Illuminating druggable dark matter in RNA virus replication using in situ cryo-EM

Viral proteins typically exist in the context of complex virions or in the even more complex host cells in which they replicate. Hence, meaningful insights into virus protein structure often need to account for this context. Various flavors of in situ cryo-electron microscopy (cryo-EM), such as cryo-electron tomography, are key methods for the contextual study of virus protein structure in pleomorphic virions and host cells. Here, we review recent in situ cryo-EM work on three selected phenomena in RNA virus replication: the maturation and nuclear entry of HIV-1, the membrane-bound replication organelles of positive-sense RNA viruses, and the membrane-less viral factories of negative-sense RNA viruses. We highlight cases where the imaged phenomena are targets of novel antiviral drugs (such as the recently approved antiretroviral Lenacapavir), drug candidates, and antiviral strategies. Finally, we discuss recent technical advances that extend the reach of in situ cryo-EM in virology.