Function and horizontal transfer of the small terminase subunit of the tailed bacteriophage Sf6 DNA packaging nanomotor

Leavitt, Justin C.; Gilcrease, Eddie B.; Wilson, Kassandra; Casjens, Sherwood

doi:10.1016/j.virol.2013.02.023

Cited by 40 publications

(42 citation statements)

References 98 publications

(180 reference statements)

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“…5C). Indeed, for several head-full packaging phages, the pac site for DNA cleavage to generate termini for packaging is located in the small terminase-coding sequence (39). An alternative possible determinant of packaging orientation is the terminal gp3 proteins, covalently bound to each 5= end of the DNA.…”

Section: Discussionmentioning

confidence: 99%

An RNA Domain Imparts Specificity and Selectivity to a Viral DNA Packaging Motor

Wang

Jardine

Grimes

2015

J Virol

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During assembly, double-stranded DNA viruses, including bacteriophages and herpesviruses, utilize a powerful molecular motor to package their genomic DNA into a preformed viral capsid. An integral component of the packaging motor in the Bacillus subtilis bacteriophage 29 is a viral genome-encoded pentameric ring of RNA (prohead RNA [pRNA]). pRNA is a 174-base transcript comprised of two domains, domains I and II. Early studies initially isolated a 120-base form (domain I only) that retains high biological activity in vitro; hence, no function could be assigned to domain II. Here we define a role for this domain in the packaging process. DNA packaging using restriction digests of 29 DNA showed that motors with the 174-base pRNA supported the correct polarity of DNA packaging, selectively packaging the DNA left end. In contrast, motors containing the 120-base pRNA had compromised specificity, packaging both left-and right-end fragments. The presence of domain II also provides selectivity in competition assays with genomes from related phages. Furthermore, motors with the 174-base pRNA were restrictive, in that they packaged only one DNA fragment into the head, whereas motors with the 120-base pRNA packaged several fragments into the head, indicating multiple initiation events. These results show that domain II imparts specificity and stringency to the motor during the packaging initiation events that precede DNA translocation. Heteromeric rings of pRNA demonstrated that one or two copies of domain II were sufficient to impart this selectivity/stringency. Although 29 differs from other doublestranded DNA phages in having an RNA motor component, the function provided by pRNA is carried on the motor protein components in other phages. During viral assembly, double-stranded DNA (dsDNA) viruses, such as the tailed bacteriophages and herpesviruses, package their genomic DNA into a preformed viral shell (prohead) (1, 2). This process is driven by a viral genome-encoded molecular motor that assembles at a unique vertex of the prohead and utilizes the energy from ATP binding and hydrolysis to translocate DNA. During this process, the DNA is compacted to a nearly crystalline density and the motor must overcome the barriers of electrostatic repulsion and entropy that oppose this process. Indeed, the dsDNA viral packaging motors are among the most powerful molecular motors yet characterized (3).The packaging process involves distinct phases (Fig. 1A), including (i) initiation, which is comprised of assembly events that culminate in a prohead-motor-DNA substrate complex primed for packaging; (ii) translocation, whereby the DNA is driven into the head in an ATP-dependent manner; and (iii) termination, when DNA translocation ceases and the DNA-filled head is stabilized while the transient parts of the motor are readied for disassembly (2). While the mechanics of translocation are repeated over thousands of mechanochemical cycles, it is the singular events of the initiation phase that serve to provide the specificity for...

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Section: Discussionmentioning

confidence: 99%

An RNA Domain Imparts Specificity and Selectivity to a Viral DNA Packaging Motor

Wang

Jardine

Grimes

2015

J Virol

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“…Procedures were performed as previously described for isolation of wild-type (wt) P22 (c1-7 13-amH101) (16) and virions containing wild-type (cI60) or truncated (b221) genome lengths (17). The His73Leu change in the P22 tail needle protein (gp26) was created by recombinantly replacing the His73 codon with CTC in the P22 prophage of Salmonella enterica strain UB-1790, as described previously (18,19). HSV-1 capsids containing wild-type (KOS strain) or truncated (recombinant R7023 F strain [20]) genomes were isolated as previously described (9).…”

Section: Methodsmentioning

confidence: 99%

Exploring the Balance between DNA Pressure and Capsid Stability in Herpesviruses and Phages

