Construction and Crystal Structure of Recombinant STNV Capsids

Lane, S.W.; Dennis, C.A.; Lane, C.L.; Trinh, Chi H.; Rizkallah, P.J.; Stockley, Peter G.; Phillips, Simon E. V.

doi:10.1016/j.jmb.2011.07.062

Cited by 40 publications

(63 citation statements)

References 37 publications

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“…This suggests that the viral ssDNA ordering is dictated by VP structure. Icosahedrally ordered RNA has been found in a number of viruses, including flock house virus, MS2, and satellite tobacco necrosis virus (STNV) (88)(89)(90). In MS2 and STNV, the capsid protein is involved in extensive contacts with ordered nucleotides (89,90).…”

Section: Resultsmentioning

confidence: 99%

“…Icosahedrally ordered RNA has been found in a number of viruses, including flock house virus, MS2, and satellite tobacco necrosis virus (STNV) (88)(89)(90). In MS2 and STNV, the capsid protein is involved in extensive contacts with ordered nucleotides (89,90). For these viruses, structural, biochemical, and biophysical studies with RNA aptamers indicate that homologous (not identical) repeated RNA sequences, which occur in stem-loop structures, likely dictate the icosahedral ordering of observed nucleotides in the capsid structures (91)(92)(93)(94)(95).…”

Section: Resultsmentioning

confidence: 99%

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Structural Characterization of H-1 Parvovirus: Comparison of Infectious Virions to Empty Capsids

Halder

Nam

Govindasamy

et al. 2013

J Virol

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bThe structure of single-stranded DNA (ssDNA) packaging H-1 parvovirus (H-1PV), which is being developed as an antitumor gene delivery vector, has been determined for wild-type (wt) virions and noninfectious (empty) capsids to 2.7-and 3.2-Å resolution, respectively, using X-ray crystallography. The capsid viral protein (VP) structure consists of an ␣-helix and an eightstranded anti-parallel ␤-barrel with large loop regions between the strands. The ␤-barrel and loops form the capsid core and surface, respectively. In the wt structure, 600 nucleotides are ordered in an interior DNA binding pocket of the capsid. This accounts for ϳ12% of the H-1PV genome. The wt structure is identical to the empty capsid structure, except for side chain conformation variations at the nucleotide binding pocket. Comparison of the H-1PV nucleotides to those observed in canine parvovirus and minute virus of mice, two members of the genus Parvovirus, showed both similarity in structure and analogous interactions. This observation suggests a functional role, such as in capsid stability and/or ssDNA genome recognition for encapsulation. The VP structure differs from those of other parvoviruses in surface loop regions that control receptor binding, tissue tropism, pathogenicity, and antibody recognition, including VP sequences reported to determine tumor cell tropism for oncotropic rodent parvoviruses. These structures of H-1PV provide insight into structural features that dictate capsid stabilization following genome packaging and three-dimensional information applicable for rational design of tumor-targeted recombinant gene delivery vectors. H -1 parvovirus (H-1PV) is a member of the rodent subgroup of the Parvovirus genus of the single-stranded DNA (ssDNA) Parvoviridae (1). H-1PV was first isolated from rats transplanted with HEP-1, a human liver adenocarcinoma cell line (2), and also from aborted human fetuses (3). Recombinant vectors based on H-1PV and a number of other rodent parvoviruses, including minute virus of mice (MVM) and LuIII, are promising candidates for antitumor delivery vectors, particularly for cytoreductive and immunogene therapy approaches (reviewed in references 4 and 5). The inherent oncotropism of these autonomous parvoviruses is based on their dependence on cellular proliferation factors expressed during the S phase and the differentiated state of the host cell (6, 7). The rodent parvoviruses display oncopreferential cytotoxic activity in vitro and also possess an oncosuppressive potential, inhibiting the formation of spontaneous and chemical or virus-induced tumors in vitro and in vivo (8-13). These viruses can also persistently infect their natural hosts, do not integrate their genome into cellular chromosomes, and are not associated with human disease (reviewed in reference 4).Recombinant rodent parvovirus vectors targeted for tumor therapy utilize a double-edged strategy that takes advantage of their inherent oncotropism and selective cytotoxicity plus their ability to deliver therapeutic genes that code for ...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Structural Characterization of H-1 Parvovirus: Comparison of Infectious Virions to Empty Capsids

Halder

Nam

Govindasamy

et al. 2013

J Virol

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“…Removal of this region in STNV prevents assembly (39), whereas in other viruses it promotes RNA-independent assembly of T = 1 capsids (40). Here we have used smFCS assays to probe the roles of RNA in STNV assembly at nanomolar concentrations, showing that it follows a two-stage PS-mediated mechanism, thus preventing assembly around noncognate RNAs.…”

