The alphavirus nonstructural protein 2 NTPase induces a host translational shut-off through phosphorylation of eEF2 via cAMP-PKA-eEF2K signaling.

Chikungunya virus (CHIKV) is a reemerging alphavirus. Since 2005, it has infected millions of people during outbreaks in Africa, Asia, and South/Central America. CHIKV replication depends on host cell factors at many levels and is expected to have a profound effect on cellular physiology. To obtain more insight into host responses to infection, stable isotope labeling with amino acids in cell culture and liquid chromatography-tandem mass spectrometry were used to assess temporal changes in the cellular phosphoproteome during CHIKV infection. Among the ~3,000 unique phosphorylation sites analyzed, the largest change in phosphorylation status was measured on residue T56 of eukaryotic elongation factor 2 (eEF2), which showed a >50-fold increase at 8 and 12 h p.i. Infection with other alphaviruses (Semliki Forest, Sindbis and Venezuelan equine encephalitis virus (VEEV)) triggered a similarly strong eEF2 phosphorylation. Expression of a truncated form of CHIKV or VEEV nsP2, containing only the N-terminal and NTPase/helicase domains (nsP2-NTD-Hel), sufficed to induce eEF2 phosphorylation, which could be prevented by mutating key residues in the Walker A and B motifs of the NTPase domain. Alphavirus infection or expression of nsP2-NTD-Hel resulted in decreased cellular ATP levels and increased cAMP levels. This did not occur when catalytically inactive NTPase mutants were expressed. The wild-type nsP2-NTD-Hel inhibited cellular translation independent of the C-terminal nsP2 domain, which was previously implicated in directing the virus-induced host shut-off for Old World alphaviruses. We hypothesize that the alphavirus NTPase activates a cellular adenylyl cyclase resulting in increased cAMP levels, thus activating PKA and subsequently eukaryotic elongation factor 2 kinase. This in turn triggers eEF2 phosphorylation and translational inhibition. We conclude that the nsP2-driven increase of cAMP levels contributes to the alphavirus-induced shut-off of cellular protein synthesis that is shared between Old and New World alphaviruses. MS Data are available via ProteomeXchange with identifier PXD009381.

Molecular characterization of the RNA-protein complex directing -2/-1 programmed ribosomal frameshifting during arterivirus replicase expression.

Programmed ribosomal frameshifting (PRF) is a mechanism used by arteriviruses like porcine reproductive and respiratory syndrome virus (PRRSV) to generate multiple proteins from overlapping reading frames within its RNA genome. PRRSV employs -1 PRF directed by RNA secondary and tertiary structures within its viral genome (canonical PRF), as well as a noncanonical -1 and -2 PRF that are stimulated by the interactions of PRRSV nonstructural protein 1ß (nsp1ß) and host protein poly(C)-binding protein (PCBP) 1 or 2 with the viral genome. Together, nsp1ß and one of the PCBPs act as transactivators that bind a C-rich motif near the shift site to stimulate -1 and -2 PRF, thereby enabling the ribosome to generate two frameshift products that are implicated in viral immune evasion. How nsp1ß and PCBP associate with the viral RNA genome remains unclear. Here, we describe the purification of the nsp1ß:PCBP2:viral RNA complex on a scale sufficient for structural analysis using small-angle X-ray scattering and stochiometric analysis by analytical ultracentrifugation. The proteins associate with the RNA C-rich motif as a 1:1:1 complex. The monomeric form of nsp1ß within the complex differs from previously reported homodimer identified by X-ray crystallography. Functional analysis of the complex via mutational analysis combined with RNA-binding assays and cell-based frameshifting reporter assays reveal a number of key residues within nsp1ß and PCBP2 that are involved in complex formation and function. Our results suggest that nsp1ß and PCBP2 both interact directly with viral RNA during formation of the complex to coordinate this unusual PRF mechanism.

A novel role for poly(C) binding proteins in programmed ribosomal frameshifting.

Translational control through programmed ribosomal frameshifting (PRF) is exploited widely by viruses and increasingly documented in cellular genes. Frameshifting is induced by mRNA secondary structures that compromise ribosome fidelity during decoding of a heptanucleotide 'slippery' sequence. The nsp2 PRF signal of porcine reproductive and respiratory syndrome virus is distinctive in directing both -2 and -1 PRF and in its requirement for a trans-acting protein factor, the viral replicase subunit nsp1ß. Here we show that the the trans-activation of frameshifting is carried out by a protein complex composed of nsp1ß and a cellular poly(C) binding protein (PCBP). From the results of in vitro translation and electrophoretic mobility shift assays, we demonstrate that a PCBP/nsp1ß complex binds to a C-rich sequence downstream of the slippery sequence and here mimics the activity of a structured mRNA stimulator of PRF. This is the first description of a role for a trans-acting cellular protein in PRF. The discovery broadens the repertoire of activities associated with poly(C) binding proteins and prototypes a new class of virus-host interactions.

Temporal SILAC-based quantitative proteomics identifies host factors involved in chikungunya virus replication.

