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A viral genome packaging ring-ATPase is a flexibly coordinated pentamer

ABSTRACT: Multi-subunit ring-ATPases carry out a myriad of biological functions, including genome packaging in viruses. Though the basic structures and functions of these motors have been well-established, the mechanisms of ATPase firing and motor coordination are poorly understood. Here, using single-molecule fluorescence, we determine that the active bacteriophage T4 DNA packaging motor consists of five subunits of gp17. By systematically doping motors with an ATPase-defective subunit and selecting single motors containing a precise number of active or inactive subunits, we find that the packaging motor can tolerate an inactive subunit. However, motors containing one or more inactive subunits exhibit fewer DNA engagements, a higher failure rate in encapsidation, reduced packaging velocity, and increased pausing. These findings suggest a DNA packaging model in which the motor, by re-adjusting its grip on DNA, can skip an inactive subunit and resume DNA translocation, suggesting that strict coordination amongst motor subunits of packaging motors is not crucial for function. In viruses, multi-subunit ring-ATPases are involved in genome packaging. Here, using single-molecule techniques, the authors determine that the active bacteriophage T4 DNA packaging motor is a pentamer and show that the motor can tolerate inactive subunits, suggesting that strict coordination between the subunits is not crucial.

SUBMITTER: Dai L

PROVIDER: S-EPMC8589836 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Multi-segmented viruses often multimerize their genomic segments to ensure efficient and stoichiometric packaging of the correct genetic cargo. In the bipartite Nodaviridae family, genome heterodimerization is also observed and conserved among different species. However, the nucleotide composition and biological function for this heterodimer remain unclear. Using Flock House virus as a model system, we developed a next-generation sequencing approach ("XL-ClickSeq") to probe heterodimer site sequences. We identified an intermolecular base-pairing site which contributed to heterodimerization in both wild-type and defective virus particles. Mutagenic disruption of this heterodimer site exhibited significant deficiencies in genome packaging and encapsidation specificity to viral genomic RNAs. Furthermore, the disruption of this intermolecular interaction directly impacts the thermostability of the mature virions. These results demonstrate that the intermolecular RNA-RNA interactions within the encapsidated genome of an RNA virus have an important role on virus particle integrity and thus may impact its transmission to a new host.IMPORTANCEFlock House virus is a member of Nodaviridae family of viruses, which provides a well-studied model virus for non-enveloped RNA virus assembly, cell entry, and replication. The Flock House virus genome consists of two separate RNA molecules, which can form a heterodimer upon heating of virus particles. Although similar RNA dimerization is utilized by other viruses (such as retroviruses) as a packaging mechanism and is conserved among Nodaviruses, the role of heterodimerization in the Nodavirus replication cycle is unclear. In this research, we identified the RNA sequences contributing to Flock House virus genome heterodimerization and discovered that such RNA-RNA interaction plays an essential role in virus packaging efficiency and particle integrity. This provides significant insight into how the interaction of packaged viral RNA may have a broader impact on the structural and functional properties of virus particles.

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A viral genome packaging ring-ATPase is a flexibly coordinated pentamer

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