Identification of Interactions between Sindbis Virus Capsid Protein and Cytoplasmic vRNA as Novel Virulence Determinants

<div><p>Alphaviruses are arthropod-borne viruses that represent a significant threat to public health at a global level. While the formation of alphaviral nucleocapsid cores, consisting of cargo nucleic acid and the viral capsid protein, is an essential molecular process of infection, the precise interactions between the two partners are ill-defined. A CLIP-seq approach was used to screen for candidate sites of interaction between the viral Capsid protein and genomic RNA of Sindbis virus (SINV), a model alphavirus. The data presented in this report indicates that the SINV capsid protein binds to specific viral RNA sequences in the cytoplasm of infected cells, but its interaction with genomic RNA in mature extracellular viral particles is largely non-specific in terms of nucleotide sequence. Mutational analyses of the cytoplasmic viral RNA-capsid interaction sites revealed a functional role for capsid binding early in infection. Interaction site mutants exhibited decreased viral growth kinetics; however, this defect was not a function of decreased particle production. Rather mutation of the cytoplasmic capsid-RNA interaction sites negatively affected the functional capacity of the incoming viral genomic RNAs leading to decreased infectivity. Furthermore, cytoplasmic capsid interaction site mutants are attenuated in a murine model of neurotropic alphavirus infection. Collectively, the findings of this study indicate that the identified cytoplasmic interactions of the viral capsid protein and genomic RNA, while not essential for particle formation, are necessary for genomic RNA function early during infection. This previously unappreciated role of capsid protein during the alphaviral replication cycle also constitutes a novel virulence determinant.</p></div

Mutation of the SINV C:R interaction sites negatively affects incoming viral genomic RNA function.

<p><b>A</b>) The RNA decay profiles of the incoming viral genomic RNAs of the individual SINV C:R interaction site mutants and parental wild type virus were determined by qRT-PCR analysis as described in the materials and methods. Plotted is the relative abundance of the incoming viral genomic RNAs (y-axis) with regards to time (x-axis). Regression analysis was utilized to determine the RNA decay profile (as shown with the solid line) and the dashed lines represent the 95% confidence intervals of the aforementioned regression. <b>B</b>) The half-lives of the individual genomic RNAs as determined using the calculations reported in Dolken et al., as determined by the first point at which the relative abundance has reached 0.5. C) The levels of Nanoluciferase activity for wild type parental virus and the nt10100 and nt10400 C:R interaction site mutants were determined as reported in the materials and methods at the indicated times post infection. All quantitative data in this figure represents the mean of at least three independent biological replicates. Comparative analysis was performed using variable bootstrapping, as described in the materials and methods, with the error bar representing the standard deviation of the mean. Statistical significance, as indicated on the individual panels above, are the p-Values obtained from Student’s t-test.</p

Proposed model of SINV C:R interaction site function.

<p>After receptor mediated endocytosis the acidification of the endosome resulting in release of the nucleocapsid core into the host cytoplasm. Disassembly of the nucleocapsid core occurs shortly after endosomal release; and SINV capsid protein remains bound to the C:R interaction sites following disassembly. Retention of Capsid:RNA binding at the C:R interaction sites enables evasion of the host RNA decay machinery and rapid assembly of the host translational machinery. Collectively, these interactions lead to the efficient establishment of viral infection, including but not limited to the modulation of the host innate immune response resulting in viral disease and pathogenesis. Disruption of the C:R interaction sites (lower pathway), leading to the ablation of Capsid:RNA interactions, results in RNA instability and a reduction of genomic RNA function early during infection.</p

Analysis and mutation of the SINV C:R interaction sites.

<p>A) Magnified regions of the viral genomic RNA with special emphasis on the individual C:R interaction sites prioritized during this study. Plotted on the left y-axis are the Z-scores previously reported in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006473#ppat.1006473.g001" target="_blank">Fig 1</a>. The right y-axis indicates the prevalence of single nucleotide polymorphisms (SNPS) across a curated set of SINV genomic RNAs; higher values indicate increased base identity variation at a particular nucleotide but are not informative as to relative base conservation. Nucleotide position is reported on the x-axis. B) The individual nucleotide sequences of the C:R interaction sites are described in regards to parental sequence (top) and mutant sequence (bottom). The mutated nucleotides are highlighted in red. C) Quantitative analysis of the individual C:R interaction sites following mutation indicates that Capsid:RNA binding is significantly reduced relative to parental virus. Data shown is the mean of three independent biological replicates, with the error bar representing the standard deviation of the mean.</p

Sindbis virus capsid protein associates with viral RNA in the cytoplasm of infected cells at discrete interaction sites.

