Hybrid brome mosaic virus RNAs express and are packaged in tobacco mosaic virus coat protein in vivo

Sacher, R. F.; French, Ron; Ahlquist, Paul

doi:10.1016/0042-6822(88)90049-9

Cited by 35 publications

(31 citation statements)

References 27 publications

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“…Peptides from the A and P22 N proteins require helical conformations for specific RNA binding, consistent with extensive mutagenesis studies of the A domain (35) and as shown for the HIV Rev-RRE interaction (9). Arginine-rich domains of brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV) coat proteins are necessary for specific binding and encapsidation of genomic RNAs (24,36), and both adopt helical conformations. Although specific RNA-binding sites for the coat proteins have not been identified, NMR studies with a CCMV peptide indicate that its helical structure is stabilized in the presence of oligophosphates (37), similar to the increased stability of the Rev a-helix upon RNA binding (23).…”

Section: Discussionmentioning

confidence: 81%

“…Gel shift experiments were performed with A and P22 N peptides, having 35% and 15% helical contents, respectively, and with a set of wild-type and mutant A and P22 boxB RNAs. The A peptide bound tightly to its wild-type hairpin but weakly to RNAs with substitutions in the loop or closing base pair or to (24), and splicing factors PRP6 (25) and U2AF 65-kDa subunit (26). Numbers in parentheses refer to amino acid positions within the proteins.…”

Section: Resultsmentioning

confidence: 99%

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Structural variety of arginine-rich RNA-binding peptides.

Tan

Frankel²

1995

Proc. Natl. Acad. Sci. U.S.A.

190

184

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Arginine-rich domains are used by a variety of RNA-binding proteins to recognize specific RNA hairpins. It has been shown previously that a 17-aa arginine-rich peptide from the human immunodeficiency virus Rev protein binds specifically to its RNA site when the peptide is in an a-helical conformation. Here (2), and in the third primarily to the phosphate backbone (3). Important contacts also are made to anticodon loop nucleotides (1, 2). The cocrystal structure of an R17 phage coat protein-RNA complex shows important contacts to bulge and loop nucleotides of an RNA hairpin (4). These studies provide detailed views of RNA-protein interactions and include rather different types of protein structures. In other RNA-binding proteins, conserved sequence motifs are found that suggest similar structures (5, 6). One motif, the arginine-rich motif, consists of a short region of basic amino acids (-8-20 residues in length) particularly rich in arginine. This motif is found in bacterial antiterminators, ribosomal proteins, coat proteins from RNA viruses, the human immunodeficiency virus (HIV) Tat and Rev proteins (7), and the bovine immunodeficiency virus (BIV) Tat protein (8).Studies with model peptides from HIV Rev, HIV Tat, and BIV Tat have shown that isolated arginine-rich domains can bind RNA with high affinity and recognize features of the RNA similar to the intact proteins (8-13). In HIV Rev, a 17-aa peptide binds specifically to RRE RNA when the peptide is in an a-helical conformation and requires six amino acids (four arginines, one threonine, and one asparagine) for binding (9).NMR studies indicate that Rev peptide binding stabilizes the structure of an internal RNA loop, forming G-G and G-A base pairs and two looped-out bases (14, 15). In HIV Tat, a single arginine residue in the arginine-rich domain is largely responsible for recognition of a bulge region in TAR RNA (12), and arginine, even as the free amino acid, binds to TAR in the sameThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. manner as in the Tat peptide (16), suggesting that a defined peptide conformation may not be needed for recognition. An NMR model of the TAR-arginine complex suggests that the arginine guanidinium group hydrogen bonds to a guanine base in the major groove and to two phosphates and that the complex is stabilized by a base triple interaction between a U in the bulge and an AU base pair above the bulge (17). Recent NMR studies of the equine infectious anemia virus (EIAV) Tat protein (18) and of a peptide containing the HIV Tat arginine-rich domain fused to the core regulatory domain of EIAV Tat (19) have suggested that these arginine-rich domains have a tendency to form a-helices, but it is not known whether they are helical when bound to RNA.To better define the structural repertoire of the argininerich RNA-binding motif, we have examined the RNA-binding and confo...

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Section: Discussionmentioning

confidence: 81%

Section: Resultsmentioning

confidence: 99%

Structural variety of arginine-rich RNA-binding peptides.

Tan

Frankel²

1995

Proc. Natl. Acad. Sci. U.S.A.

190

184

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“…Here we showed that the Nterminal basic region plays a key role in HCV assembly in the CFS. Interestingly, this region is also known to bind RNA, which is thought to nucleate capsid assembly for a number of viruses (5,12,13,55,62). Conversely, we found that regions outside the N terminus (i.e., distal to amino acid 82) did not influence HCV capsid assembly per se.…”

Section: Discussionmentioning

confidence: 99%

Identification of Residues in the Hepatitis C Virus Core Protein That Are Critical for Capsid Assembly in a Cell-Free System

