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SummaryRubella virus (RUB) assembles its replication complexes (RCs) in modified organelles of endolysosomal origin, known as cytopathic vacuoles (CPVs). These peculiar structures are key elements of RUB factories, where rough endoplasmic reticulum, mitochondria, and Golgi are recruited. Bicistronic RUB replicons expressing an antibiotic resistance gene either in the presence or the absence of the RUB capsid (C) gene were used to study the structure of RCs in transfected cells. Confocal microscopy showed that the RUB replicase components P90 and P150 localized to CPVs, as did double-stranded RNA (dsRNA), a marker for RNA synthesis. Electron microscopy (EM) showed that replicons generated CPVs containing small vesicles and large vacuoles, similar to CPVs from RUB-infected cells and that the replicase proteins were sufficient for organelle recruitment. Some of these CPVs contained straight membranes. When cross-sectioned, these rigid membranes appeared to be sheets of closely packed proteins. Immuno-EM revealed that these sheets, apparently in contact with the cytosol, contained both P150 and P90, as well as dsRNA, and thus could be two-dimensional arrays of functional viral replicases. Labelling of dsRNA after streptolysin-O permeabilization showed that replication of viral genome takes place on the cytoplasmic side of CPVs. When present, C accumulated around CPVs. Mitochondrial protein P32 was detected within modified CPVs, the first demonstration of involvement of this protein, which interacts with C, with RCs.

Summary More than any other methodology, transmission electron microscopy (TEM) has contributed to our understanding of the architecture and organization of cells. With current detection limits approaching atomic resolution, it will ultimately become possible to ultrastructurally image intracellular macromolecular assemblies in situ. Presently, however, methods to unambiguously identify proteins within the crowded environment of the cell’s interior are lagging behind. We describe a novel approach, metal-tagging TEM (METTEM) that allows detection of intracellular proteins in mammalian cells with high specificity, exceptional sensitivity and at molecular scale resolution. In live cells treated with gold salts, proteins bearing a small metal-binding tag will form 1-nm gold nanoclusters, readily detectable in electron micrographs. The applicability and strength of METTEM is demonstrated by a study of Rubella virus replicase and capsid proteins, which revealed virus-induced cell structures not seen before.

The rubella virus capsid protein (C) has been shown to complement a lethal deletion (termed ⌬NotI) in P150 replicase protein. To investigate this phenomenon, we generated two lines of Vero cells that stably expressed either C (C-Vero cells) or C lacking the eight N-terminal residues (C⌬8-Vero cells), a construct previously shown to be unable to complement ⌬NotI. In C-Vero cells but not Vero or C⌬8-Vero cells, replication of a wild-type (wt) replicon expressing the green fluorescent protein (GFP) reporter gene (RUBrep/GFP) was enhanced, and replication of a replicon with ⌬NotI (RUBrep/GFP-⌬NotI) was rescued. Surprisingly, replicons with deleterious mutations in the 5 and 3 cis-acting elements were also rescued in C-Vero cells. Interestingly, the C⌬8 construct localized to the nucleus while the C construct localized in the cytoplasm, explaining the lack of enhancement and rescue in C⌬8-Vero cells since rubella virus replication occurs in the cytoplasm. Enhancement and rescue in C-Vero cells were at a basic step in the replication cycle, resulting in a substantial increase in the accumulation of replicon-specific RNAs. There was no difference in translation of the nonstructural proteins in C-Vero and Vero cells transfected with the wt and mutant replicons, demonstrating that enhancement and rescue were not due to an increase in the efficiency of translation of the transfected replicon transcripts. In replicon-transfected C-Vero cells, C and the P150 replicase protein associated by coimmunoprecipitation, suggesting that C might play a role in RNA replication, which could explain the enhancement and rescue phenomena. A unifying model that accounts for enhancement of wt replicon replication and rescue of diverse mutations by the rubella virus C protein is proposed.Rubella virus (RUB), the causative agent of rubella and congenital rubella syndrome, is the sole member of the genus Rubivirus in the family Togaviridae of animal viruses (for a review, see reference 11). The rubella virus genome is a singlestranded, plus-polarity RNA of 9,762 nucleotides (nts) in length that contains two open reading frames (ORFs). The 5Ј-proximal ORF, the nonstructural protein ORF (NS-ORF), encodes two nonstructural proteins involved in virus RNA replication, P150 and P90 (the gene order is 5Ј-P150-P90-3Ј within the ORF), while the 3Ј-proximal ORF, the structural protein ORF (SP-ORF), encodes the three virion proteins, the capsid protein (C) and envelope glycoproteins E1 and E2 (5Ј-C-E2-E1-3Ј within the ORF). The NS-ORF is translated from the genomic RNA, while the SP-ORF is translated from a subgenomic (SG) RNA consisting of roughly the 3Ј third of the genomic RNA. Both of these RNA species are transcribed from a genome-length RNA of minus polarity in infected cells.The rubella virus C protein is a multifunctional protein. C is involved in several intermolecular interactions. First, it contains a motif between residues 28 and 56 (C is 300 amino acids [aa] in length) that binds the genomic RNA (16). Recent characterization has revealed th...

