Altered Rous sarcoma virus Gag polyprotein processing and its effects on particle formation.
ABSTRACT Proteolytic processing of the Rous sarcoma virus (RSV) Gag precursor was altered in vivo through the introduction of amino acid substitutions into either the polyprotein cleavage junctions or the PR coding sequence. Single amino acid substitutions (V(P2)S and P(P4)G), which are predicted from in vitro peptide substrate cleavage data to decrease the rate of release of PR from the Gag polyprotein, were placed in the NC portion of the NC-PR junction. These substitutions do not affect the efficiency of release of virus-like particles from COS cells even though recovered particles contain significant amounts of uncleaved Pr76gag in addition to mature viral proteins. Single amino acid substitutions (A(P3)F and S(P1)Y), which increase the rate of PR release from Gag, also do not affect budding of virus-like particles from cells. Substitution of the inefficiently cleaved MA-p2 junction sequence in Gag by eight amino acids from the rapidly cleaved NC-PR sequence resulted in a significant increase in cleavage at the new MA-p2 junction, but again without an effect on budding. However, decreased budding was observed when the A(P3)F or S(P1)Y substitution was included in the NC-PR junction sequence between the MA and p2 proteins. A budding defect was also caused by substitution into Gag of a PR subunit containing three amino acid substitutions (R105P, G106V, and S107N) in the substrate binding pocket that increase the catalytic activity of PR. The defect appears to be the result of premature proteolytic processing that could be rescued by inactivating PR through substitution of a serine for the catalytic aspartic acid residue. This budding defect was also rescued by single amino acid substitutions in the NC-PR cleavage site which decrease the rate of release of PR from Gag. A similar budding defect was caused by replacing the Gag PR with two PR subunits covalently linked by four glycine residues. In contrast to the defect caused by the triply substituted PR, the budding defect observed with the linked PR dimer could not be rescued by NC-PR cleavage site mutations, suggesting that PR dimerization is a limiting step in the maturation process. Overall, these results are consistent with a model in which viral protein maturation occurs after PR subunits are released from the Gag polyprotein.
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ABSTRACT: The retroviral Gag protein plays the central role in the assembly process and can form membrane-enclosed, virus-like particles in the absence of any other viral products. These particles are similar to authentic virions in density and size. Three small domains of the human immunodeficiency virus type 1 (HIV-1) Gag protein have been previously identified as being important for budding. Regions that lie outside these domains can be deleted without any effect on particle release or density. However, the regions of Gag that control the size of HIV-1 particles are less well understood. In the case of Rous sarcoma virus (RSV), the size determinant maps to the CA (capsid) and adjacent spacer sequences within Gag, but systematic mapping of the HIV Gag protein has not been reported. To locate the size determinants of HIV-1, we analyzed a large collection of Gag mutants. To our surprise, all mutants with defects in the MA (matrix), CA, and the N-terminal part of NC (nucleocapsid) sequences produced dense particles of normal size, suggesting that oncoviruses (RSV) and lentiviruses (HIV-1) have different size-controlling elements. The most important region found to be critical for determining HIV-1 particle size is the p6 sequence. Particles lacking all or small parts of p6 were uniform in size distribution but very large as measured by rate zonal gradients. Further evidence for this novel function of p6 was obtained by placing this sequence at the C terminus of RSV CA mutants that produce heterogeneously sized particles. We found that the RSV-p6 chimeras produced normally sized particles. Thus, we present evidence that the entire p6 sequence plays a role in determining the size of a retroviral particle.Journal of Virology 06/1998; 72(6):4667-77. · 4.65 Impact Factor
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ABSTRACT: Two types of Rous sarcoma virus (RSV) group-antigen protein (Gag) virus like particles (VLPs), full-length Gag (Gag701) and RSV protease domain (PR)-deleted mutant (Gag577) were expressed in silkworm larvae. Gag577 was secreted into hemolymph efficiently using wild type bacmid (WT), cysteine protease-deficient bacmid (CP(-)), cysteine protease and chitinase-deficient bacmid (CP(-)Chi(-)) bacmids, but comparatively Gag701 secretion levels were low. VLPs were purified on 10-60% (v/v) sucrose density gradient by ultracentrifugation and their structures confirmed under electron microscope. When hPRR and RSV Gag577 were co-expressed in silkworm larvae, human prorenin receptor (hPRR) was displayed on the surface of RSV VLPs, which was detected by Western blotting and immunoelectron microscopy. Moreover, binding of hPRR localized on the surface of VLPs to human prorenin was confirmed by ELISA. These results indicate that active hPRR was displayed on the surface of RSV VLPs, which can be utilized for drug discovery of hPRR blockers to prevent nephropathy. Moreover, this transmembrane protein display system using RSV Gag in silkworm larvae is applicable to expression of intact transmembrane proteins and binding assay of transmembrane proteins to its ligands, especially for transmembrane proteins which cannot be purified from membrane fractions in active states.Journal of Biotechnology 09/2011; 155(2):185-92. DOI:10.1016/j.jbiotec.2011.07.008 · 2.88 Impact Factor
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ABSTRACT: Proteolytic processing of viral polyproteins is essential for retrovirus infectivity. Retroviral proteases (PR) become activated during or after assembly of the immature, non-infectious virion. They cleave viral polyproteins at specific sites, inducing major structural rearrangements termed maturation. Maturation converts retroviral enzymes into their functional form, transforms the immature shell into a metastable state primed for early replication events, and enhances viral entry competence. Not only cleavage at all PR recognition sites, but also an ordered sequence of cleavages is crucial. Proteolysis is tightly regulated, but the triggering mechanisms and kinetics and pathway of morphological transitions remain enigmatic. Here, we outline PR structures and substrate specificities focusing on HIV PR as a therapeutic target. We discuss design and clinical success of HIV PR inhibitors, as well as resistance development towards these drugs. Finally, we summarize data elucidating the role of proteolysis in maturation and highlight unsolved questions regarding retroviral maturation. Copyright © 2015 Elsevier Inc. All rights reserved.Virology 03/2015; 479-480. DOI:10.1016/j.virol.2015.03.021 · 3.28 Impact Factor