Deletion of Highly Conserved Arginine-Rich RNA Binding Motif in Cowpea Chlorotic Mottle Virus Capsid Protein Results in Virion Structural Alterations and RNA Packaging Constraints

Panneerselvam, A.; Apte, Swapna; Wilkens, Stephan; Rao, A. L. N.

doi:10.1128/jvi.79.6.3277-3288.2005

Cited by 32 publications

(23 citation statements)

References 35 publications

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“…Past structural and biochemical studies indicate that the N-terminal region of the capsid protein is located inside the closed and swollen forms of WT-CCMV and inside the closed form of SS-CCMV (5,42,50,54,55). However, protease mass mapping experiments reveal that in solution the N terminus of the capsid subunit is readily accessible to trypsin.…”

Section: Vol 80 2006 Structure and Dynamics Of Salt-stable Ccmv 3587mentioning

confidence: 99%

Enhanced Local Symmetry Interactions Globally Stabilize a Mutant Virus Capsid That Maintains Infectivity and Capsid Dynamics

Speir

Bothner

et al. 2006

J Virol

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Structural transitions in viral capsids play a critical role in the virus life cycle, including assembly, disassembly, and release of the packaged nucleic acid. Cowpea chlorotic mottle virus (CCMV) undergoes a well-studied reversible structural expansion in vitro in which the capsid expands by 10%. The swollen form of the particle can be completely disassembled by increasing the salt concentration to 1 M. Remarkably, a single-residue mutant of the CCMV N-terminal arm, K42R, is not susceptible to dissociation in high salt (salt-stable CCMV [SS-CCMV]) and retains 70% of wild-type infectivity. We present the combined structural and biophysical basis for the chemical stability and viability of the SS-CCMV particles. A 2.7-Å resolution crystal structure of the SS-CCMV capsid shows an addition of 660 new intersubunit interactions per particle at the center of the 20 hexameric capsomeres, which are a direct result of the K42R mutation. Protease-based mapping experiments of intact particles demonstrate that both the swollen and closed forms of the wild-type and SS-CCMV particles have highly dynamic N-terminal regions, yet the SS-CCMV particles are more resistant to degradation. Thus, the increase in SS-CCMV particle stability is a result of concentrated tethering of subunits at a local symmetry interface (i.e., quasi-sixfold axes) that does not interfere with the function of other key symmetry interfaces (i.e., fivefold, twofold, quasi-threefold axes). The result is a particle that is still dynamic but insensitive to high salt due to a new series of bonds that are resistant to high ionic strength and preserve the overall particle structure.Assembly, stability, and disassembly of icosahedral virus particles are coordinated by protein-protein and protein-nucleic acid interactions. Cowpea chlorotic mottle virus (CCMV) provides a model system for examining these interactions. The advantages of this system include in vivo and in vitro assembly assays (4, 7, 12, 55), a reversible transition between distinctly different closed and expanded forms (6), a high-resolution crystal structure of the closed particle (42), an electron cryomicroscopy image reconstruction of the swollen particle (42), and a general understanding of the requirements for assembly and stabilization of the capsid (6,53,55,56).CCMV is a small RNA plant virus that belongs to the Bromovirus genus in the Bromoviridae family. The viral genome is composed of four (positive-sense) single-stranded RNA molecules that are encapsidated in three morphologically identical particles (8,21,27). RNA1 and RNA2 encode proteins involved in RNA-dependent RNA replication and are packaged in separate particles. RNA3, encoding the movement and capsid proteins, and RNA 4, a subgenomic RNA of RNA 3 encoding just the capsid protein, are copackaged in a third particle (2). The structure of the CCMV virion has been determined to a 3.2-Å resolution (42) revealing a Tϭ3 truncated icosahedron having a diameter of 286 Å and composed of 180 identical protein subunits arranged as protruding ...

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Section: Vol 80 2006 Structure and Dynamics Of Salt-stable Ccmv 3587mentioning

confidence: 99%

Enhanced Local Symmetry Interactions Globally Stabilize a Mutant Virus Capsid That Maintains Infectivity and Capsid Dynamics

Speir

Bothner

et al. 2006

J Virol

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“…Additionally, the capsid protein of cowpea chlorotic mottle virus has an arginine-rich motif similar to that of HIV-1 Rev, which allows recognition and packaging of specific RNAs (Annamalai et al, 2005). However, HIV-1 Rev has never been found in virus particles.…”

Section: Rev and Genome Packagingmentioning

confidence: 99%

Rev: beyond nuclear export

Groom

Anderson

Lever

2009

Journal of General Virology

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Rev remains a hot topic. In this review, we revisit the insights that have been gained into the control of gene expression by the retroviral protein Rev and speculate on where current research is leading. We outline what is known about the role of Rev in translation and encapsidation and how these are linked to its more traditional role of nuclear export, underlining the multifaceted nature of this small viral protein. We discuss what more is to be learned in these fields and why continuing research on these 116 amino acids and understanding their function is still important in devising methods to combat AIDS.

