Positive-strand RNA viruses, the largest genetic class of viruses, include numerous important pathogens such as Zika virus. These viruses replicate their RNA genomes in novel, membrane-bounded mini-organelles, but the organization of viral proteins and RNAs in these compartments has been largely unknown. We used cryo-electron tomography to reveal many previously unrecognized features of Flock house nodavirus (FHV) RNA replication compartments. These spherular invaginations of outer mitochondrial membranes are packed with electron-dense RNA fibrils and their volumes are closely correlated with RNA replication template length. Each spherule’s necked aperture is crowned by a striking cupped ring structure containing multifunctional FHV RNA replication protein A. Subtomogram averaging of these crowns revealed twelve-fold symmetry, concentric flanking protrusions, and a central electron density. Many crowns were associated with long cytoplasmic fibrils, likely to be exported progeny RNA. These results provide new mechanistic insights into positive-strand RNA virus replication compartment structure, assembly, function and control.DOI: http://dx.doi.org/10.7554/eLife.25940.001

The poliovirus 3 noncoding region (3 NCR) is necessary for efficient virus replication. A poliovirus mutant, PV⌬3NCR, with a deletion of the entire 3 NCR, yielded a virus that was capable of synthesizing viral RNA, albeit with a replication defect caused by deficient positive-strand RNA synthesis compared to wild-type virus. We detected multiple ribonucleoprotein (RNP) complexes in extracts from poliovirus-infected HeLa cells formed with a probe corresponding to the 5 end of poliovirus negative-strand RNA (the complement of the genomic 3 NCR), and the levels of these RNP complexes increased during the course of viral infection. Previous studies have identified RNP complexes formed with the 3 end of poliovirus negative-strand RNA, including one that contains a 36-kDa protein later identified as heterogeneous nuclear ribonucleoprotein C (hnRNP C). We report here that the 5 end of poliovirus negative-strand RNA is capable of interacting with endogenous hnRNP C, as well as with poliovirus nonstructural proteins. Further, we demonstrate that the addition of recombinant purified hnRNP C proteins can stimulate virus RNA synthesis in vitro and that depletion of hnRNP C proteins in cultured cells results in decreased virus yields and a correspondingly diminished accumulation of positive-strand RNAs. We propose that the association of hnRNP C with poliovirus negative-strand termini acts to stabilize or otherwise promote efficient positive-strand RNA synthesis.Picornaviruses are a group of small, positive-sense RNA viruses that replicate in the host cell cytoplasm. Their genomes are characterized by highly structured 5Ј noncoding regions (NCRs) containing a cruciform RNA structure (termed stemloop I, or cloverleaf) necessary for viral-RNA replication and an internal ribosome entry site (IRES) that allows translation of a virus polyprotein from the single open reading frame of genomic RNA. The virus polyprotein is cleaved by encoded viral proteinases (30) to yield the mature structural and nonstructural proteins, including the RNA-dependent RNA polymerase 3D. Due to the limited size of the genome, picornaviruses have evolved to utilize host cell factors in concert with their own virus-encoded proteins and RNA secondary structures to efficiently drive the replication cycle. Poliovirus, an example of the viruses using such a combination of host and viral functions, is the causative agent of paralytic poliomyelitis and the most extensively studied picornavirus. Cellular proteins play an important role in the replication of the poliovirus RNA genome (19, 37). For example, poly(rC) binding protein 2 (PCBP2) binds to the stem-loop I RNA cruciform structure at the 5Ј end of the genome. Binding of PCBP2 to this region is required for viral-RNA replication, and disruption of the capability of the protein to bind to stem-loop I disrupts RNA replication (55). During the synthesis of poliovirus negativestrand RNA intermediates, it has been proposed that the 5Ј and 3Ј ends of poliovirus positive-sense RNA communicate via interactions fo...

The hnRNP C heterotetramer [(C13)C2] binds RNA polymerase II transcripts in the nucleus, along with other proteins of the core hnRNP complex, and plays an important role in mRNA biogenesis and transport. Infection of HeLa cells with poliovirus causes hnRNP C to relocalize from the nucleus, where it is normally retained during interphase, to the cytoplasm. We have proposed that in the cytoplasm, the protein isoforms of hnRNP C participate in the recognition of viral specific RNAs by the poliovirus replication proteins and/or in the assembly of membrane-bound RNA replication complexes. In SK-OV-3 cells, which express reduced levels of hnRNP C compared to HeLa cells or 293 cells, the kinetics of poliovirus replication are delayed. hnRNP C is also re-localized from the nucleus to the cytoplasm in SK-OV-3 cells infected with poliovirus. Increased expression of hnRNP C in SK-OV-3 cells by transient transfection increases the rate of virus production and overall yield over that seen in mock-transfected cells. We propose that hnRNP C interacts with poliovirus RNA and replication proteins to increase the efficiency of viral genomic RNA synthesis.

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