Efficiency and selectivity of RNA packaging by Rous sarcoma virus Gag deletion mutants

Self Cite

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.

Section: Discussionmentioning

confidence: 99%

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

Nelle

Wills

1996

Self Cite

“…Because the amount of Gag protein in virus samples was slightly higher for HIVgptA14-15 than for HIVgpt ( Fig. 1B), it appears that HIVgptA14-15 reduced both the specificity of viral RNA packaging (as determined by the reduced ratio of genomic RNA versus spliced RNA [23]) and the amount of viral RNA encapsidated (as determined by the ratio of viral RNA to Gag protein).…”

Section: Rna Encapsidation Into Wt and Nc Mutant Hiv Particlesmentioning

confidence: 97%

“…The specificity with which retroviral full-length genomic RNAs are encapsidated suggests that specific sequences are required for RNA selection. These cis-acting sequences, called packaging (Psi), or encapsidation, signals, have been identified in avian, murine, and primate retroviruses (13,14,17,19,23). For Moloney murine leukemia virus (M-MuLV) and human immunodeficiency virus (HIV), packaging signals are located between splice sites and therefore are not contained in the subgenomic mRNAs.…”

mentioning

confidence: 99%

Nucleocapsid protein effects on the specificity of retrovirus RNA encapsidation

Zhang

Barklis

1995

134

We have analyzed the roles of Gag protein nucleocapsid (NC) domains in the packaging or encapsidation of retroviral RNAs into virus particles. We found that mutation of both zinc finger motifs of the human immunodeficiency virus (HIV) NC domain reduced but did not eliminate encapsidation of the HIV viral RNA. However, the NC mutations also resulted in a three-to fourfold reduction in the specificity of RNA encapsidation, as determined by comparison of virus-associated genomic and spliced RNA levels. As a complementary approach, we replaced the NC domain of Moloney murine leukemia virus (M-MuLV) with that of HIV.

“…At the moment, we do not know when during the viral replication cycle this second function is needed. It is not required for RNA incorporation into virions, since replacement of the first 84 residues of MA with the small membrane-binding domain of Src does not prevent genome packaging (36); furthermore, MA binds RNA very weakly and nonspecifically (21). The block to infectivity probably is not due to an inability of the mutants to incorporate the Gag-Pol fusion protein, since reverse transcriptase activity was detected in the noninfectious viruses.…”

Section: Discussionmentioning

confidence: 99%

Evidence for a second function of the MA sequence in the Rous sarcoma virus Gag protein

Parent

Wilson

Resh

et al. 1996

Self Cite

During retrovirus assembly, Gag proteins bind to the inner leaflet of the plasma membrane to initiate the budding process. The molecular basis of this protein-lipid interaction is poorly understood. For the human immunodeficiency virus type 1 Gag protein, we recently reported that the membrane-binding domain resides within the N-terminal 31 amino acids and consists of two components: myristate and a cluster of basic residues, which together promote membrane binding in vitro and budding in vivo (W. Zhou, L. J. Parent, J. W. Wills, and M. D. Resh, J. Virol. 68:2556-2569, 1994). The positively charged residues associate electrostatically with acidic phospholipids to stabilize membrane binding, while myristate provides membrane-binding energy via hydrophobic interactions. Here we demonstrate that the human immunodeficiency virus type 1 Gag membranebinding domain can fully replace the membrane-targeting function of the N-terminal 100 residues of the nonmyristylated Rous sarcoma virus (RSV) Gag protein. To further explore the importance of myristate and basic residues in membrane binding, we developed a gain-of-function assay whereby budding was restored to defective mutants of RSV Gag. Detailed mutational analysis revealed that the position, number, and context of charged residues are crucial to budding. Myristate provides additional membrane-binding energy, which is critical when a Gag protein is near the threshold of stable membrane association. Finally, viruses with altered matrix (MA) proteins that are noninfectious, even though they produce particles with high efficiency, were identified. Thus, we present the first evidence that the RSV MA sequence plays two distinct roles, membrane binding during particle assembly and a second, as yet undefined function required for viral infectivity.