The Structure of the Phage T4 DNA Packaging Motor Suggests a Mechanism Dependent on Electrostatic Forces

101

In herpesviruses and many bacterial viruses, genome-packaging is a precisely mediated process fulfilled by a virally encoded molecular machine called terminase that consists of two protein components: A DNA-recognition component that defines the specificity for packaged DNA, and a catalytic component that provides energy for the packaging reaction by hydrolyzing ATP. The terminase docks onto the portal protein complex embedded in a single vertex of a preformed viral protein shell called procapsid, and pumps the viral DNA into the procapsid through a conduit formed by the portal. Here we report the 1.65 Å resolution structure of the DNA-recognition component gp1 of the Shigella bacteriophage Sf6 genomepackaging machine. The structure reveals a ring-like octamer formed by interweaved protein monomers with a highly extended fold, embracing a tunnel through which DNA may be translocated. The N-terminal DNA-binding domains form the peripheral appendages surrounding the octamer. The central domain contributes to oligomerization through interactions of bundled helices. The C-terminal domain forms a barrel with parallel beta-strands. The structure reveals a common scheme for oligomerization of terminase DNA-recognition components, and provides insights into the role of gp1 in formation of the packaging-competent terminase complex and assembly of the terminase with the portal, in which ring-like protein oligomers stack together to form a continuous channel for viral DNA translocation.terminase | DNA-packaging | oligomer | bacteriophage | protein:DNA interaction I n many tailed double-stranded DNA (dsDNA) bacteriophages and herpesvirus, the newly synthesized viral DNA in the infected host cell is present as a concatemer that is comprised of multiple copies of unit-length genome. The concatemeric DNA is packaged into a preformed capsid precursor termed procapsid through a channel formed by a portal protein complex embedded in a unique vertex of the procapsid (1-5). The DNApackaging process involves a precisely coordinated sequence of molecular events, driven by a molecular machine called terminase consisting of two proteins: A DNA-recognition component and a catalytic component, also known as the "small" and "large" subunit, respectively. The terminase specifically binds to concatemeric viral DNA through the DNA-recognition component and cleaves it using the catalytic component, generating a new terminus for the DNA. This terminus is threaded through the portal protein channel embedded in the procapsid, and the catalytic component pumps the DNA into the procapsid in an ATP-dependent manner. When an appropriate amount of DNA is inserted, the terminase cuts the DNA again, dissociates from the procapsid, and can start the next cycle of DNA-packaging. Many of these features in DNA-packaging are also shared by adenoviruses (6). The x-ray structures of the catalytic component from bacteriophage T4 and the portals from various phages were previously described (7-10). However, it is not known how the terminases are assembled and h...

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Crystal structure of the DNA-recognition component of the bacterial virus Sf6 genome-packaging machine

Zhao

Finch

Sequeira

et al. 2010

101

“…High-resolution structures of all of the conserved motor components found among tailed dsDNA bacteriophages have been determined (2)(3)(4)(5). The small bacteriophage T4 terminase protein gp16 is required for cutting and packaging the replicative DNA concatemer in vivo but is nonessential and inhibitory for linear DNA packaging in vitro (6).…”

mentioning

confidence: 99%

Compression of the DNA substrate by a viral packaging motor is supported by removal of intercalating dye during translocation

Dixit¹,

Ray

Black³

2012

Viral genome packaging into capsids is powered by high-forcegenerating motor proteins. In the presence of all packaging components, ATP-powered translocation in vitro expels all detectable tightly bound YOYO-1 dye from packaged short dsDNA substrates and removes all aminoacridine dye from packaged genomic DNA in vivo. In contrast, in the absence of packaging, the purified T4 packaging ATPase alone can only remove up to ∼1/3 of DNA-bound intercalating YOYO-1 dye molecules in the presence of ATP or ATP-γ-S. In sufficient concentration, intercalating dyes arrest packaging, but rare terminase mutations confer resistance. These distant mutations are highly interdependent in acquiring function and resistance and likely mark motor contact points with the translocating DNA. In stalled Y-DNAs, FRET has shown a decrease in distance from the phage T4 terminase C terminus to portal consistent with a linear motor, and in the Y-stem DNA compression between closely positioned dye pairs. Taken together with prior FRET studies of conformational changes in stalled Y-DNAs, removal of intercalating compounds by the packaging motor demonstrates conformational change in DNA during normal translocation at low packaging resistance and supports a proposed linear "DNA crunching" or torsional compression motor mechanism involving a transient gripand-release structural change in B form DNA.DNA structure | terminase inhibitors N ucleic acid translocation into an empty procapsid is a conserved capsid assembly mechanism found among diverse DNA and RNA viruses (1). High-resolution structures of all of the conserved motor components found among tailed dsDNA bacteriophages have been determined (2-5). The small bacteriophage T4 terminase protein gp16 is required for cutting and packaging the replicative DNA concatemer in vivo but is nonessential and inhibitory for linear DNA packaging in vitro (6). Thus, T4 DNA translocation in vitro can be driven by a two-component motor consisting of a prohead portal ring channel dodecamer situated at a single packaging vertex that is docked during packaging with gp17 terminase-ATPase. A linear packaging motor mechanism proposed that a terminase to portal DNA grip-and-release driven by a motor protein conformational change drives DNA into the prohead by a DNA compression motor stroke (7-10).It has long been known that acridine dyes inhibit bacteriophage development at concentrations below those that inhibit growth of the bacterial host. Phage mutations that confer resistance to such dyes arise through different mechanisms. Among these are mutations in the phage T4 terminase gene 17, called ac and q, that confer acridine and quinacrine resistance (11). All 9-aminoacridine (9AA) treatments have been shown by electron microscopy to arrest DNA packaging in vivo; however, whether the site of action of 9AA is the DNA, the active site of the packaging enzyme, or the enzyme DNA complex-in fact, acridines are known to inactivate protease active sites or virion proteins associated with DNA (reviewed by Black et al., in r...

“…Furthermore, bacteriophages provide enzymes for digesting bacterial cell walls, mechanical means for puncturing cell membranes, and a tube for delivering the DNA genome into the host cytoplasm. In addition, the special vertex may be required for genome packaging into preformed proheads, as in HSV (15) or in most bacteriophages (18,19). Yet another function of special vertices can be to nucleate prohead assembly in some bacteriophages (20).…”

mentioning

confidence: 99%

An icosahedral algal virus has a complex unique vertex decorated by a spike

Cherrier

Kostyuchenko

Xiao

et al. 2009

Self Cite

Paramecium bursaria Chlorella virus-1 is an icosahedrally shaped, 1,900-Å-diameter virus that infects unicellular eukaryotic green algae. A 5-fold symmetric, 3D reconstruction using cryoelectron microscopy images has now shown that the quasiicosahedral virus has a unique vertex, with a pocket on the inside and a spike structure on the outside of the capsid. The pocket might contain enzymes for use in the initial stages of infection. The unique vertex consists of virally coded proteins, some of which have been identified. Comparison of shape, size, and location of the spike with similar features in bacteriophages T4 and P22 suggests that the spike might be a cell-puncturing device. Similar asymmetric features may have been missed in previous analyses of many other viruses that had been assumed to be perfectly icosahedral.5-fold averaging ͉ cryoelectron microscopy reconstruction ͉ Paramecium bursaria Chlorella virus-1 ͉ cell entry ͉ specialized vertex protein