Rotaviruses (RV) are considered to be the most common cause of viral gastroenteritis in young children and infants worldwide. Before the recent developments, studies on rotavirus biology have always suffered from the lack of an effective reverse genetics (RG) system to generate recombinant rotaviruses and study the precise role of the viral proteins in the context of RV infection. In the first part of this work, we aimed to develop a helper-virus dependent reverse genetics system applicable to all genomic segments (gs). Our new approach is based on the use of a selective marker (i.e. EGFP) that could in principle be used with any gs. A T7-driven genome segment-like RNA encoding EGFP fused to the viral protein gene of interest separated either by the Tobacco Etch Virus protease (TEVp) cleavage site or self-cleaving peptide 2A is expressed in virus infected cells. In vivo processing of the fusion protein yields the fully functional viral protein and EGFP, which will be used as marker to identify cells sustaining recombinant RV replication. In order to increase the availability of the recombinant segment to be packaged, we harnessed the CRISPR/Csy4 fused to NSP5 to shuttle the virus-like mRNA into the RV viral factories. Infection-sorting cycles were repeated several times to isolate reassortant RVs. However, despite many efforts, we encountered some difficulties in rescuing the recombinant rotaviruses (rRV). In the second part of this work, we took advantage of the newly developed plasmid-only based RG system for the generation of recombinant rotaviruses to study the role of NSP5 hyperphosphorylation in the context of RV infection. Using an NSP5 trans-complementing cell line we generated and characterised several rRVs with mutations in NSP5. We demonstrated that a rRV lacking NSP5 was completely unable to assemble viroplasms and to replicate, confirming its pivotal role in rotavirus replication. Furthermore, we investigated the mechanism of NSP5 hyperphosphorylation during RV infection using NSP5 phosphorylation-negative rRV strains, corroborating its role into the assembly and maintaining the architecture of the RV viral factories. Ultimately, here we present the first genome editing of a double stranded RNA (dsRNA) virus harnessing the CRISPR/Csy4 endoribonuclease. The editing was achieved shuttling the Csy4 by fusing it with the NSP5, to viroplasms, the site of RV genome replication and virus assembly. Taking advantage of the fully tractable RG system we generated several rRVs directing the Csy4 target sequence into the different RV genome segments. After a single round of infection of cells expressing the NSP5-Csy4 enzyme with rRV strains, carrying the Csy4 target sequence into the gs5 (gs5*), we observed the generation of a deleted version of gs5* lacking 27 and 42 nucleotides. We proved that this was the direct cause of Csy4-mediated cleavage activity as the 3’ site of both edited segments coincides with the Csy4 cleavage into the viroplasmic environment and was only achieved with the active nuclease but not with a catalytically inactive mutant. Furthermore, we took advantage of this precise in vivo editing of the RV genome to address an important question related to the virus replication cycle: the role of the secondary transcription by newly made transcriptionally active dual-layered intermediate particles. We imaged for the first time the product of secondary transcription events in living cell and our results suggest that this step of viral replication strongly contributes to the overall production of viral protein at late hours post infection. A defined and consistent editing of a dsRNA viral genome can pave the way to shed light on the still underexplored and poorly understood replication steps of rotaviruses.

The potentials of reverse genetics for Rotaviruses: from viral mechanisms to genome editing / Papa, Guido. - (2019 Oct 04).

The potentials of reverse genetics for Rotaviruses: from viral mechanisms to genome editing

Papa, Guido
2019

Abstract

Rotaviruses (RV) are considered to be the most common cause of viral gastroenteritis in young children and infants worldwide. Before the recent developments, studies on rotavirus biology have always suffered from the lack of an effective reverse genetics (RG) system to generate recombinant rotaviruses and study the precise role of the viral proteins in the context of RV infection. In the first part of this work, we aimed to develop a helper-virus dependent reverse genetics system applicable to all genomic segments (gs). Our new approach is based on the use of a selective marker (i.e. EGFP) that could in principle be used with any gs. A T7-driven genome segment-like RNA encoding EGFP fused to the viral protein gene of interest separated either by the Tobacco Etch Virus protease (TEVp) cleavage site or self-cleaving peptide 2A is expressed in virus infected cells. In vivo processing of the fusion protein yields the fully functional viral protein and EGFP, which will be used as marker to identify cells sustaining recombinant RV replication. In order to increase the availability of the recombinant segment to be packaged, we harnessed the CRISPR/Csy4 fused to NSP5 to shuttle the virus-like mRNA into the RV viral factories. Infection-sorting cycles were repeated several times to isolate reassortant RVs. However, despite many efforts, we encountered some difficulties in rescuing the recombinant rotaviruses (rRV). In the second part of this work, we took advantage of the newly developed plasmid-only based RG system for the generation of recombinant rotaviruses to study the role of NSP5 hyperphosphorylation in the context of RV infection. Using an NSP5 trans-complementing cell line we generated and characterised several rRVs with mutations in NSP5. We demonstrated that a rRV lacking NSP5 was completely unable to assemble viroplasms and to replicate, confirming its pivotal role in rotavirus replication. Furthermore, we investigated the mechanism of NSP5 hyperphosphorylation during RV infection using NSP5 phosphorylation-negative rRV strains, corroborating its role into the assembly and maintaining the architecture of the RV viral factories. Ultimately, here we present the first genome editing of a double stranded RNA (dsRNA) virus harnessing the CRISPR/Csy4 endoribonuclease. The editing was achieved shuttling the Csy4 by fusing it with the NSP5, to viroplasms, the site of RV genome replication and virus assembly. Taking advantage of the fully tractable RG system we generated several rRVs directing the Csy4 target sequence into the different RV genome segments. After a single round of infection of cells expressing the NSP5-Csy4 enzyme with rRV strains, carrying the Csy4 target sequence into the gs5 (gs5*), we observed the generation of a deleted version of gs5* lacking 27 and 42 nucleotides. We proved that this was the direct cause of Csy4-mediated cleavage activity as the 3’ site of both edited segments coincides with the Csy4 cleavage into the viroplasmic environment and was only achieved with the active nuclease but not with a catalytically inactive mutant. Furthermore, we took advantage of this precise in vivo editing of the RV genome to address an important question related to the virus replication cycle: the role of the secondary transcription by newly made transcriptionally active dual-layered intermediate particles. We imaged for the first time the product of secondary transcription events in living cell and our results suggest that this step of viral replication strongly contributes to the overall production of viral protein at late hours post infection. A defined and consistent editing of a dsRNA viral genome can pave the way to shed light on the still underexplored and poorly understood replication steps of rotaviruses.
Burrone, O.R.
Papa, Guido
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.11767/103709
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