<p><b>A</b>) A schematic drawing of the genomic RNA of SINV. Protein coding and noncoding regions are labeled. For reference the graphs in panels B through D are aligned with this schematic. <b>B</b>) Coverage of anti-Capsid CLIP-seq libraries derived from purified, mature infectious viral particles. Depth of coverage is represented by the y-axis, with the x-axis representing nucleotide position. <b>C</b>) Depth of coverage for anti-Capsid libraries derived from cytoplasmic fractions. Plotted on the y-axis is the depth of coverage of the anti-Capsid libraries following subtractive analysis, with the x-axis representing nucleotide position. <b>D</b>) Statistical analysis of the data presented in panel C, with the y-axis indicating the Z-score of all represented sequences. The x-axis represents the nucleotide position, with all panels above being aligned with the indicated nucleotide numbering intervals.</p

Mutation of the individual SINV C:R interaction site mutants alters SINV disease and mortality in murine models of infection.

<p><b>A</b>) Adult wild type mice were infected with either parental SINV AR86 (N = 4), or one of the individual SINV C:R interaction site mutants, SINV.nt9300 (N = 6); SINV.nt10100 (N = 4); SINV.nt10400 (N = 6), via footpad injection. The infected animals were monitored daily for weight and disease presentation. Plotted in this panel is the percent survival for the experimentally infected animals with respect to time. Statistical analysis, via Kaplan-Meier, is reported inset to the panel. <b>B</b>) The mean weight, as plotted as percent of starting weight, with respect to time for the animals described in panel A. Plotted values represents the means of the individual animals and the error bar represents the standard deviation of the means. Animals lost to mortality were culled from further measurements. <b>C</b>) The titer of central nervous tissue of parental wild type SINV or the highly attenuated SINV.nt10100 mutant infected mice. Plotted are the individual titers of 8 experimentally infected mice harvested at 3-days post infection. Statistical significance, as indicated on the individual panels above, are the p-Values obtained from Student’s t-test.</p

Mutation of the individual SINV C:R interaction sites increases the induction of type-I interferons.

<p>The amount of soluble Type-I Interferons produced during individual SINV C:R interaction site mutant relative to that of parental wild type virus at 24 hours post infection as determined using a tissue culture model of IFN induction. All quantitative data in this figure represents the mean of three independent biological replicates, with the error bar representing the standard deviation of the mean. Statistical significance, as indicated on the individual panels above, are the p-Values obtained from Student’s t-test.</p

Analysis of SINV C:R interaction site mutant growth kinetics and infectivity.

<p><b>A</b>) The one-step growth kinetics of the individual SINV C:R interaction site mutants and parental wild type virus were assessed in HEK293 cells. <b>B</b>) Quantitative analysis of the SINV C:R interaction site mutants and parental wild type virus in regards to the number of infectious units (left y-axis), and the total number of viral particles produced as measured by qRT-PCR (right y-axis). <b>C</b>) The infectivity of the individual SINV C:R interaction site mutants and parental wild type virus as reported as the ratio of total particles per infectious unit as determined using BHK-21 cells. All quantitative data in this figure represents the mean of three independent biological replicates, the error bar representing the standard deviation of the mean. Statistical significance, as indicated on the individual panels above, are the p-Values obtained from Student’s t-test.</p

Assessment of SINV C:R interaction site mutant RNA synthesis and gene expression.

<p><b>A</b>) Quantitative analysis of the three viral RNA species generated during SINV infection of HEK293 cells at 12-hours post infection at an MOI of 10 infectious units per cell. The copy numbers of the Genomic, Subgenomic, and Minus strand RNAs were quantified using qRT-PCR. Data in this panel represents the mean of three independent biological replicates, the error bar representing the standard deviation of the mean. <b>B</b>) Metabolic labeling of infected HEK293 cells at the indicated times. Infected cells were metabolically labeled for a period of two hours immediately preceding the time indicated in the figure. The migratory positions of nsP2, Capsid, and the viral glycoproteins; and host Actin, are indicated to the right of the panel. Data shown is representative of several independent biological replicates and technical replicates.</p