Identification of potential accessory proteins encoded by the bovine rotavirus strain RF

Rotaviruses (RVs) are a major cause of acute gastroenteritis in infants and young children worldwide, accounting for ~215,000 deaths annually, mostly in developing countries. Likewise, RV-associated enteritis in young calves and piglets has a significant economic impact on livestock production as a result of the high morbidity and mortality caused. The RV genome comprises 11 segments of dsRNA encoding structural and non-structural proteins that are essential for virus replication. However, the coding capacity of individual RV genes varies between strains with evidence demonstrating expression of accessory proteins from alternative open reading frames. Work in this thesis aimed to identify accessory proteins encoded by the bovine RV strain RF and investigate their role in pathogenicity. Additionally, lack of a plasmid-only reverse genetics system to artificially engineer and mutate an infectious RV has limited studies of its genome. Thus, a plasmid-only reverse genetics system was established for the RF strain that was used in proof-of-principle experiments to tag viral protein NSP3 with SARS-CoV-2 spike peptides. In vitro transcription and translation assays led to the identification of an unknown polypeptide of ~100 kDa in the RF gene segment 1 (VP1). Bioinformatic analyses identified highly conserved AUG codons corresponding to putative alternative translation initiation sites. Specifically, in the cell free system, mutation of AUG12 (M130) and AUG14 (M146) decreased the expression of the unknown polypeptide, suggesting that both AUGs were utilised for downstream translation initiation. In contrast, in cells transfected with VP1-EGFP tagged constructs, only AUG12 (M130) was utilised for the expression of the unknown polypeptide. Thus, a novel VP1 isoform named VP1-N129 was identified. In the context of RV infection, only the leucine substitution at M130 residue (M130L) affected virus production whereas substitution of methionine to valine (M130V) had no effect on the viral rescue nor on replication. Neither M146L nor M146V mutation affected the viral rescue or replication kinetics. To assess the impact of M130 and M146 mutations on the viral polymerase activity, attempts were made to develop a ‘mini-genome’ assay. Preliminary results from this assay showed no effect on the polymerase function, although further experiments are needed to improve the sensitivity of the assay. In conclusion, a novel N-terminally truncated isoform of VP1 has been identified but further studies are required to confirm its role as a potential accessory protein in RV infection

Towards the development of a mini-genome assay for species A Rotaviruses

RNA virus polymerases carry out multiple functions necessary for successful genome replication and transcription. A key tool for molecular studies of viral RNA-dependent RNA polymerases (RdRps) is a ‘minigenome’ or ‘minireplicon’ assay, in which viral RdRps are reconstituted in cells in the absence of full virus infection. Typically, plasmids expressing the viral polymerase protein(s) and other co-factors are co-transfected along with a plasmid expressing an RNA encoding a fluorescent or luminescent reporter gene flanked by viral untranslated regions containing cis-acting elements required for viral RdRp recognition. This reconstitutes the viral transcription/replication machinery and allows viral RdRp activity to be measured as a correlate of reporter protein signal. Here we report on the development of a ‘first-generation’ plasmid-based minigenome assay for species A rotavirus, using a firefly luciferase reporter gen

Using species A rotavirus reverse genetics to engineer chimeric viruses expressing SARS-CoV-2 spike epitopes:Heterologous viral peptide expression by rotavirus A

