Viral Genomic DNA Packaging Machinery

Abstract

Tailed double-stranded DNA bacteriophage employs a protein terminase motor to package their genome into a preformed protein shell-a system shared with eukaryotic dsDNA viruses such as herpesviruses. DNA packaging motor proteins represent excellent targets for antiviral therapy, with Letermovir, which binds Cytomegalovirus terminase, already licensed as an effective prophylaxis. In the realm of bacterial viruses, these DNA packaging motors comprise three protein constituents: the portal protein, small terminase and large terminase. The portal protein guards the passage of DNA into the preformed protein shell and acts as a protein interaction hub throughout viral assembly. Small terminase recognises the viral DNA and recruits large terminase, which in turn pumps DNA in an ATP-dependent manner. Large terminase also cleaves DNA at the termination of packaging. Multiple high-resolution structures of each component have been resolved for different phages, but it is only more recently that the field has moved towards cryo-EM reconstructions of protein complexes. In conjunction with highly informative single-particle studies of packaging kinetics, these structures have begun to inspire models for the packaging process and its place among other DNA machines.

Keywords: ATPase; Bacteriophage; DNA translocation; Large terminase; Nuclease; Portal protein; Small terminase.

Figure 1. dsDNA bacteriophage assembly.

A) Small terminase recruits large terminase to the viral genomic DNA B) Large terminase docks onto the portal vertex of the preformed protein prohead C) Large terminase powers packaging of DNA into the prohead using ATP hydrolysis D) the prohead expands into a mature capsid and DNA is cleaved after one genome length has been packaged E) the terminase complex dissociates and is replaced by neck and tail proteins to form a mature virion

Figure 2

DNA packaging strategies by ds DNA phage A) Φ29 like phages package protein capped genome lengths B) cos phage package cos site capped genome units C) pac phage package in excess of one complete genome length

Figure 3. The T4 portal protein BDB 3JA7.

A) The portal is positioned at the unique prohead vertex and coordinates binding of the motor B) Portal proteins from dodecameric rings C) The portal protein domain architecture

Figure 4

Small terminase architecture A) P74-26 small terminase. PDB 6V1I B) p22 small terminase. PDB 3P9A C) The HK97 complex in complex with DNA. Two unfolded regions fold into helices on DNA binding. PDB 8POP

Figure 5

Large Terminase Structure A) Ribbon diagram of bacteriophage Sf6 large terminase, PDB code 4IFE. B) The nuclease active site of bacteriophage SF6, coordinating Manganese ions, PDB code 5C15. C) The ATPase active site of the Sf6 large terminase coordinating ATP, PDB code 4IFE

Figure 6. Kinetic cycle of Φ29.

Timeline of the burst/dwell cycle, adapted from (Chistol et al. 2012). Five sequential ATP hydrolysis events produce just four translocation steps of 2.5 bp, followed by sequential ADP release and ATP binding.

Figure 7. Coordination of ATP hydrolysis.

On ATP hydrolysis, conformational change within a single large terminase subunit repositions the trans acting arginine finger into the adjacent ATP active site catalysing a subsequent hydrolysis event. This occurs sequentially around the ring.

Figure 8. Comparison of complete packaging complexes of ds DNA phage.

Cryo-EM reconstructions are shown for: A) Φ29 EMDB 22441 fitted PDB 7JQQ, B) T4 EMDB 1572 fitted PDB 3EZK, C) HK97 EMDB 16653 fitted PDB 6Z6D

Figure 9. Schematic of the Φ29 packaging mechanism.

The ATPase domains of the large terminase pentameric ring sequentially transition from a cracked helix to planar arrangement in agreement with the dwell- burst kinetic cycle