Reverse genetics: Unlocking the secrets of negative sense RNA viral pathogens

Figure 1 Reverse genetic approaches for the study of viral pathogenesis. This figure demonstrates a T7 driven minigenome system (A) and a polI dependent virus rescue system for influenza A virus (B). In (A) the pCAGGS plasmids enable synthesis of T7 polymerase and the viral polymerase complex; nucleoprotein (N), phosphoprotein (P) and large polymerase (L). T7 drives transcription of the minigenome for intracellular synthesis of negative-sense RNA (-veRNA) genome analogues. The minigenome contains Luciferase flanked by leader and trailer sequences that contain gene start (GS) and gene end (GE) sequences essential for polymerase function. To ensure correct processing of the trailer sequence the hepatitis δ ribozyme is included prior to the T7 terminator sequence. The viral proteins encapsidate the RNA analogue and facilitate transcription, which results in Luciferase production and increase in luciferase indicates minigenome transcription. In (B) eight plasmids PB1, PB2, PA, HA, NP, NA, M, NS, representing the influenza A virus genome, are transfected into 293T cells and each of the negative sense viral RNA segments (-veRNA) are produced by polI driven transcription. From the same plasmid, viral messenger RNAs are transcribed in an ambisense direction by polII. Capped and polyadenylated mRNAs are exported from the nucleus into the cytoplasm. Some viral mRNAs are spliced before export, such as M2 and NEP, and translation of viral mRNA occurs on ribosomes attached to endoplasmic reticulum or on ribosomes free in the cytoplasm. Viral proteins in the cytoplasm return into the nucleus and facilitate replication of the viral genome, which is transcribed to produce more viral mRNA and exported from the nucleus. Viral membrane proteins are modified and transported to the apical membrane of the cell. The RNPs are transported to the plasma membrane containing HA and NA in association with M1 and NEP, and 8 RNPs are packaged into progeny virions for neuraminidase-mediated release. HA: Hemagglutinin; NA: Neuraminidase; PB: Polymerase basic; PA : Acidic polymerase; RNPs: Ribonucleoprotein complexes; NS: Non-structural.