Specific Anchoring and Local Translation of Poxviral ATI mRNA at Cytoplasmic Inclusion Bodies.

On-site translation of mRNAs provides an efficient means of subcellular protein localization. In eukaryotic cells, the transport of cellular mRNAs to membraneless sites usually occurs prior to translation and involves specific sequences known as zipcodes that interact with RNA binding and motor proteins. Poxviruses replicate in specialized cytoplasmic factory regions where DNA synthesis, transcription, translation, and virion assembly occur. Some poxviruses embed infectious virus particles outside of factories in membraneless protein bodies with liquid gel-like properties known as A-type inclusions (ATIs) that are comprised of numerous copies of the viral 150-kDa ATI protein. Here, we demonstrate by fluorescent in situ hybridization that these inclusions are decorated with ATI mRNA. On-site translation is supported by the localization of a translation initiation factor eIF4E and by ribosome-bound nascent chain ribopuromycylation. Nascent peptide-mediated anchoring of ribosome-mRNA translation complexes to the inclusions is suggested by release of the mRNA by puromycin, a peptide chain terminator. Following puromycin washout, relocalization of ATI mRNA at inclusions depends on RNA and protein synthesis but requires neither microtubules nor actin polymerization. Further studies show that the ATI mRNAs remain near the sites of transcription in the factory regions when stop codons are introduced near the N terminus of the ATI or large truncations are made at the N or C termini. Instead of using a zipcode, we propose that ATI mRNA localization is mediated by ribosome-bound nascent ATI polypeptides that interact with ATI protein in inclusions and thereby anchor the complex for multiple rounds of mRNA translation.IMPORTANCE Poxvirus genome replication, transcription, translation, and virion assembly occur at sites within the cytoplasm known as factories. Some poxviruses sequester infectious virions outside of the factories in inclusion bodies comprised of numerous copies of the 150-kDa ATI protein, which can provide stability and protection in the environment. We provide evidence that ATI mRNA is anchored by nascent peptides and translated at the inclusion sites rather than in virus factories. Association of ATI mRNA with inclusion bodies allows multiple rounds of local translation and prevents premature ATI protein aggregation and trapping of virions within the factory.

Human Host Range Restriction of the Vaccinia Virus C7/K1 Double Deletion Mutant Is Mediated by an Atypical Mode of Translation Inhibition.

Replication of vaccinia virus in human cells depends on the viral C7 or K1 protein. A previous human genome-wide short interfering RNA (siRNA) screen with a C7/K1 double deletion mutant revealed SAMD9 as a principal host range restriction factor along with additional candidates, including WDR6 and FTSJ1. To compare their abilities to restrict replication, the cellular genes were individually inactivated by CRISPR/Cas9 mutagenesis. The C7/K1 deletion mutant exhibited enhanced replication in each knockout (KO) cell line but reached wild-type levels only in SAMD9 KO cells. SAMD9 was not depleted in either WDR6 or FTSJ1 KO cells, suggesting less efficient alternative rescue mechanisms. Using the SAMD9 KO cells as controls, we verified a specific block in host and viral intermediate and late protein synthesis in HeLa cells and demonstrated that the inhibition could be triggered by events preceding viral DNA replication. Inhibition of cap-dependent and -independent protein synthesis occurred primarily at the translational level, as supported by DNA and mRNA transfection experiments. Concurrent with collapse of polyribosomes, viral mRNA was predominantly in 80S and lighter ribonucleoprotein fractions. We confirmed the accumulation of cytoplasmic granules in HeLa cells infected with the C7/K1 deletion mutant and further showed that viral mRNA was sequestered with SAMD9. RNA granules were still detected in G3BP KO U2OS cells, which remained nonpermissive for the C7/K1 deletion mutant. Inhibition of cap-dependent and internal ribosome entry site-mediated translation, sequestration of viral mRNA, and failure of PKR, RNase L, or G3BP KO cells to restore protein synthesis support an unusual mechanism of host restriction.IMPORTANCE A dynamic relationship exists between viruses and their hosts in which each ostensibly attempts to exploit the other's vulnerabilities. A window is opened into the established condition, which evolved over millennia, if loss-of-function mutations occur in either the virus or host. Thus, the inability of viral host range mutants to replicate in specific cells can be overcome by identifying and inactivating the opposing cellular gene. Here, we investigated a C7/K1 host range mutant of vaccinia virus in which the cellular gene SAMD9 serves as the principal host restriction factor. Host restriction was triggered early in infection and manifested as a block in translation of viral mRNAs. Features of the block include inhibition of cap-dependent and internal ribosome entry site-mediated translation, sequestration of viral RNA, and inability to overcome the inhibition by inactivation of protein kinase R, ribonuclease L, or G3 binding proteins, suggesting a novel mechanism of host restriction.

Identification of Vaccinia Virus Replisome and Transcriptome Proteins by Isolation of Proteins on Nascent DNA Coupled with Mass Spectrometry.

