Abstract
The viral RNA (vRNA) genome of influenza viruses is replicated by the RNA-dependent RNA polymerase (RNAP) via a complementary RNA (cRNA) intermediate. The vRNA promoter can adopt multiple conformations when bound by the RNAP. However, the dynamics, determinants, and biological role of these conformations are unknown; further, little is known about cRNA promoter conformations. To probe the RNA conformations adopted during initial replication, we monitored single, surface-immobilized vRNA and cRNA initiation complexes in real-time. Our results show that, while the 3′ terminus of the vRNA promoter exists in dynamic equilibrium between pre-initiation and initiation conformations, the cRNA promoter exhibited very limited dynamics. Two residues in the proximal 3′ region of the cRNA promoter (residues absent in the vRNA promoter) allowed the cRNA template strand to reach further into the active site, limiting promoter dynamics. Our results highlight promoter-dependent differences in influenza initiation mechanisms, and advance our understanding of virus replication.
Highlights
Influenza is one of the world’s major uncontrolled diseases
In order to test whether these two states exist independently or interconvert within individual molecules, and to recover the timescales of any dynamics, we developed a total-internal reflection fluorescence (TIRF) microscopy assay for realtime observations of immobilized RNAP-promoter viral RNA (vRNA) complexes for extended periods
We discovered distinct differences in dynamics between short synthetic 3 vRNA and complementary RNA (cRNA) templates, highlighting differences in the initiation mechanisms between the two promoters
Summary
Annual influenza epidemics are estimated to cause up to 650 000 deaths worldwide, while intermittent pandemics have led to the deaths of many millions of people. Influenza is caused by the influenza virus, an enveloped virus with a single-stranded, negative-sense, segmented viral RNA (vRNA) genome. Each vRNA segment is bound by the viral RNA-dependent-RNA-polymerase (RNAP) and viral nucleoprotein (NP) to form a ribonucleoprotein complex (vRNP) [1,2]. In vRNPs, the 13 nucleotides at the 5 end and 12 nucleotides at the 3 end of each vRNA segment are highly conserved, forming the viral promoter, which is bound by the RNAP [3,4]. The remaining part of the pseudo-circularized vRNA segment is bound by multiple copies of NP arranged in a helical, flexible rod-shape
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