Klein

Dellos

Lingappa

2005

J Virol

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Significant advances have been made in understanding hepatitis C virus (HCV) replication through development of replicon systems. However, neither replicon systems nor standard cell culture systems support significant assembly of HCV capsids, leaving a large gap in our knowledge of HCV virion formation. Recently, we established a cell-free system in which over 60% of full-length HCV core protein synthesized de novo in cell extracts assembles into HCV capsids by biochemical and morphological criteria. Here we used mutational analysis to identify residues in HCV core that are important for capsid assembly in this highly reproducible cell-free system. We found that basic residues present in two clusters within the N-terminal 68 amino acids of HCV core played a critical role, while the uncharged linker domain between them was not. Furthermore, the aspartate at position 111, the region spanning amino acids 82 to 102, and three serines that are thought to be sites of phosphorylation do not appear to be critical for HCV capsid formation in this system. Mutation of prolines important for targeting of core to lipid droplets also failed to alter HCV capsid assembly in the cell-free system. In addition, wild-type HCV core did not rescue assembly-defective mutants. These data constitute the first systematic and quantitative analysis of the roles of specific residues and domains of HCV core in capsid formation.Hepatitis C virus (HCV) is a major public health concern worldwide. Two percent of the world's population is infected with the virus (54), which is the major cause of non-A, non-B hepatitis and which often leads to cirrhosis of the liver or hepatocellular carcinoma (42). While current treatments have improved, they are poorly tolerated, not completely efficacious, and not available in all settings (31). Development of better treatments and vaccines has been hampered by the lack of virus replication systems. Standard cell culture systems do not support HCV replication, and the chimpanzee is the only animal model capable of being infected and yielding high HCV titers (23). These limitations have been partly overcome by the development of HCV replicon systems, which support autonomous replication of HCV RNA (2,10,36,37). Replicon systems have allowed specific requirements of HCV RNA replication to be studied; however, HCV particles, or even nucleocapsids, are not produced in these systems (9,

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“…2). Some plant viruses, such as brome mosaic virus, apparently require the assembly of virus particles for both types of movement process to occur (16). However, this is not the case in all plant virus systems.…”

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confidence: 97%

Systemic movement of an RNA plant virus determined by a point substitution in a 5' leader sequence.

Petty¹,

Edwards²,

Jackson³

1990

Proc. Natl. Acad. Sci. U.S.A.

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The ability of viruses to move through infected plants is an important determinant of host range and pathogenicity. We have investigated the genetic basis for the inability of the Type strain of barley stripe mosaic hordeivirus to undergo long-range systemic movement in the tobacco Nicotiana benthamiana. We show that, in this model system, a short open reading frame in the 5' leader of the smallest viral genomic RNA prevents long-range vascular movement. As predicted by the ribosome scanning model, the leader open reading frame decreases the efficiency with which the 5' proximal gene is translated in vitro. Thus, systemic pathogenicity in this system may be determined by the efficiency of translation of a viral gene in vivo and is not determined by the primary sequence of the encoded protein.The mechanisms by which plant viruses move from cell to cell and through the vasculature of infected plants are currently unknown. However, these processes generally require the interaction of specific viral gene products with the host and are an important constraint on viral host range and pathogenicity (1,2 Fig. 1) were prepared by digestion of full-length cloned cDNA with Kpn I and Hpa I, treatment with T4 DNA polymerase to render the ends flush, and recircularization of the large plasmid fragments according to standard procedures (9). Chimeric recombinants were constructed by exchanging 5' and 3' regions of y cDNA at the conserved HincIl and Pst I sites using yb deletion mutants of each strain. The 372-nucleotide (nt) direct repeat (4, 7) was removed from a full-length Type strain y cDNA clone by digestion with,Nru I and recircularization of the large plasmid fragment (see Fig. 1). This deletion mutant was then used to construct both the Nru I site cDNA chimeras and the oligonucleotide-directed mutations in the Type strain genetic background. In vitro transcription and plant inoculation with the mutant RNAs were carried out as described (4).A full-length cDNA clone of RNA y from the CV17 strain of BSMV was isolated in the transcription vector pBSMY(3'-T7) as described previously, except that the T7a Band-Aid oligonucleotide was used in place of T7y (for details, see ref.8). Sequencing of the cDNA clones using universal M13 primers was as described (8). Oligonucleotide-directed mutations were made by the procedure of Kunkel (10) as described (4) and confirmed by sequencing.$ Analysis of Systemic Movement. The systemic movement of BSMV in N. benthamiana was assessed visually by the appearance of disease symptoms and by detection of viral coat protein antigen in extracts from inoculated and systemically infected leaves (or their equivalent in the case of nonmoving strains and mutants) 12 days after inoculation. Protein extracts prepared from N. benthamiana leaves were fractionated by electrophoresis in SDS/15% polyacrylamide gels and blotted onto nitrocellulose, and viral coat protein was detected with anti-BSMV antiserum as described (4).In Vitro Translation. For in vitro translation, capped in vitro transcripts of...

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Hybrid brome mosaic virus RNAs express and are packaged in tobacco mosaic virus coat protein in vivo

Cited by 35 publications

References 27 publications

Structural variety of arginine-rich RNA-binding peptides.

Structural variety of arginine-rich RNA-binding peptides.

Identification of Residues in the Hepatitis C Virus Core Protein That Are Critical for Capsid Assembly in a Cell-Free System

Systemic movement of an RNA plant virus determined by a point substitution in a 5' leader sequence.

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