Poxviruses replicate within the cytoplasm and encode proteins for DNA and mRNA synthesis. To investigate poxvirus replication and transcription from a new perspective, we incorporated 5-ethynyl-2'-deoxyuridine (EdU) into nascent DNA in cells infected with vaccinia virus (VACV). The EdU-labeled DNA was conjugated to fluor- or biotin-azide and visualized by confocal, superresolution, and transmission electron microscopy. Nuclear labeling decreased dramatically after infection, accompanied by intense labeling of cytoplasmic foci. The nascent DNA colocalized with the VACV single-stranded DNA binding protein I3 in multiple puncta throughout the interior of factories, which were surrounded by endoplasmic reticulum. Complexes containing EdU-biotin-labeled DNA cross-linked to proteins were captured on streptavidin beads. After elution and proteolysis, the peptides were analyzed by mass spectrometry to identify proteins associated with nascent DNA. The known viral replication proteins, a telomere binding protein, and a protein kinase were associated with nascent DNA, as were the DNA-dependent RNA polymerase and intermediate- and late-stage transcription initiation and elongation factors, plus the capping and methylating enzymes. These results suggested that the replicating pool of DNA is transcribed and that few if any additional viral proteins directly engaged in replication and transcription remain to be discovered. Among the host proteins identified by mass spectrometry, topoisomerases IIα and IIß and PCNA were noteworthy. The association of the topoisomerases with nascent DNA was dependent on expression of the viral DNA ligase, in accord with previous proteomic studies. Further investigations are needed to determine possible roles for PCNA and other host proteins detected.IMPORTANCE Poxviruses, unlike many well-characterized animal DNA viruses, replicate entirely within the cytoplasm of animal cells, raising questions regarding the relative roles of viral and host proteins. We adapted newly developed procedures for click chemistry and iPOND (Isolation of proteins on nascent DNA) to investigate vaccinia virus (VACV), the prototype poxvirus. Nuclear DNA synthesis ceased almost immediately following VACV infection, followed swiftly by the synthesis of viral DNA within discrete cytoplasmic foci. All viral proteins known from genetic and proteomic studies to be required for poxvirus DNA replication were identified in the complexes containing nascent DNA. The additional detection of the viral DNA-dependent RNA polymerase and intermediate and late transcription factors provided evidence for a temporal coupling of replication and transcription. Further studies are needed to assess the potential roles of host proteins, including topoisomerases IIα and IIß and PCNA, which were found associated with nascent DNA.

Poxvirus decapping enzymes enhance virulence by preventing the accumulation of dsRNA and the induction of innate antiviral responses.

Poxvirus replication involves synthesis of double-stranded RNA (dsRNA), which can trigger antiviral responses by inducing phosphorylation-mediated activation of protein kinase R (PKR) and stimulating 2'5'-oligoadenylate synthetase (OAS). PKR inactivates the translation initiation factor eIF2α via phosphorylation, while OAS induces the endonuclease RNase L to degrade RNA. We show that poxvirus decapping enzymes D9 and D10, which remove caps from mRNAs, inhibit these antiviral responses by preventing dsRNA accumulation. Catalytic site mutations of D9 and D10, but not of either enzyme alone, halt vaccinia virus late protein synthesis and inhibit virus replication. Infection with the D9-D10 mutant was accompanied by massive mRNA reduction, cleavage of ribosomal RNA, and phosphorylation of PKR and eIF2α that correlated with a ∼ 15-fold increase in dsRNA compared to wild-type virus. Additionally, mouse studies show extreme attenuation of the mutant virus. Thus, vaccinia virus decapping, in addition to targeting mRNAs for degradation, prevents dsRNA accumulation and anti-viral responses.

Colocalization of transcription and translation within cytoplasmic poxvirus factories coordinates viral expression and subjugates host functions.

Poxviruses are large DNA viruses that include the causal agent of human smallpox and vaccinia virus. Poxviruses replicate in cytoplasmic foci known as DNA factories. Here we show that a virus-encoded transcription factor, viral mRNA, cellular RNA-binding protein heterodimer G3BP/Caprin-1 (p137), translation initiation factors eIF4E and eIF4G, and ribosomal proteins are concentrated in the same subdomains of cytoplasmic DNA factories. Furthermore, a cell coinfected with two recombinant vaccinia viruses expressing a virus core protein fused to cyan or yellow fluorescent protein displayed separate cyan and yellow factories, indicating that each factory formed from a single genome and was the site of transcription and translation as well as DNA replication. Hijacking of the host translation apparatus within the factory likely enhances the efficiency of virus replication and contributes to the suppression of host protein synthesis, thereby facilitating poxvirus subjugation of the cell.

Vaccinia virus intermediate stage transcription is complemented by Ras-GTPase-activating protein SH3 domain-binding protein (G3BP) and cytoplasmic activation/proliferation-associated protein (p137) individually or as a heterodimer.

Transcription of the DNA genome of vaccinia virus occurs in the cytoplasm and is temporally programmed by early, intermediate, and late stage-specific transcription factors in conjunction with a viral multisubunit RNA polymerase. The RNA polymerase, capping enzyme, and three factors (VITF-1, VITF-2, and VITF-3) are sufficient for in vitro transcription of a DNA template containing an intermediate stage promoter. Vaccinia virus intermediate transcription factor (VITF)-1 and -3 are virus-encoded, whereas VITF-2 was partially purified from extracts of uninfected HeLa cells. Using purified and recombinant viral proteins, we showed that the HeLa cell factor was required for transcription of linear or nicked circular templates but not of super coiled DNA. HeLa cell polypeptides of approximately 110 and 66 kDa copurified with VITF-2 activity through multiple chromatographic steps. The polypeptides were separated by SDS-polyacrylamide gel electrophoresis and identified by mass spectrometry as Ras-GTPase-activating protein SH3 domain-binding protein (G3BP) and p137, recently named cytoplasmic activation/proliferation-associated protein-1. The co-purification of the two polypeptides with transcription-complementing activity was confirmed with specific antibodies, and their association with each other was demonstrated by affinity chromatography of tagged recombinant forms. Furthermore, recombinant G3BP and p137 expressed individually or together in mammalian or bacterial cells complemented the activity of the viral RNA polymerase and transcription factors. The involvement of cellular proteins in transcription of intermediate stage genes may regulate the transition between early and late phases of vaccinia virus replication.