African Swine Fever Virus NP868R Capping Enzyme Promotes Reovirus Rescue during Reverse Genetics by Promoting Reovirus Protein Expression, Virion Assembly, and RNA Incorporation into Infectious Virions

Abstract

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. 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.

In Vivo Live Imaging of Oncolytic Mammalian Orthoreovirus Expressing NanoLuc Luciferase in Tumor Xenograft Mice

“…Prototype reporter MRVs harboring a chloramphenicol acetyltransferase gene inserted into the MRV S2 gene segment, or a green fluorescent protein gene inserted into the S4 gene segment, replicate only in permissive cell lines expressing S2 or S4 gene products, respectively (12,16). Autonomous replicating MRVs harboring iLOV or UnaG fluorescent genes in the S1 gene segment lack expression of an intact 1 capsid protein, which affects MRV infectivity (17,18). Use of these reporter MRVs for oncolytic research is restricted due to a lack of essential viral proteins.…”

Characterization of a replicating mammalian orthoreovirus with tetracysteine tagged μNS for live cell visualization of viral factories

“…Much of the work defining VFs has been done using transient transfection of plasmids expressing μNS and other virus proteins, primarily because it has thus far been difficult to visualize these proteins during infection in live cells. While researchers have been able to add fluorescent tags to viral proteins in other viruses and recover recombinant virus, the MRV genome has only recently been made amenable to reverse genetics technologies (16-19). Utilizing this technology, a virus in which the coding region within the S4 gene segment was replaced by the EGFP gene has been recovered, however, this virus replicates only when propagated in cells expressing the S4 gene product σ3 (16).…”

“…Utilizing this technology, a virus in which the coding region within the S4 gene segment was replaced by the EGFP gene has been recovered, however, this virus replicates only when propagated in cells expressing the S4 gene product σ3 (16). Both replicating and non-replicating recombinant viruses have also been recovered with fluorescent proteins iLOV and UnaG independently expressed downstream of the N-terminal half of viral spike protein, σ1 (σ1-N), as well as a fusion of σ1-N with UnaG which could infect cells but was unable to replicate in the absence of wildtype MRV (19, 20). In addition, recombinant reoviruses in which small protein coding sequences (6His, HA-tag, and 3HA-tag) were added to the σ1 protein have been recovered (21).…”

Characterization of a Replicating Mammalian Orthoreovirus with Tetracysteine-Tagged μNS for Live-Cell Visualization of Viral Factories

“…While researchers have been able to add fluorescent tags to viral proteins in other viruses and recover recombinant virus, the MRV genome has only recently been made amenable to reverse-genetics technologies (16)(17)(18)(19). Utilizing this technology, a virus in which the coding region within the S4 gene segment was replaced by the enhanced green fluorescent protein (EGFP) gene has been recovered; however, this virus replicates only when propagated in cells expressing the S4 gene product 3 (16).…”

“…Utilizing this technology, a virus in which the coding region within the S4 gene segment was replaced by the enhanced green fluorescent protein (EGFP) gene has been recovered; however, this virus replicates only when propagated in cells expressing the S4 gene product 3 (16). Both replicating and nonreplicating recombinant viruses have also been recovered with fluorescent proteins iLOV and UnaG independently expressed downstream of the N-terminal half of viral spike protein 1 ( 1-N), as well as a fusion of 1-N with UnaG, which could infect cells but was unable to replicate in the absence of wild-type MRV (19,20). In addition, recombinant reoviruses in which small protein-coding sequences (6His, hemagglutinin [HA] tag, and 3HA tag) were added to the 1 protein have been recovered (21).…”