Himanshu Sharma

Cellular homeostasis is sustained by balancing the cell growth and quiescence in response to specific environmental cues. While the mechanisms governing the transition from growth to dormancy are reasonably well understood, the processes underlying exit from dormancy remain poorly characterised. Using an integrated approach employing bacterial genetics, Cryo-EM, and molecular microbiology, we investigated this phenomenon by studying the cross-talk between two antagonistic pathways, ribosome biogenesis that promotes cell growth, and stringent response that induces dormancy. We show that the exit from dormancy requires a conserved Era GTPase whose synthesis selectively rises during onset of exponential growth phase in Escherichia coli. Era not only accelerates the maturation of head and platform regions of 30S ribosomal subunit but also dislodges RelA from 70S ribosome. This derepresses the 70S ribosome from RelA-mediated inhibition, and promotes protein synthesis driving the active cell growth. This study uncovers that temporal epistatic interaction between Era and RelA governs cellular resuscitation in bacteria. Given that the dormant bacterial populations contribute to antibiotic tolerance, understanding this regulatory axis offers new insights for resensitising the recalcitrant dormant bacteria to antibiotics.Competing Interest StatementThe authors have declared no competing interest.Anusandhan National Research Foundation, SPR/2023/000151, CRG/2022/005662, CRG/2019/003385Ignite Life Science Foundation, https://ror.org/03g42sh82, ACORN-AMR/2023/004, IGNITE/FG-OC/2021/005Ministry of Education, GoI, MoE-STARS/STARS-2/2023-1032Department of Atomic Energy, RTI 4008Department of Biotechnology, DBT/PR12422/MED/31/287/2014European Commission, 101087571

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.