we interpret this to mean that it compensates for the defects in L3. Randomized mutations adjacent to G213 maintained the wild-type base composition but not its sequence. The kissing-loop sequence at end of L3, postulated to function in genome dimerization, was not required for infectivity but was selected for over time. Finally, a deletion of L3 was constructed and found to be poorly infectious.

SUBMITTER: Doria-Rose NA

PROVIDER: S-EPMC110144 | biostudies-literature | 1998 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

In vivo selection of Rous sarcoma virus mutants with randomized sequences in the packaging signal.

Doria-Rose N A NA Vogt V M VM

Journal of virology 19981001 10

Retrovirus genomes contain a sequence at the 5' end which directs their packaging into virions. In Rous sarcoma virus, previous studies have identified important segments of the packaging signal, Psi, and support elements of a secondary-structure prediction. To further characterize this sequence, we used an in vivo selection strategy to test large collections of mutants. We generated pools of full-length viral DNA molecules with short stretches of random sequence in Psi and transfected each pool ...[more]

PMID: 9733847

Similar Datasets

Project description:UnlabelledPurified retroviral Gag proteins can assemble in vitro to form immature virus-like particles (VLPs). By cryoelectron tomography, Rous sarcoma virus VLPs show an organized hexameric lattice consisting chiefly of the capsid (CA) domain, with periodic stalk-like densities below the lattice. We hypothesize that the structure represented by these densities is formed by amino acid residues immediately downstream of the folded CA, namely, the short spacer peptide SP, along with a dozen flanking residues. These 24 residues comprise the SP assembly (SPA) domain, and we propose that neighboring SPA units in a Gag hexamer coalesce to form a six-helix bundle. Using in vitro assembly, alanine scanning mutagenesis, and biophysical analyses, we have further characterized the structure and function of SPA. Most of the amino acid residues in SPA could not be mutated individually without abrogating assembly, with the exception of a few residues near the N and C termini, as well as three hydrophilic residues within SPA. We interpret these results to mean that the amino acids that do not tolerate mutations contribute to higher-order structures in VLPs. Hydrogen-deuterium exchange analyses of unassembled Gag compared that of assembled VLPs showed strong protection at the SPA region, consistent with a higher-order structure. Circular dichroism revealed that a 29mer SPA peptide shifts from a random coil to a helix in a concentration-dependent manner. Analytical ultracentrifugation showed concentration-dependent self-association of the peptide into a hexamer. Taken together, these results provide strong evidence for the formation of a critical six-helix bundle in Gag assembly.ImportanceThe structure of a retrovirus like HIV is created by several thousand molecules of the viral Gag protein, which assemble to form the known hexagonal protein lattice in the virus particle. How the Gag proteins pack together in the lattice is incompletely understood. A short segment of Gag known to be critical for proper assembly has been hypothesized to form a six-helix bundle, which may be the nucleating event that leads to lattice formation. The experiments reported here, using the avian Rous sarcoma virus as a model system, further define the nature of this segment of Gag, show that it is in a higher-order structure in the virus particle, and provide the first direct evidence that it forms a six-helix bundle in retrovirus assembly. Such knowledge may provide underpinnings for the development of antiretroviral drugs that interfere with virus assembly.

Dataset Information

In vivo selection of Rous sarcoma virus mutants with randomized sequences in the packaging signal.

Publications

In vivo selection of Rous sarcoma virus mutants with randomized sequences in the packaging signal.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets