In vitro characterisation of the MS2 RNA polymerase complex reveals host factors that modulate emesviral replicase activity

Abstract

The RNA phage MS2 is one of the most important model organisms in molecular biology and virology. Despite its comprehensive characterisation, the composition of the RNA replication machinery remained obscure. Here, we characterised host proteins required to reconstitute the functional replicase in vitro. By combining a purified replicase sub-complex with elements of an in vitro translation system, we confirmed that the three host factors, EF-Ts, EF-Tu, and ribosomal protein S1, are part of the active replicase holocomplex. Furthermore, we found that the translation initiation factors IF1 and IF3 modulate replicase activity. While IF3 directly competes with the replicase for template binding, IF1 appears to act as an RNA chaperone that facilitates polymerase readthrough. Finally, we demonstrate in vitro formation of RNAs containing minimal motifs required for amplification. Our work sheds light on the MS2 replication machinery and provides a new promising platform for cell-free evolution.

Estimation of Nanoparticle’s Surface Electrostatic Potential in Solution Using Acid–Base Molecular Probes. III. Experimental Hydrophobicity/Hydrophilicity and Charge Distribution of MS2 Virus Surface

“…MS2 is an icosahedral bacteriophage infecting Gram-negative bacteria (Escherichia coli) − and consisting of a protein outer layer (capsid) and encapsulated internal 3569 nucleotides long single stranded RNA genome partly bound to the capsid. − The capsid protects the genomic RNA; it consists of one maturation protein and 178 copies of the coat protein (arranged as 89 dimers), which self-organize in a shell structure forming the capsid. It is known that the diameter of the capsid is approximately 28 nm, and its thickness is about 2.5 nm.…”

Design principles and applications of synthetic self‐replicating RNAs

“…The two members of the Fiersviridae , Emesvirus zinderi (MS2) and Qubevirus (Qβ), two of the smallest known +ssRNA viruses to date, have genome sizes of 3.6 kb and respectively 4.2 kb, thus approximately only twice as large as large satellite viruses like the Hepatits delta virus (HDV, 1.7 kb) (Fiers et al, 1976; Flores et al, 2011; Schuppli et al, 2000). They encode for RdRP subunits that, together with three E. coli host factors, form the active RdRP complexes capable of replication of genomic and synthetic RNAs (Blumenthal & Carmichael, 1979; Kita et al, 2008; Wagner et al, 2022). Larger RNA viruses, for example, from the families of Togaviridae (e.g., VEEV) and Flaviviridae (e.g., Hepacivirus HCV) (9.0–13 kb) encode the fully active RdRP complex in their genome, split across multiple genes in a single polyprotein ORF (Chase, 2022; Weaver et al, 2004).…”

“…(II) All three srRNAs encode for the respective RdRP subunit only. Thus, they still require three additional host factors—elongation factors EF‐Tu, EF‐Ts, and ribosomal protein S1—to assemble the fully active RdRP complex, which are readily provided in E. coli based in vitro translation systems (Blumenthal & Carmichael, 1979; Wagner et al, 2022). This minimizes the gene load on the srRNA required for self‐replication.…”

Complete Virion Simulated: All-Atom Model of an MS2 Bacteriophage with Native Genome

“…There are no other molecules in this virus. Dynamically, however, infecting bacteria involves a complicated mechanism that includes the bacterial pilus and a still unknown process of extracting the viral RNA from the capsid and transferring it to the bacteria. − The capsid itself remains largely intact outside the bacteria. Therefore, the detailed all-atom dynamic model of MS2 will help to elucidate this mechanism as well as aid in various engineering methodologies for this virus.…”