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Rotaviruses pose a significant threat to young children. To identify novel pro- and anti-rotavirus host factors, we performed genome-wide CRISPR/Cas9 screens using rhesus rotavirus and African green monkey cells. Genetic deletion of either SERPINB1 or TMEM236, the top two antiviral factors, in MA104 cells increased virus titers in a rotavirus strain independent manner. Using this information, we optimized the existing rotavirus reverse genetics systems by combining SERPINB1 knockout MA104 cells with a C3P3-G3 helper plasmid. We improved the recovery efficiency and rescued several low-titer rotavirus reporter and mutant strains that prove difficult to rescue otherwise. Furthermore, we demonstrate that TMEM236 knockout in Vero cells supported higher yields of two live-attenuated rotavirus vaccine strains than the parental cell line and represents a more robust vaccine-producing cell substrate. Collectively, we developed a third-generation optimized rotavirus reverse genetics system and generated gene-edited Vero cells as a new substrate for improving rotavirus vaccine production., (© 2024. The Author(s).)

Reoviruses, like many eukaryotic viruses, contain an inverted 7-methylguanosine (m7G) cap linked to the 5' nucleotide of mRNA. The traditional functions of capping are to promote mRNA stability, protein translation, and concealment from cellular proteins that recognize foreign RNA. To address the role of mRNA capping during reovirus replication, we assessed the benefits of adding the African swine fever virus NP868R capping enzyme during reovirus rescue. C3P3, a fusion protein containing T7 RNA polymerase and NP868R, was found to increase protein expression 5- to 10-fold compared to T7 RNA polymerase alone while enhancing reovirus rescue from the current reverse genetics system by 100-fold. Surprisingly, RNA stability was not increased by C3P3, suggesting a direct effect on protein translation. A time course analysis revealed that C3P3 increased protein synthesis within the first 2 days of a reverse genetics transfection. This analysis also revealed that C3P3 enhanced processing of outer capsid μ1 protein to μ1C, a previously described hallmark of reovirus assembly. Finally, to determine the rate of infectious-RNA incorporation into new virions, we developed a new recombinant reovirus S1 gene that expressed the fluorescent protein UnaG. Following transfection of cells with UnaG and infection with wild-type virus, passage of UnaG through progeny was significantly enhanced by C3P3. These data suggest that capping provides nontraditional functions to reovirus, such as promoting assembly and infectious-RNA incorporation. IMPORTANCE Our findings expand our understanding of how viruses utilize capping, suggesting that capping provides nontraditional functions to reovirus, such as promoting assembly and infectious-RNA incorporation, in addition to enhancing protein translation. Beyond providing mechanistic insight into reovirus replication, our findings also show that reovirus reverse genetics rescue is enhanced 100-fold by the NP868R capping enzyme. Since reovirus shows promise as a cancer therapy, efficient reovirus reverse genetics rescue will accelerate production of recombinant reoviruses as candidates to enhance therapeutic potency. NP868R-assisted reovirus rescue will also expedite production of recombinant reovirus for mechanistic insights into reovirus protein function and structure., (Copyright © 2017 American Society for Microbiology.)

It is regularly thought that human complex disorder susceptibility genes show differences in gene expression between normal and pathologic tissues. Thus, differences of transcript amounts could be indicative of complex disorder susceptibility loci and, therefore, be used for the discovery or the validation of human susceptibility genes to complex disorders/traits. Whether human complex disorder susceptibility genes effectively display differences in transcript amounts was tested by meta-analysis of the published literature comparing transcript amounts of well-validated human susceptibility genes to complex traits/disorders. A total of 94 gene-disease associations, which were studied in at least three independent studies and showed strong evidence of positive association, were analyzed. For 23 out of these 94 well-validated gene-disease associations, 120 gene expression studies comparing normal and pathologic human tissues were found. For 60 out of these 120 gene expression studies, the difference of level expression between normal and pathologic human tissues was statistically significant. This result was highly significant, as only 6 significant results were expected randomly under the null hypothesis (P < 10(-112)). A large excess of replication studies were also found, which were in agreement with the original report (P = 6 x 10(-4)). However, the overall level of expression change between normal and pathologic human tissues was relatively moderate, because only 36 (60%) and 19 (31.6%) out of the 62 statistically significant gene expression studies reached 2- or 3-fold changes in expression level, respectively. The present meta-analysis confirms statistical differences of expression levels between normal and pathologic human tissues for human susceptibility genes to complex traits/disorders. However, the levels of differences in transcript amounts appear to be relatively weak. These findings rationalize the use of gene expression for the discovery/validation of human susceptibility genes, but the weak differences of expression typically found should be taken into account for the design of such studies.