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“…Confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM) were performed as described previously (3,6). For rhodamine treatment, healthy leaves of N. benthamiana were infiltrated with rhodamine 123 (Invitrogen), diluted in distilled water according to the manufacturer's recommendation.…”

Section: Methodsmentioning

confidence: 99%

Replication-Coupled Packaging Mechanism in Positive-Strand RNA Viruses: Synchronized Coexpression of Functional Multigenome RNA Components of an Animal and a Plant Virus in Nicotiana benthamiana Cells by Agroinfiltration

Panneerselvam¹,

Rofail²,

DeMason

et al. 2008

J Virol

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Flock house virus (FHV), a bipartite RNA virus of insects and a member of the Nodaviridae family, shares viral replication features with the tripartite brome mosaic virus (BMV), an RNA virus that infects plants and is a member of the Bromoviridae family. In BMV and FHV, genome packaging is coupled to replication, a widely conserved mechanism among positive-strand RNA viruses of diverse origin. To unravel the events that modulate the mechanism of replication-coupled packaging, in this study, we have extended the transfer DNA (T-DNA)-based agroinfiltration system to express functional genome components of FHV in plant cells (Nicotiana benthamiana). Replication, intracellular membrane localization, and packaging characteristics in agroinfiltrated plant cells revealed that T-DNA plasmids of FHV were biologically active and faithfully mimicked complete replication and packaging behavior similar to that observed for insect cells. Complementation with homologous replicase (with respect to CP) failed to enhance packaging specificity. Taken together, we propose that the transcription of CP mRNA from homologous replication and its translation must be synchronized to confer packaging specificity.Flock house virus (FHV), a member of the family Nodaviridae, and brome mosaic virus (BMV), the type member of the Bromovirus genus, are multicomponent positive-strand RNA viruses of animals and plants, respectively (44, 52). FHV was first isolated from the New Zealand grass grub Costelytra zealandica (52). The 4.5-kb single-strand positive-sense RNA genome of FHV is divided between two capped and nonpolyadenylated RNAs copackaged into a single nonenveloped icosahedral virion with a T ϭ 3 symmetry (50). Genomic RNA 1 (F1) is a 3,107-nucleotide (nt) sequence that encodes a 112-kDa RNA-dependent RNA polymerase (RdRp or protein A) that is necessary and sufficient for FHV RNA replication (8,27,42). In addition, F1 also encodes a 387-nt subgenomic RNA 3 (sgF3) sequence that corresponds to that of the 3Ј terminus of F1 (16,22). sgF3 encodes two proteins, b1 and b2. Protein b1 has no recognized function, and b2 is the designated suppressor of RNA silencing activity in Drosophila melanogaster S2 cells (33). Genomic RNA 2 (F2) is a 1,400-nt sequence that encodes the 43-kDa viral capsid protein (CP) precursor ␣ required for the assembly of FHV provirions (49). Each provirion consists of 180 subunits of protein ␣ arranged with a T ϭ 3 quasi-equivalent symmetry and the two genomic RNAs.Provirions are not infectious unless they undergo an autocatalytic maturation process, which results in the cleavage of protein ␣ into protein ␤ (38 kDa) and protein ␥ (5 kDa) (23, 51). sgF3 has been shown to trans activate F2 replication (20). The replication of FHV occurs on the outer mitochondrial membranes (36). An unusual and remarkable feature associated with FHV is its ability to cross the kingdom barrier and to infect a wide variety of cells, including cells of insects (23), mammals (8), yeasts (41), and several species of monocotyledonous and dicotyledon...

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Deletion of Highly Conserved Arginine-Rich RNA Binding Motif in Cowpea Chlorotic Mottle Virus Capsid Protein Results in Virion Structural Alterations and RNA Packaging Constraints

Cited by 32 publications

References 35 publications

Enhanced Local Symmetry Interactions Globally Stabilize a Mutant Virus Capsid That Maintains Infectivity and Capsid Dynamics

Enhanced Local Symmetry Interactions Globally Stabilize a Mutant Virus Capsid That Maintains Infectivity and Capsid Dynamics

Rev: beyond nuclear export

Replication-Coupled Packaging Mechanism in Positive-Strand RNA Viruses: Synchronized Coexpression of Functional Multigenome RNA Components of an Animal and a Plant Virus in Nicotiana benthamiana Cells by Agroinfiltration

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