Murine norovirus (MNV), identified in 2003, is the only norovirus which replicates efficiently in tissue culture and as a result has been used extensively as a model for human noroviruses, a major cause of acute gastroenteritis. The current report describes the generation of a new approach to reverse genetics recovery of genetically defined MNV that relies on the transfection of in vitro transcribed capped RNA directly into cells. The use of the recently developed ScriptCap post-transcriptional enzymatic capping system, followed by optimized Neon mediated electroporation of the highly permissive RAW 264.7 cells, resulted in the rapid and robust recovery of infectious MNV. Transfection of cells capable of supporting virus replication but not permissive to virus infection, namely human or hamster kidney cells, also resulted in robust recovery of infectious virus without subsequent amplification by multiple rounds of re-infection. This latter system may provide a reproducible method to measure the specific infectivity of mutant norovirus RNA allowing the accurate quantitation of the effect of mutations on norovirus replication.

Long noncoding RNAs (lncRNAs) play diverse roles in biological processes. Aedes aegypti ( Ae . aegypti ), a blood-sucking mosquito, is the principal vector responsible for replication and transmission of arboviruses including dengue, Zika, and Chikungunya virus. Systematic identification and developmental characterisation of Ae . aegypti lncRNAs are still limited. We performed genome-wide identification of lncRNAs, followed by developmental profiling of lncRNA in Ae . aegypti . We identified a total of 4,689 novel lncRNA transcripts, of which 2,064, 2,076, and 549 were intergenic, intronic, and antisense respectively. Ae . aegypti lncRNAs share many characteristics with other species including low expression, low GC content, short in length, and low conservation. Besides, the expression of Ae . aegypti lncRNAs tend to be correlated with neighbouring and antisense protein-coding genes. A subset of lncRNAs shows evidence of maternal inheritance; hence, suggesting potential role of lncRNAs in early-stage embryos. Additionally, lncRNAs show higher tendency to be expressed in developmental and temporal specific manner. The results from this study provide foundation for future investigation on the function of Ae . aegypti lncRNAs.

Using a cell-based assay for RNA synthesis by the RNA-dependent RNA polymerase (RdRp) of noroviruses, we previously observed that VP1, the major structural protein of the human GII.4 norovirus, enhanced the GII.4 RdRp activity but not that of the related murine norovirus (MNV) or other unrelated RNA viruses (C. V. Subba-Reddy, I. Goodfellow, and C. C. Kao, J. Virol. 85:13027–13037, 2011). Here, we examine the mechanism of VP1 enhancement of RdRp activity and the mechanism of mouse norovirus replication. We determined that the GII.4 and MNV VP1 proteins can enhance cognate RdRp activities in a concentration-dependent manner. The VP1 proteins coimmunoprecipitated with their cognate RdRps. Coexpression of individual domains of VP1 with the viral RdRps showed that the VP1 shell domain (SD) was sufficient to enhance polymerase activity. Using SD chimeras from GII.4 and MNV, three loops connecting the central β-barrel structure were found to be responsible for the species-specific enhancement of RdRp activity. A differential scanning fluorimetry assay showed that recombinant SDs can bind to the purified RdRpsin vitro. An MNV replicon with a frameshift mutation in open reading frame 2 (ORF2) that disrupts VP1 expression was defective for RNA replication, as quantified by luciferase reporter assay and real-time quantitative reverse transcription-PCR (qRT-PCR).Trans-complementation of VP1 or its SD significantly recovered the VP1 knockout MNV replicon replication, and the presence or absence of VP1 affected the kinetics of viral RNA synthesis. The results document a regulatory role for VP1 in the norovirus replication cycle, further highlighting the paradigm of viral structural proteins playing additional functional roles in the virus life cycle.

All members of the Caliciviridae family of viruses produce a subgenomic RNA during infection. The subgenomic RNA typically encodes only the major and minor capsid proteins, but in murine norovirus (MNV), the subgenomic RNA also encodes the VF1 protein, which functions to suppress host innate immune responses. To date, the mechanism of norovirus subgenomic RNA synthesis has not been characterized. We have previously described the presence of an evolutionarily conserved RNA stem-loop structure on the negative-sense RNA, the complementary sequence of which codes for the viral RNA-dependent RNA polymerase (NS7). The conserved stem-loop is positioned 6 nucleotides 3= of the start site of the subgenomic RNA in all caliciviruses. We demonstrate that the conserved stem-loop is essential for MNV viability. Mutant MNV RNAs with substitutions in the stem-loop replicated poorly until they accumulated mutations that revert to restore the stem-loop sequence and/or structure. The stem-loop sequence functions in a noncoding context, as it was possible to restore the replication of an MNV mutant by introducing an additional copy of the stem-loop between the NS7-and VP1-coding regions. Finally, in vitro biochemical data suggest that the stem-loop sequence is sufficient for the initiation of viral RNA synthesis by the recombinant MNV RNA-dependent RNA polymerase, confirming that the stem-loop forms the core of the norovirus subgenomic promoter. IMPORTANCENoroviruses are a significant cause of viral gastroenteritis, and it is important to understand the mechanism of norovirus RNA synthesis. Here we describe the identification of an RNA stem-loop structure that functions as the core of the norovirus subgenomic RNA promoter in cells and in vitro. This work provides new insights into the molecular mechanisms of norovirus RNA synthesis and the sequences that determine the recognition of viral RNA by the RNA-dependent RNA polymerase. N oroviruses, members of the Caliciviridae family of small positive-sense RNA viruses, are a major cause of viral gastroenteritis in the developed world (1, 2). Despite their impact, noroviruses remain poorly characterized, as an in-depth understanding of the molecular mechanisms of norovirus genome translation and replication has been hampered by the inability to culture human norovirus (3). Murine norovirus (MNV) is a model system with which to understand the norovirus life cycle (4), as it replicates in cultured cells (5) and a number of tractable reverse genetics systems are available (6-9). Studies of MNV have therefore led to a number of significant advances in the understanding of the molecular mechanism of norovirus genome translation and replication (reviewed in reference 10).The norovirus RNA genome typically carries three open reading frames (ORFs) (11). MNV also has an additional ORF in the region overlapping the VP1-coding region (12, 13) (Fig. 1A). All members of the Caliciviridae family of small positive-sense RNA viruses synthesize a shorter-than-genome-length, subgenomic RNA (sgRNA)...