Investigation of canonical and noncanonical transcription during coronavirus replication.

Abstract

Coronaviruses express their structural and accessory genes through the process of discontinuous transcription. The mechanism involves the transcription regulatory sequence (TRS) leader (L), which is located at the 5′ end of the genome, and a homologous TRS Body (B) sequence which are located upstream of each of the structural and accessory genes. These TRS are believed to act as stalling signals for the viral replication transcription complex (RTC), which transcribes the genome in a 3′ to 5′ direction. When the RTC stalls a template switching event occurs and allows the RTC to “Jump” from the TRS-B to the TRS-L and then continue transcribing the 5′ untranslated region (UTR). This produces a nested set of subgenomic mRNAs (sgmRNA) which include the downstream sequence from the TRS-B, where the template switch occurred and the 5′ UTR. Early work in transmissible gastroenteritis virus (TGEV) highlighted the importance of homology between the TRS-B and TRS-L in sgmRNA expression, with all TRSs in TGEV showing 100% homology. However, work in infectious bronchitis virus (IBV) uncovered viable sgmRNA produced from TRS-Bs without 100% homology. This included TRS-B with substantial differences termed noncanonical sgmRNA such as open reading frame (ORF) 7 located near the 3′ end of the genome. The aim of this project was to determine whether IBV, TGEV or Porcine respiratory coronavirus (PRCV), encode noncanonical TRS-B which may provide the viruses additional coding potential. A secondary aim of this project was to determine how changes to the TRS-B sequence and flanking regions impact sgmRNA expression. To satisfy the first aim ribosomal profiling and long read sequencing were used. The ribosomal profiling data did not uncover any signs of noncanonical genes being transcribed or translated. However, multiple sequences were found in the long read sequencing dataset which would have originated from noncanonical TRS-Bs. For the second aim several rIBVs (recombinant IBV) were produced with mutations within the TRS-B and flanking regions which were aimed to alter the expression of a nonessential gene. The levels of gene expression of the relevant gene as well as genes with nearby TRS-Bs were quantified. It was found that despite efforts to increase, decrease or abolish expression, the rIBVs only showed decreased expression although there was evidence of additional products. These experiments were first conducted in an ex vivo chicken organ culture and later confirmed in an in vivo animal experiment. This work demonstrated the first use of ribosomal profiling on TGEV and PRCV while indicating that the control of expression from TRS-Bs is far more complex than previously thought. These finding will add to our understanding of the model of discontinuous transcription which could indicate an increased coding capacity as well as additional methods of sgmRNA regulation in the coronavirus genome.