Last week, a paper was published in J. Virology comparing the effectiveness of various NanoLuc luciferase alphavirus reporter constructs. The authors of the study, Chengqun Sun et al. from the University of Pittsburgh, placed these reporter genes in three different locations in the genome of several alphaviruses and compared the effect on their ability to replicate in vitro and in vivo. They also assessed the ability of the luciferase genes to persist during infection of cultured cells and in a mouse model. They showed that the size and location of the reporter had a significant effect on successful replication and persistence. They also showed that the reporters could potentially be integrated at different positions within the genome to study different aspects of viral pathogenesis.
What are Alphaviruses?
Alphaviruses are mosquito-borne pathogens that cause infectious arthritis and encephalitis in humans and animals. They have a positive-sense, single-stranded RNA genome encoding four non structural (nsP1-4) and three structural proteins (viral capsid and envelope proteins). The non-structural genes are expressed early in infection and control replication of viral RNA; the structural proteins are synthesized later in infection as viral particles are synthesized and released from the infected cell.
Location, Location, SIZE
The J Virology paper reports the results of positioning either firefly (61KDa) or NanoLuc (19kDa) luciferase reporters as a fusion with the non-structural protein nsP3, as a cleavable element between two of the structural proteins (TaV), or downstream of the last structural gene (DP—a position typically used for previous alphavirus reporters). NanoLuc luciferase constructs were much more stable both in vivo and in vitro than firefly constructs. Firefly luciferase was rapidly lost when positioned downstream of structural genes or as an auto-cleavable element between the capsid and envelope proteins. For both luciferases, the reporter position as a fusion with nsP3 or as a cleavable element between the capsid and envelope genes performed better than the DP constructs.
NanoLuc nsP3 and TaV constructs were similar in virulence to the parent virus in and animal model whereas firefly luciferase constructs were attenuated regardless of the reporter position. To determine whether the size of the construct was the critical factor in this attenuation, the authors also tested GFP constructs, which are similar in size to NanoLuc luciferase. The GFP construct results were similar to those using the NanoLuc constructs, suggesting that the reduced reporter size is a critical factor affecting attenuation.
Reporter Gene and Viral Persistence
In vitro studies in cell culture showed that the NanoLuc reporter was retained throughout passage, decreasing less than twofold after 10 passages, while the firefly signal returned to background levels after 6 passages. In an in vivo model, the TaV NanoLuc construct was most virulent, causing 100% mortality like the parent virus, but with a longer survival time. DP-NanoLuc and all firefly vectors were less virulent. GFP vectors again showed similar mortality results to NanoLuc.
Finally, in vivo imaging of NanoLuc and firefly constructs in mice showed that by 24 and 48h post-infection, the firefly luciferase signal was only detectable at the site of injection. However the NanoLuc signal was detectable at many sites throughout the body and continued to increase up to 48 hours, indicating clearly the value of a stably integrated reporter with a bright signal.
In the discussion the authors point out that both the reporter size and location are factors in persistence, retention and virulence. Results with the downstream constructs showed that position is a clearly attenuating factor, regardless of reporter used, and that the brightness of NanoLuc gives more sensitive measurement of replication in vivo and in vitro.
This is the second study in recent months showing how the size and brightness of NanoLuc luciferase can result in generation of small RNA viruses that more closely resemble wildtype behavior and virulence than larger reporter constructs. The first study involved insertion of the reporter into “influenza virus. In both these studies, the brightness of the reporter resulted in more sensitivite detection, both in vivo and in vitro.
Here’s the Paper
Sun C, Gardner CL, Watson AM, Ryman KD, & Klimstra WB (2014). Stable, High-Level Expression of Reporter Proteins from Improved Alphavirus Expression Vectors To Track Replication and Dissemination during Encephalitic and Arthritogenic Disease. Journal of virology, 88 (4), 2035-46 PMID: 24307590Sun, C., et al (2014) Stable, High-Level Expression of Reporter Proteins from Improved Alphavirus Expression Vectors To Track Replication and Dissemination during Encephalitic and Arthritogenic Disease. J. Virol. 88(4), 2035-2046.