Chikungunya virus (CHIKV) is an arthropod-borne reemerging human pathogen that generally causes a severe persisting arthritis. Since 2005, the virus has infected millions of people during outbreaks in Africa, Indian Ocean Islands, Asia, and South/Central America. Many steps of the replication and expression of CHIKV's 12-kb RNA genome are highly dependent on cellular factors, which thus constitute potential therapeutic targets. SILAC and LC-MS/MS were used to define the temporal dynamics of the cellular response to infection. Using samples harvested at 8, 10, and 12 h postinfection, over 4700 proteins were identified and per time point 2800-3500 proteins could be quantified in both biological replicates. At 8, 10, and 12 h postinfection, 13, 38, and 106 proteins, respectively, were differentially expressed. The majority of these proteins showed decreased abundance. Most subunits of the RNA polymerase II complex were progressively degraded, which likely contributes to the transcriptional host shut-off observed during CHIKV infection. Overexpression of four proteins that were significantly downregulated (Rho family GTPase 3 (Rnd3), DEAD box helicase 56 (DDX56), polo-like kinase 1 (Plk1), and ubiquitin-conjugating enzyme E2C (UbcH10) reduced susceptibility of cells to CHIKV infection, suggesting that infection-induced downregulation of these proteins is beneficial for CHIKV replication. All MS data have been deposited in the ProteomeXchange with identifier PXD001330 (http://proteomecentral.proteomexchange.org/dataset/PXD001330).

Transactivation of programmed ribosomal frameshifting by a viral protein.

Programmed -1 ribosomal frameshifting (-1 PRF) is a widely used translational mechanism facilitating the expression of two polypeptides from a single mRNA. Commonly, the ribosome interacts with an mRNA secondary structure that promotes -1 frameshifting on a homopolymeric slippery sequence. Recently, we described an unusual -2 frameshifting (-2 PRF) signal directing efficient expression of a transframe protein [nonstructural protein 2TF (nsp2TF)] of porcine reproductive and respiratory syndrome virus (PRRSV) from an alternative reading frame overlapping the viral replicase gene. Unusually, this arterivirus PRF signal lacks an obvious stimulatory RNA secondary structure, but as confirmed here, can also direct the occurrence of -1 PRF, yielding a third, truncated nsp2 variant named "nsp2N." Remarkably, we now show that both -2 and -1 PRF are transactivated by a protein factor, specifically a PRRSV replicase subunit (nsp1ß). Embedded in nsp1ß's papain-like autoproteinase domain, we identified a highly conserved, putative RNA-binding motif that is critical for PRF transactivation. The minimal RNA sequence required for PRF was mapped within a 34-nt region that includes the slippery sequence and a downstream conserved CCCANCUCC motif. Interaction of nsp1ß with the PRF signal was demonstrated in pull-down assays. These studies demonstrate for the first time, to our knowledge, that a protein can function as a transactivator of ribosomal frameshifting. The newly identified frameshifting determinants provide potential antiviral targets for arterivirus disease control and prevention. Moreover, protein-induced transactivation of frameshifting may be a widely used mechanism, potentially including previously undiscovered viral strategies to regulate viral gene expression and/or modulate host cell translation upon infection.

Efficient -2 frameshifting by mammalian ribosomes to synthesize an additional arterivirus protein.

Programmed -1 ribosomal frameshifting (-1 PRF) is a gene-expression mechanism used to express many viral and some cellular genes. In contrast, efficient natural utilization of -2 PRF has not been demonstrated previously in eukaryotic systems. Like all nidoviruses, members of the Arteriviridae (a family of positive-stranded RNA viruses) express their replicase polyproteins pp1a and pp1ab from two long ORFs (1a and 1b), where synthesis of pp1ab depends on -1 PRF. These polyproteins are posttranslationally cleaved into at least 13 functional nonstructural proteins. Here we report that porcine reproductive and respiratory syndrome virus (PRRSV), and apparently most other arteriviruses, use an additional PRF mechanism to access a conserved alternative ORF that overlaps the nsp2-encoding region of ORF1a in the +1 frame. We show here that this ORF is translated via -2 PRF at a conserved G_GUU_UUU sequence (underscores separate ORF1a codons) at an estimated efficiency of around 20%, yielding a transframe fusion (nsp2TF) with the N-terminal two thirds of nsp2. Expression of nsp2TF in PRRSV-infected cells was verified using specific Abs, and the site and direction of frameshifting were determined via mass spectrometric analysis of nsp2TF. Further, mutagenesis showed that the frameshift site and an unusual frameshift-stimulatory element (a conserved CCCANCUCC motif 11 nucleotides downstream) are required to direct efficient -2 PRF. Mutations preventing nsp2TF expression impair PRRSV replication and produce a small-plaque phenotype. Our findings demonstrate that -2 PRF is a functional gene-expression mechanism in eukaryotes and add another layer to the complexity of arterivirus genome expression.

Non-invasive prenatal diagnosis of beta-thalassemia and sickle-cell disease using pyrophosphorolysis-activated polymerization and melting curve analysis.

OBJECTIVE: The aim of this study was to develop a pyrophosphorolysis-activated polymerization (PAP) assay for non-invasive prenatal diagnosis (NIPD) of ß-thalassemia major and sickle-cell disease (SCD). PAP is able to detect mutations in free fetal DNA in a highly contaminating environment of maternal plasma DNA. METHODS: Pyrophosphorolysis-activated polymerization primers were designed for 12 informative SNPs, genotyped by melting curve analysis (MCA) in both parents. The PAP assay was tested in a series of 13 plasma DNA samples collected from pregnant women. A retrospective NIPD was performed in a couple at risk for SCD. RESULTS: All PAP reactions were optimized and able to detect <3% target gDNA in a background of >97% wildtype gDNA. In all 13 cases, the paternal allele was detected by PAP in maternal plasma at 10 to 18 weeks of gestation. For the couple at risk, PAP showed presence of the normal paternal SNP allele in maternal plasma, which was confirmed by results of the chorionic villus sampling analysis. CONCLUSIONS: In contrast to other methods used for NIPD, the combined PAP and MCA analysis detecting the normal paternal allele is also applicable for couples at risk carrying the same mutation, provided that a previously born child is available for testing to determine the linkage to the paternal SNPs.