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Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

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Viral Capsid Trafficking along Treadmilling Tubulin Filaments in Bacteria

Vorrapon Chaikeeratisak¹, Kanika Khanna², Katrina T Nguyen²

¹Division of Biological Sciences, University of California, San Diego, San Diego, CA 92093, USA; Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok, 10330, Thailand.

|June 15, 2019

Abstract:

Cargo trafficking along microtubules is exploited by eukaryotic viruses, but no such examples have been reported in bacteria. Several large Pseudomonas phages assemble a dynamic, tubulin-based (PhuZ) spindle that centers replicating phage DNA sequestered within a nucleus-like structure. Here, we show that capsids assemble on the membrane and then move rapidly along PhuZ filaments toward the phage nucleus for DNA packaging. The spindle rotates the phage nucleus, distributing capsids around its surface. PhuZ filaments treadmill toward the nucleus at a constant rate similar to the rate of capsid movement and the linear velocity of nucleus rotation. Capsids become trapped along mutant static PhuZ filaments that are defective in GTP hydrolysis. Our results suggest a transport and distribution mechanism in which capsids attached to the sides of filaments are trafficked to the nucleus by PhuZ polymerization at the poles, demonstrating that the phage cytoskeleton evolved cargo-trafficking capabilities in bacteria.

Insights

Bacterial viruses use a dynamic cytoskeleton (PhuZ) for cargo transport. Capsids move along PhuZ filaments to the phage nucleus for DNA packaging, demonstrating evolved bacterial cytoskeleton capabilities.

Area of Science:

Microbiology
Structural Biology
Molecular Biology

Background:

Eukaryotic viruses utilize microtubule-based cargo trafficking.
No bacterial examples of such mechanisms were previously reported.
Large Pseudomonas phages possess a tubulin-based (PhuZ) spindle for DNA organization.

Purpose of the Study:

To investigate the mechanism of capsid transport and DNA packaging in Pseudomonas phages.
To elucidate the role of the PhuZ cytoskeleton in bacterial viral replication.
To demonstrate cargo-trafficking capabilities evolved within the bacterial cytoskeleton.

Main Methods:

Observation of capsid assembly on the membrane and movement along PhuZ filaments.
Analysis of phage nucleus rotation and capsid distribution.
Investigation of PhuZ filament dynamics, including treadmilling and GTP hydrolysis.
Study of capsid behavior on mutant PhuZ filaments defective in GTP hydrolysis.

Main Results:

Capsids assemble on the membrane and are rapidly trafficked along PhuZ filaments to the phage nucleus.
The PhuZ spindle rotates the phage nucleus, facilitating capsid distribution.
PhuZ filament polymerization drives capsid movement and nucleus rotation.
Capsids are immobilized on static PhuZ mutants, indicating the importance of filament dynamics.

Conclusions:

Phage capsids are transported via a mechanism involving PhuZ polymerization at the poles.
This demonstrates that the bacterial cytoskeleton has evolved cargo-trafficking functions.
PhuZ-based transport is crucial for DNA packaging and viral replication in these bacteria.

Read full analysis

Keywords:

PhuZ Pseudomonas phage capsid distribution capsid trafficking giant phage nuclear rotation phage nucleus treadmilling

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