A rubella virus (RUB) replicon was constructed by replacing the 3' proximal structural protein ORF (SP-ORF) in Robo402, a RUB infectious cDNA clone, with a reporter gene, green fluorescent protein (GFP). This replicon, RUBrep/GFP, mimics naturally occurring RUB defective-interfering (DI) RNAs generated during serial undiluted passage that maintain the 5' proximal nonstructural protein ORF (NS-ORF) but contain deletions in the SP-ORF. Following transfection of Vero cells with in vitro RNA transcripts from RUBrep/GFP, replicon replication occurred and the replicon was amplified and spread to other cells in the presence of standard helper virus. GFP expression was a much more sensitive indicator of replicon replication than was Northern analysis to detect replicon-specific RNAs. Most of a series of RUBrep/GFP constructs with deletions in the NS-ORF not only were incapable of self-replication, but were not amplified by standard helper virus. The only exception was a construct with an in-frame deletion between two NotI sites that removed nucleotides 1685-2192 of the genome; this construct did not express GFP by itself, but did express GFP in the presence of standard helper RUB and was spread to other cells. Thus, with the exception of this region, the NS-ORF is required in cis for amplification of RUB replicons by standard helper virus, explaining the selection of DI RNAs that maintain the NS-ORF. Surprisingly, when the NotI deletion was introduced into Robo402, a viable virus resulted that replicated only threefold less efficiently than did Robo402 virus. Thus, the NotI region of the NS-ORF is not necessary for virus replication. This deletion covers a region of the NS-ORF without predicted function, which therefore may function as a spacer or hinge between functional domains. Nevertheless, it was an unexpected finding that a small virus such as RUB could dispense with approximately 10% of its genome.

Rubella virus (RUB) replicons with an in-frame deletion of 507 nucleotides between two NotI sites in the P150 nonstructural protein (⌬NotI) do not replicate (as detected by expression of a reporter gene encoded by the replicon) but can be amplified by wild-type helper virus (Tzeng et al., Virology 289:63-73, 2001). Surprisingly, virus with ⌬NotI was viable, and it was hypothesized that this was due to complementation of the NotI deletion by one of the virion structural protein genes. Introduction of the capsid (C) protein gene into ⌬NotI-containing replicons as an in-frame fusion with a reporter gene or cotransfection with both ⌬NotI replicons and RUB replicon or plasmid constructs containing the C gene resulted in replication of the ⌬NotI replicon, confirming the hypothesis that the C gene was the structural protein gene responsible for complementation and demonstrating that complementation could occur either in cis or in trans. Approximately the 5 one-third of the C gene was necessary for complementation. Mutations that prevented translation of the C protein while minimally disturbing the C gene sequence abrogated complementation, while synonymous codon mutations that changed the C gene sequence without affecting the amino acid sequence at the 5 end of the C gene had no effect on complementation, indicating that the C protein, not the C gene RNA, was the moiety responsible for complementation. Complementation occurred at a basic step in the virus replication cycle, because ⌬NotI replicons failed to accumulate detectable virus-specific RNA.Rubella virus (RUB) is the sole member of the genus Rubivirus in the family Togaviridae (for a review, see reference 7). The RUB virion consists of a genomic, single-stranded RNA enclosed in a quasispherical capsid composed of multiple copies of the viral capsid protein, C, which is in turn surrounded by a lipid bilayer envelope in which are embedded two virus glycoproteins, E1 and E2. The RUB genome is 9,762 uncleotides (nt) in length, of positive-polarity, and contains two long open reading frames (ORFs). The 5Ј-proximal ORF, or nonstructural protein ORF (NS-ORF), is translated from the genome RNA into a 240-kDa precursor that is proteolytically cleaved at a single site by a virus-encoded protease into two products: an N-terminal product of 150 kDa (P150) and a C-terminal product of 90 kDa (P90). The 3Ј-proximal ORF, or structural protein ORF (SP-ORF), which is translated from a subgenomic (SG) RNA, encodes the virion proteins in the order 5Ј-C-E2-E1-3Ј; processing of these proteins is mediated by the cellular enzyme signal endopeptidase.The RUB NS proteins function in viral RNA replication. From predictions based on computer alignment with sequences from other viruses, P150 contains (from N terminus to C terminus) a methyltransferase domain, a Y domain, a proline hinge domain, an X domain, and a protease domain that catalyzes the cleavage of the NS precursor; P90 contains helicase and RNA-dependent RNA polymerase (RDRP) domains (12). Of these, the activities of the proteas...

The rubella virus (RUB) nonstructural protein (NS) open reading frame (ORF) encodes a polypeptide ‫؍‬ 4.1°C). The infectivity of an RUB infectious cDNA clone containing the mutations D1210A/D1217A was decreased by ϳ20-fold in comparison to the wild-type (wt) clone, and these mutations rapidly reverted to the wt sequence. The NS protease containing these mutations was less efficient at precursor cleavage than the wt NS protease at 35°C, and the mutant NS protease was temperature sensitive at 39°C, confirming that the Ca 2؉ -binding loop played a structural role in the NS protease and was specifically required for optimal stability under physiological conditions.

In late 2014, Zika virus (ZIKV; Flaviviridae, Flavivirus) emerged as a significant arboviral disease threat in the Western hemisphere. Aedes aegypti and Aedes albopictus have been considered the principal vectors of ZIKV in the New World due to viral isolation frequency and vector competence assessments. Limited reports of Culex transmission potential have highlighted the need for additional vector competence assessments of North American Culex species. Accordingly, North American Culex pipiens and Culex quinquefasciatus were orally exposed and intrathoracically inoculated with the African prototype ZIKV strain and currently circulating Asian lineage ZIKV strains to assess infection, dissemination, and transmission potential. Results indicated that these two North American Culex mosquito species were highly refractory to oral infection with no dissemination or transmission observed with any ZIKV strains assessed. Furthermore, both Culex mosquito species intrathoracically inoculated with either Asian or African lineage ZIKVs failed to expectorate virus in saliva. These in vivo results were further supported by the observation that multiple mosquito cell lines of Culex species origin demonstrated significant growth restriction of ZIKV strains compared with Aedes-derived cell lines. In summation, no evidence for the potential of Cx. pipiens or Cx. quinquefasciatus to serve as a competent vector for ZIKV transmission in North America was observed.