Identification of RNA-Protein Interaction Networks Involved in the Norovirus Life Cycle

Abstract

Human noroviruses are one of the major causes of acute gastroenteritis in the developed world, yet our understanding of their molecular mechanisms of genome translation and replication lags behind that for many RNA viruses. Due to the nonculturable nature of human noroviruses, many related members of the Caliciviridae family of small RNA viruses are often used as model systems to dissect the finer details of the norovirus life cycle. Murine norovirus (MNV) has provided one such system with which to study the basic mechanisms of norovirus translation and replication in cell culture. In this report we describe the use of riboproteomics to identify host factors that interact with the extremities of the MNV genome. This network of RNA-protein interactions contains many well-characterized host factors, including PTB, La, and DDX3, which have been shown to play a role in the life cycle of other RNA viruses. By using RNA coimmunoprecipitation, we confirmed that a number of the factors identified using riboproteomics are associated with the viral RNA during virus replication in cell culture. We further demonstrated that RNA inhibition-mediated knockdown of the intracellular levels of a number of these factors inhibits or slows norovirus replication in cell culture, allowing identification of new intracellular targets for this important group of pathogens. The genomes of small positive-strand RNA viruses must not only encode the viral proteins required for viral genome replication and infectious virus production, but they must also contain cis-acting RNA sequences and structures that play critical roles in the virus life cycle (40). Host cell factors that interact with these RNA elements play important roles in all aspects of the virus life cycle (49); consequently, the manipulation of these interactions via the introduction of mutations into the viral genome is a viable approach to rational attenuation (24,26,57,78). In addition, the modification of these RNA-protein interactions by using small-molecule or peptide inhibitors has been shown to provide antiviral activity, at least in cell culture (8,19).Noroviruses, first identified in 1972 by Albert Kapikian (33), are now accepted as the major cause of viral gastroenteritis in the developed world (21). Reports indicate that in excess of 23 million cases occur each year in the United States, resulting in a significant economic impact. Now over 20 years since the first full genome sequence of a norovirus became available (77), investigators are still unable to efficiently propagate human noroviruses in cell culture (15, 37). Recent data have indicated that the viral RNA purified from the feces of individuals infected with Norwalk virus, the prototype human norovirus, can replicate when transfected into immortalized cells in culture (23). These data clearly demonstrate that one of the major blocks in the ability of human noroviruses to replicate in cell culture is the ability of the virus to enter and spread from cell to cell. It is also clear that the intracellular environ...

Norovirus Genome Circularization and Efficient Replication Are Facilitated by Binding of PCBP2 and hnRNP A1

“…Since both PCBP2-His and His-hnRNP A1 recombinant proteins were able to stabilize the 5=-3=-end interactions of the MNV-1 genomic RNA in vitro, the abilities of both proteins to interact directly with the 5= and 3= ends of the MNV-1 genomic RNA were further analyzed. PCBP2 has been previously identified as a component of an RNP formed on the 5= and the 3= ends of the NV (14,17) and the MNV-1 genome (14, 39), and hnRNP A1 was recently reported to bind to the MNV-1 3= UTR (45). Therefore, the ability of the recombinant His-hnRNP A1 to bind to the 5= or the 3= end of the MNV-1 genomic RNA was confirmed by EMSA (Fig.…”

“…A spacer sequence consisting of 45 N nucleotides between the 5= and 3= RNA sequences was used to facilitate RNA folding (33). A folding prediction with a ⌬G of Ϫ70.6 was obtained, where all stem-loop structures predicted previously and confirmed by biochemical mapping were conserved (18,45). In addition, a base-pairing region formed from a sequence of 6 nt (64 to 68) of the 5= end (ACAAGA) and nt 7323 to 7328 within the 3= UTR (UUUUGU) was predicted (Fig.…”

“…Twelve hours posttransfection, the cells were infected with MNV-1 (MOI ϭ 5 TCID 50 /cell), and samples were harvested for RNA isolation, virus titer determination, and Western blotting at 18 h p.i. The virus titer was determined by TCID 50 , and the viral RNA copy number was quantified using RT-quantitative PCR (qPCR) as described previously (14,45). The results were plotted using Sigma Plot, and two-way analysis of variance (ANOVA) was performed as a method of statistical analysis.…”

Exploración de la interacción entre la región 5´UTR del Sugarcane Mosaic Virus (SCMV) y proteínas del hospedero maíz

“…Los factores celulares del hospedero que interactúan con estos elementos de ARN, desempeñan funciones importantes en todos los aspectos del ciclo de vida del virus [17]. En consecuencia, la manipulación de estas interacciones o la modifi cación de las interacciones RNA-proteína pueden permitir una atenuación o una actividad antiviral [22].…”

“…Existe información limitada respecto a proteínas del hospedero que interactúen con las regiones no traducidas de potyvirus, sin embargo se ha reportado la interacción de proteínas de hospedero con las regiones 5´UTR y 3´UTR no traducidas del genoma del virus Norwalk [17].…”