Species A rotavirus (RVA) vaccines based on live attenuated viruses are used worldwide in humans. The recent establishment of a reverse genetics system for rotoviruses (RVs) has opened the possibility of engineering chimeric viruses expressing heterologous peptides from other viral or microbial species in order to develop polyvalent vaccines. We tested the feasibility of this concept by two approaches. First, we inserted short SARS-CoV-2 spike peptides into the hypervariable region of the simian RV SA11 strain viral protein (VP) 4. Second, we fused the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, or the shorter receptor binding motif (RBM) nested within the RBD, to the C terminus of nonstructural protein (NSP) 3 of the bovine RV RF strain, with or without an intervening Thosea asigna virus 2A (T2A) peptide. Mutating the hypervariable region of SA11 VP4 impeded viral replication, and for these mutants, no cross-reactivity with spike antibodies was detected. To rescue NSP3 mutants, we established a plasmid-based reverse genetics system for the bovine RV RF strain. Except for the RBD mutant that demonstrated a rescue defect, all NSP3 mutants delivered endpoint infectivity titers and exhibited replication kinetics comparable to that of the wild-type virus. In ELISAs, cell lysates of an NSP3 mutant expressing the RBD peptide showed cross-reactivity with a SARS-CoV-2 RBD antibody. 3D bovine gut enteroids were susceptible to infection by all NSP3 mutants, but cross-reactivity with SARS-CoV-2 RBD antibody was only detected for the RBM mutant. The tolerance of large SARS-CoV-2 peptide insertions at the C terminus of NSP3 in the presence of T2A element highlights the potential of this approach for the development of vaccine vectors targeting multiple enteric pathogens simultaneously. IMPORTANCE We explored the use of rotaviruses (RVs) to express heterologous peptides, using SARS-CoV-2 as an example. Small SARS-CoV-2 peptide insertions (<34 amino acids) into the hypervariable region of the viral protein 4 (VP4) of RV SA11 strain resulted in reduced viral titer and replication, demonstrating a limited tolerance for peptide insertions at this site. To test the RV RF strain for its tolerance for peptide insertions, we constructed a reverse genetics system. NSP3 was C-terminally tagged with SARS-CoV-2 spike peptides of up to 193 amino acids in length. With a T2A-separated 193 amino acid tag on NSP3, there was no significant effect on the viral rescue efficiency, endpoint titer, and replication kinetics. Tagged NSP3 elicited cross-reactivity with SARS-CoV-2 spike antibodies in ELISA. We highlight the potential for development of RV vaccine vectors targeting multiple enteric pathogens simultaneously

Fibrotic Myofibroblasts Manifest Genome-Wide Derangements of Translational Control

Background: As a group, fibroproliferative disorders of the lung, liver, kidney, heart, vasculature and integument are common, progressive and refractory to therapy. They can emerge following toxic insults, but are frequently idiopathic. Their enigmatic propensity to resist therapy and progress to organ failure has focused attention on the myofibroblast–the primary effector of the fibroproliferative response. We have recently shown that aberrant beta 1 integrin signaling in fibrotic fibroblasts results in defective PTEN function, unrestrained Akt signaling and subsequent activation of the translation initiation machinery. How this pathological integrin signaling alters the gene expression pathway has not been elucidated. Results: Using a systems approach to study this question in a prototype fibrotic disease, Idiopathic Pulmonary Fibrosis (IPF); here we show organized changes in the gene expression pathway of primary lung myofibroblasts that persist for up to 9 sub-cultivations in vitro. When comparing IPF and control myofibroblasts in a 3-dimensional type I collagen matrix, more genes differed at the level of ribosome recruitment than at the level of transcript abundance, indicating pathological translational control as a major characteristic of IPF myofibroblasts. To determine the effect of matrix state on translational control, myofibroblasts were permitted to contract the matrix. Ribosome recruitment in control myofibroblasts was relatively stable. In contrast, IPF cells manifested large alterations in the ribosome recruitment pattern. Pathological studies suggest an epithelial origin for IPF myofibroblasts through the epithelial to mesenchymal transition (EMT). In accord wit

BTN3A3 evasion promotes the zoonotic potential of influenza A viruses

Spillover events of avian influenza A viruses (IAVs) to humans could represent the first step in a future pandemic1. Several factors that limit the transmission and replication of avian IAVs in mammals have been identified. There are several gaps in our understanding to predict which virus lineages are more likely to cross the species barrier and cause disease in humans1. Here, we identified human BTN3A3 (butyrophilin subfamily 3 member A3)2 as a potent inhibitor of avian IAVs but not human IAVs. We determined that BTN3A3 is expressed in human airways and its antiviral activity evolved in primates. We show that BTN3A3 restriction acts primarily at the early stages of the virus life cycle by inhibiting avian IAV RNA replication. We identified residue 313 in the viral nucleoprotein (NP) as the genetic determinant of BTN3A3 sensitivity (313F or, rarely, 313L in avian viruses) or evasion (313Y or 313V in human viruses). However, avian IAV serotypes, such as H7 and H9, that spilled over into humans also evade BTN3A3 restriction. In these cases, BTN3A3 evasion is due to substitutions (N, H or Q) in NP residue 52 that is adjacent to residue 313 in the NP structure3. Thus, sensitivity or resistance to BTN3A3 is another factor to consider in the risk assessment of the zoonotic potential of avian influenza viruses