Ribonuclease from Bacillus Acts as an Antiviral Agent against Negative- and Positive-Sense Single Stranded Human Respiratory RNA Viruses

Figure 4

Hypothetic scheme of binase influence on functional viral RNP polymerase complex formation monitored by GFP fluorescence in the cells using the influenza A virus mini genome system. For the in vitro reconstitution of viral ribonucleoprotein complex (vRNP), plasmids pHW-PB1-Hamburg, pHW-PB2-Hamburg, pHW-PA-Hamburg, and pHW-NP-Hamburg were used. They carry genes for generating the mRNAs of polymerases and NP segments of H1N1pdm virus. These plasmids were transfected into A549 cells along with pPOLI-GFP-RT plasmid, which generates a vRNA-like POLI-transcript encoding the green fluorescent protein. GFP gene in a latter construct is encoded in negative-sense and flanked by the 3′- and 5′-noncoding region of the NS RNA segment of influenza A/WSN/33 virus [2] placed between a truncated human RNA polymerase I promotor (POLI) and hepatitis delta virus ribozyme (R). The expressed subunits of the viral polymerases and nucleoprotein replicate and transcribe the influenza virus-like RNA expressed by pPOLI-GFP-RT into GFP mRNA, resulting in the detection of GFP activity in transfected cells. The described steps are labeled as follows: (1) plasmid constructs encoding vRNP subunits and GFP as a reporter protein, (2-3) transfection and internalization of plasmids into nucleus of A549 cells, (4–6) expression of viral polymerase complex, (7) maturation of GFP transcript by viral polymerase complex, and (8) GFP signaling. Possible binase actions are represented in the steps: (A) binase internalization into cytoplasm, (B) binase internalization into nucleus, (C) and (D) inhibitory effect of binase on the expression of influenza A virus mini genome system in cytoplasm and nucleus, respectively.