Site-Directed Mutagenesis of the P2 Region of the Rous Sarcoma Virus gag Gene: Effects on Gag Polyprotein Processing

Bowles, Neil E.; Bonnet, D; Mulhauser, Frank; Spahr, Pierre-François

doi:10.1006/viro.1994.1450

Cited by 10 publications

(8 citation statements)

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“…Since HIV-1 protease is activated during budding (44,45), this putative decrease might cause the assembling and budding Gag to be prematurely cleaved, resulting in poor or aberrant budding. This potential mechanism has been previously proposed for other assembly defects (4,8,11). In support of this, the decreased production of bovine leukemia virus-like particles by an L-domain mutant is exacerbated by the presence of active protease (70).…”

Section: Our Results Demonstrate That the Nc Region Of Pr55supporting

confidence: 66%

“…In support of this, the decreased production of bovine leukemia virus-like particles by an L-domain mutant is exacerbated by the presence of active protease (70). Furthermore, certain Rous sarcoma virus assembly and budding mutants with changes in p2b (the L-domain-containing region of its Gag) also can be complemented by inactivation of protease, especially those with decreased but not blocked assembly phenotypes (8,73). Also, deletions in integrase can cause a decrease in HIV-1 budding that can be rescued by protease inactivation (11).…”

Section: Our Results Demonstrate That the Nc Region Of Pr55mentioning

confidence: 93%

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Elimination of Protease Activity Restores Efficient Virion Production to a Human Immunodeficiency Virus Type 1 Nucleocapsid Deletion Mutant

Ott

Coren

Chertova

et al. 2003

J Virol

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The nucleocapsid (NC) region of human immunodeficiency virus type 1 (HIV-1) Gag is required for specific genomic RNA packaging. To determine if NC is absolutely required for virion formation, we deleted all but seven amino acids from NC in a full-length NL4-3 proviral clone. This construct, DelNC, produced approximately four-to sixfold fewer virions than did the wild type, and these virions were noninfectious (less than 10 ؊6 relative to the wild type) and severely genomic RNA deficient. Immunoblot and high-pressure liquid chromatography analyses showed that all of the mature Gag proteins except NC were present in the mutant virion preparations, although there was a modest decrease in Gag processing. DelNC virions had lower densities and were more heterogeneous than wild-type particles, consistent with a defect in the interaction assembly or I domain. Electron microscopy showed that the DelNC virions displayed a variety of aberrant morphological forms. Inactivating the protease activity of DelNC by mutation or protease inhibitor treatment restored virion production to wild-type levels. DelNC-protease mutants formed immature-appearing particles that were as dense as wild-type virions without incorporating genomic RNA. Therefore, protease activity combined with the absence of NC causes the defect in DelNC virion production, suggesting that premature processing of Gag during assembly causes this effect. These results show that HIV-1 can form particles efficiently without NC.

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Section: Our Results Demonstrate That the Nc Region Of Pr55supporting

confidence: 66%

Section: Our Results Demonstrate That the Nc Region Of Pr55mentioning

confidence: 93%

Elimination of Protease Activity Restores Efficient Virion Production to a Human Immunodeficiency Virus Type 1 Nucleocapsid Deletion Mutant

Ott

Coren

Chertova

et al. 2003

J Virol

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“…An additional protein that could impact budding kinetics is viral protease (PR). PR has been suggested to play a role in the budding process, though the mechanism is mostly unknown (15,16,39). Alternatively, the observed two budding kinetics may represent different Gag protein assembly pathways.…”

Section: Discussionmentioning

confidence: 99%

Directly Monitoring Individual Retrovirus Budding Events Using Atomic Force Microscopy

Gladnikoff

Rousso

2008

Biophysical Journal

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Retrovirus budding is a key step in the virus replication cycle. Nonetheless, very little is known about the underlying mechanism of budding, primarily due to technical limitations preventing visualization of bud formation in real time. Methods capable of monitoring budding dynamics suffer from insufficient resolution, whereas other methods, such as electron microscopy, do not have the ability to operate under physiological conditions. Here we applied atomic force microscopy to real-time visualization of individual Moloney murine leukemia virus budding events. By using a single-particle analysis approach, we were able to observe distinct patterns in budding that otherwise remain transparent. We find that bud formation follows at least two kinetically distinct pathways. The majority of virions (74%) are produced in a slow process (>45 min), and the remaining particles (26%) assemble via a fast process (<25 min). Interestingly, repetitive budding from the same site was seen to occur in only two locations. This finding challenges the hypothesis that viral budding occurs from distinct sites and suggests that budding is not restricted laterally. In this study, we established a method to monitor the fine dynamics of the virus budding process. Using this single-particle analysis to study mutated viruses will enable us to gain additional insight into the mechanisms of viral budding.

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“…How the mutants may affect protease activity during virion maturation is not clear. Analysis of RSV mutants with alterations in p2, particularly the polyproline stretch, led to the suggestion that p2 might play a role in controlling protease activation directly (Bowles et al ., 1994). However, the primary effect of the PPPY mutations might be on assembly; in both RSV and M‐PMV, mutations in the L‐domain could still block virion budding even when the viral protease was inactivated (Wills et al ., 1994; Yasuda and Hunter, 1998).…”

Section: Discussionmentioning

confidence: 99%

Mutations altering the Moloney murine leukemia virus p12 Gag protein affect virion production and early events of the virus life cycle

1999

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The p12 Gag protein of Moloney murine leukemia virus is a small polypeptide of unknown function, containing two proline-rich motifs. To determine its role in replication, we introduced a series of deletion and alanine-scanning substitution mutations throughout the p12 coding region of a proviral DNA, and characterized the phenotypes of the resulting mutant viruses. Complete deletion of p12 and mutations affecting the PPPY motif caused substantial reduction in the yield of virions and a modest reduction in Gag processing. Proteolytic cleavage of the R-peptide from the cytoplasmic tail of the envelope protein TM was abolished in these mutants, suggesting that the PPPY motif is crucial for the viral protease to access the TM tail. The resulting virions were non-infectious, and unable to initiate DNA synthesis in infected cells. Mutants with alterations in both the N-and C-terminal portions of p12 exhibited a distinct phenotype. The production of virions and processing of Gag, Pol and Env precursors were normal. The viruses were able to direct synthesis of linear viral DNA, but there was almost no detectable circular DNAs or LTR-LTR junction. These data suggest that p12 plays a critical role in the early events of the virus life cycle.

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Site-Directed Mutagenesis of the P2 Region of the Rous Sarcoma Virus gag Gene: Effects on Gag Polyprotein Processing

Cited by 10 publications

References 0 publications

Elimination of Protease Activity Restores Efficient Virion Production to a Human Immunodeficiency Virus Type 1 Nucleocapsid Deletion Mutant

Elimination of Protease Activity Restores Efficient Virion Production to a Human Immunodeficiency Virus Type 1 Nucleocapsid Deletion Mutant

Directly Monitoring Individual Retrovirus Budding Events Using Atomic Force Microscopy

Mutations altering the Moloney murine leukemia virus p12 Gag protein affect virion production and early events of the virus life cycle

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