RNA-binding properties of the matrix protein (p19gag) of avian sarcoma and leukemia viruses

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: 59%

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

Nelle

Wills

1996

“…DNA and RNA binding assays. The nitrocellulose filter binding assay was performed as described elsewhere (36). In brief, standard reaction mixtures (50 l) contained 10 mM Tris-Cl (pH 8.0), 20 mM NaCl, 1 mM dithiothreitol, 0.01 pmol of 32 P-labeled DNA or RNA probe (with a specific activity of 2 ϫ 10 8 to 5 ϫ 10 8 cpm/g), and various amounts of purified NC proteins.…”

Section: Methodsmentioning

confidence: 99%

“…We first used nitrocellulose filter binding to assay the nucleic acid binding activity of the NC proteins. This method is based on the finding that protein, but not RNA or DNA, binds to nitrocellulose at low to moderate ionic strength (36). Therefore, it is logical to assume that a single protein molecule bound to a radioactive RNA or DNA molecule is sufficient to cause retention of the labeled probe on the membrane.…”

Section: Construction Of Nc Mutantsmentioning

confidence: 99%

The carboxyl terminus of the human foamy virus Gag protein contains separable nucleic acid binding and nuclear transport domains

Edelmann

Strong

et al. 1996

101

The Gag protein of human foamy virus (HFV) lacks Cys-His boxes present in the nucleocapsid (NC) domains of other retroviruses; instead it contains three glycine-arginine-rich motifs (GR boxes). We have expressed the carboxyl end of HFV Gag containing the GR boxes (the NC domain equivalent) and analyzed its nucleic acid binding properties. Our results show that the NC domain of HFV Gag binds with high affinity to both RNA and DNA, in a sequence-independent manner, as determined by filter binding assays. Analysis of a mutant containing a heterologous sequence in place of GR box I indicates that this motif is required for nucleic acid binding and for viral replication. A mutant in GR box II still binds to RNA and DNA in vitro, but virus containing this mutation does not replicate and no nuclear staining of the Gag protein is found in transfected cells. Surprisingly, a revertant from this mutant that completely lacks GR box II and exhibits very little nuclear transport of Gag can readily replicate in tissue culture. This finding thus provides a direct evidence that although the sequences in GR box II can serve as a nuclear transport signal, they are not required for HFV replication and it is unlikely that nuclear localization of Gag protein plays any critical role during viral infection. Taken together, our results suggest that the Gag protein of HFV may be more analogous to the core protein of the hepatitis B virus family than to conventional retroviral Gag protein.

“…For example, the virus gag precursors of higher classes of mammals, including bovine (26), equine (10,38), simian (8), and human (9,32) species, lack the peptide corresponding to p12 of MuLV. On the other hand, there has been a report that MA(p19) of Rous sarcoma virus (RSV) binds to a specific double-stranded region in the viral RNA (16), but this finding has also been contradicted by recent experiments (37).…”

mentioning

confidence: 99%

Bovine leukemia virus matrix-associated protein MA(p15): further processing and formation of a specific complex with the dimer of the 5'-terminal genomic RNA fragment

Katoh

Kyushiki

Sakamoto

et al. 1991

The retrovirus precursor protein has an arrangement of several characteristic domains with which it achieves selective and efficient packaging of the genome RNA during particle assembly. In this study, we analyzed the composition of the bovine leukemia virus (BLV) gag proteins and examined their RNA-binding properties in gel mobility shift assays, using various genomic RNA probes synthesized in vitro. Results obtained in amino acid sequence and composition analyses indicate that the matrix-associated protein MA(p15) is further processed by the BLV protease (PR) to generate MA(plO), a short peptide of seven amino acid residues, and p4. The gag precursor is now mapped as NH2-MA(plO)-p4-CA(p24)-NC(p12)-COOH. MA(p15) formed a specific complex with the dimer RNA of the U5-5' gag region presumed to contain the BLV packaging signal but not with other RNAs. The NH2-terminal cleavage product, MA(plO), bound all RNA fragments tested, while the COOH-terminal peptides with a sequence common to mammalian type C retroviruses had little affinity for RNA. The nucleocapsid protein NC(p12) bound to RNAs nonspecifically and randomly in the presence or absence of zinc ions. These results suggest a possible interaction of the NH2 terminus of the gag precursor with the 5' terminus of the genomic RNA in an early phase of particle assembly, when the conserved structure between the MA and CA domains might be involved.