Imaging the translation of HIV and MLV viruses at a single RNA level
Résumé
New strategies emerge targeting cell functions manipulated by viruses to replicate. Murine Leukemia virus is an
excellent manipulator of cellular functions because it has minimal coding capacity with only 3 genes common to all
retroviruses (gag, pol, env). In contrast, HIV-1 (the agent responsible for AIDS) is more complex with several additional
viral proteins directly regulating viral RNA metabolism. Nevertheless, these two retroviruses perform the same
replication cycle, in which translation is a key step. To study viral translation in cells we are combining SunTag and
MS2-RNA imaging strategies. For visualization at a single-molecule level, several copies of the SunTag epitopes were
fused to the nascent Gag protein. Then, a fluorescent antibody binds to the SunTag and amplifies the signal. The
colocalization of SunTag and MS2 signals provides information on the ratio of translated/untranslated viral RNA,
translation localization, and kinetics. First, we studied fixed-cells, and images were acquired in a widefield fluorescent
microscope and the colocalization between nascent peptide and RNA dots was detected using Imaris. We have derived
all the molecular tools for the study of the translation of these two viruses. Interesting preliminary results show about
20% of MLV and 35% of HIV RNA are translated at the same time.