Section: Discussionmentioning

confidence: 99%

Revealing the density of encoded functions in a viral RNA

Patel

Dykeman

Coutts

et al. 2015

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

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We present direct experimental evidence that assembly of a single-stranded RNA virus occurs via a packaging signal-mediated mechanism. We show that the sequences of coat protein recognition motifs within multiple, dispersed, putative RNA packaging signals, as well as their relative spacing within a genomic fragment, act collectively to influence the fidelity and yield of capsid selfassembly in vitro. These experiments confirm that the selective advantages for viral yield and encapsidation specificity, predicted from previous modeling of packaging signal-mediated assembly, are found in Nature. Regions of the genome that act as packaging signals also function in translational and transcriptional enhancement, as well as directly coding for the coat protein, highlighting the density of encoded functions within the viral RNA. Assembly and gene expression are therefore direct molecular competitors for different functional folds of the same RNA sequence. The strongest packaging signal in the test fragment, encodes a region of the coat protein that undergoes a conformational change upon contact with packaging signals. A similar phenomenon occurs in other RNA viruses for which packaging signals are known. These contacts hint at an even deeper density of encoded functions in viral RNA, which if confirmed, would have profound consequences for the evolution of this class of pathogens.virus assembly | single-molecule fluorescence correlation spectroscopy | satellite tobacco necrosis virus | packaging signal

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“…1), the RNA bacteriophage MS2 of the Leviviridae family and Satellite Tobacco Necrosis virus (STNV), which is representative of a large number of plant viruses. These were chosen because our previous studies (14)(15)(16)(17)(18)(19)(20)(21)(22) have revealed potential roles played by the RNA in coat protein quasi-conformer switching, required for correct assembly of the MS2 T ¼ 3 capsid and the presence of multiple putative preferred coat protein binding sites (packaging signals) within the STNV genome, which gets packaged into a T ¼ 1 capsid in which all protein conformers are identical. The two viral coat protein architectures are distinct, the STNV CP having a positively charged N-terminal extension to its globular body which is essential for assembly (17) and partially disordered in X-ray structures, whilst the MS2 CP lacks these features.…”

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confidence: 99%

Evidence that viral RNAs have evolved for efficient, two-stage packaging

Borodavka

Tůma

Stockley

2012

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

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134

190

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Genome packaging is an essential step in virus replication and a potential drug target. Single-stranded RNA viruses have been thought to encapsidate their genomes by gradual co-assembly with capsid subunits. In contrast, using a single molecule fluorescence assay to monitor RNA conformation and virus assembly in real time, with two viruses from differing structural families, we have discovered that packaging is a two-stage process. Initially, the genomic RNAs undergo rapid and dramatic (approximately 20-30%) collapse of their solution conformations upon addition of cognate coat proteins. The collapse occurs with a substoichiometric ratio of coat protein subunits and is followed by a gradual increase in particle size, consistent with the recruitment of additional subunits to complete a growing capsid. Equivalently sized nonviral RNAs, including high copy potential in vivo competitor mRNAs, do not collapse. They do support particle assembly, however, but yield many aberrant structures in contrast to viral RNAs that make only capsids of the correct size. The collapse is specific to viral RNA fragments, implying that it depends on a series of specific RNA-protein interactions. For bacteriophage MS2, we have shown that collapse is driven by subsequent protein-protein interactions, consistent with the RNA-protein contacts occurring in defined spatial locations. Conformational collapse appears to be a distinct feature of viral RNA that has evolved to facilitate assembly. Aspects of this process mimic those seen in ribosome assembly.fluorescence correlation spectroscopy | RNA folding | RNA condensation | kinetics | hydrodynamic radius P ositive-sense, single-stranded (ss)RNA viruses are ubiquitous pathogens in all kingdoms of life (1), causing significant human disease and major financial losses (2). Therapeutic strategies are currently limited and the ideal of vaccination will only ever be practical in a minority of the human and animal viruses. In addition, recent work has highlighted the potential problems that might arise by misincorporation of nonviral RNAs in viruslike particles (VLPs) (3) that are being considered as synthetic vaccines against both pathogens and oncogenic viruses (4). A more thorough understanding of the molecular events central to viral lifecycles is therefore needed. Such studies may reveal novel strategies for therapeutic intervention.Genomic RNAs play essential roles during the viral lifecycle, adopting different metastable conformations in order to be replicated, translated and packaged into the virion (5, 6). The latter process, the final step in the production of infectious viral progeny, is the topic of the experiments described here. For isometric, nonenveloped virions, protein capsids self-assemble around their genomes, resulting in their confinement at relatively high packing densities (7). It has been proposed that this is a spontaneous process because RNA molecules are branched polymers and there is no barrier to compaction due to large persistence lengths, as seen in dsDNA phages. E...

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Construction and Crystal Structure of Recombinant STNV Capsids

Cited by 40 publications

References 37 publications

Structural Characterization of H-1 Parvovirus: Comparison of Infectious Virions to Empty Capsids

Structural Characterization of H-1 Parvovirus: Comparison of Infectious Virions to Empty Capsids

Revealing the density of encoded functions in a viral RNA

Evidence that viral RNAs have evolved for efficient, two-stage packaging

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