The Stop Codon after the nsp3 Gene of Ross River Virus (RRV) Is Not Essential for Virus Replication in Three Cell Lines Tested, but RRV Replication Is Attenuated in HEK 293T Cells.

A recombinant Ross River virus (RRV) that contains the fluorescent protein mCherry fused to the non-structural protein 3 (nsP3) was constructed, which allowed real-time imaging of viral replication. RRV-mCherry contained either the natural opal stop codon after the nsP3 gene or was constructed without a stop codon. The mCherry fusion protein did not interfere with the viral life cycle and deletion of the stop codon did not change the replication capacity of RRV-mCherry. Comparison of RRV-mCherry and chikungunya virus-mCherry infections, however, showed a cell type-dependent delay in RRV-mCherry replication in HEK 293T cells. This delay was not caused by differences in cell entry, but rather by an impeded nsP expression caused by the RRV inhibitor ZAP (zinc finger CCCH-Type, antiviral 1). The data indicate that viral replication of alphaviruses is cell-type dependent, and might be unique for each alphavirus.