Bauer

Huffman

et al. 2015

J Virol

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We have recently shown in both herpesviruses and phages that packaged viral DNA creates a pressure of tens of atmospheres pushing against the interior capsid wall. For the first time, using differential scanning microcalorimetry, we directly measured the energy powering the release of pressurized DNA from the capsid. Furthermore, using a new calorimetric assay to accurately determine the temperature inducing DNA release, we found a direct influence of internal DNA pressure on the stability of the viral particle. We show that the balance of forces between the DNA pressure and capsid strength, required for DNA retention between rounds of infection, is conserved between evolutionarily diverse bacterial viruses (phages and P22), as well as a eukaryotic virus, human herpes simplex 1 (HSV-1). Our data also suggest that the portal vertex in these viruses is the weakest point in the overall capsid structure and presents the Achilles heel of the virus's stability. Comparison between these viral systems shows that viruses with higher DNA packing density (resulting in higher capsid pressure) have inherently stronger capsid structures, preventing spontaneous genome release prior to infection. This force balance is of key importance for viral survival and replication. Investigating the ways to disrupt this balance can lead to development of new mutation-resistant antivirals. IMPORTANCEA virus can generally be described as a nucleic acid genome contained within a protective protein shell, called the capsid. For many double-stranded DNA viruses, confinement of the large DNA molecule within the small protein capsid results in an energetically stressed DNA state exerting tens of atmospheres of pressures on the inner capsid wall. We show that stability of viral particles (which directly relates to infectivity) is strongly influenced by the state of the packaged genome. Using scanning calorimetry on a bacterial virus (phage ) as an experimental model system, we investigated the thermodynamics of genome release associated with destabilizing the viral particle. Furthermore, we compare the influence of tight genome confinement on the relative stability for diverse bacterial and eukaryotic viruses. These comparisons reveal an evolutionarily conserved force balance between the capsid stability and the density of the packaged genome.A virus can generally be described as a nucleic acid genome contained within a protective protein shell, termed the capsid. The viral replication cycle is composed of two distinct processes: delivery of the genetic material to the host (infection) and the subsequent production of new virions (replication). Between rounds of replication, the virion must be sufficiently stable to ensure that the packaged genome is retained within the capsid. Conversely, during infection it must be unstable enough to allow efficient genome release into the host cell. This balance between genome retention and release is described as viral metastability (1). Viral particles are therefore not inert structures and have not ...

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“…Genetic and biochemical studies of several packaging systems have delineated the mechanisms of genome recognition and DNA cutting (17,18). Structural studies (12,16,19) and single-molecule optical tweezers (3,13) and fluorescence spectroscopy (20,21) approaches have been used to dissect the mechanochemical steps of DNA translocation.…”

Section: Significancementioning

confidence: 99%

“…Singlemolecule techniques have greatly contributed to our understanding of the mechanistic details, especially of the elongation phase of phage phi29 DNA packaging (29,30). With regard to the initiation phase, it has been well documented that in most of the tailed phages and herpes viruses a terminase complex consisting of small and large terminase proteins recognizes the concatemeric viral genome and makes a cut to generate a dsDNA end (7,8,17,18). The following steps of initiation, however, are poorly understood.…”

Section: Direct Binding Of Dna To the Portal Leads To Efficient Packamentioning

confidence: 99%

Single-molecule packaging initiation in real time by a viral DNA packaging machine from bacteriophage T4

Vafabakhsh

Kondabagil

Earnest

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Viral DNA packaging motors are among the most powerful molecular motors known. A variety of structural, biochemical, and singlemolecule biophysical approaches have been used to understand their mechanochemistry. However, packaging initiation has been difficult to analyze because of its transient and highly dynamic nature. Here, we developed a single-molecule fluorescence assay that allowed visualization of packaging initiation and reinitiation in real time and quantification of motor assembly and initiation kinetics. We observed that a single bacteriophage T4 packaging machine can package multiple DNA molecules in bursts of activity separated by long pauses, suggesting that it switches between active and quiescent states. Multiple initiation pathways were discovered including, unexpectedly, direct DNA binding to the capsid portal followed by recruitment of motor subunits. Rapid succession of ATP hydrolysis was essential for efficient initiation. These observations have implications for the evolution of icosahedral viruses and regulation of virus assembly.virus packaging | single-molecule fluorescence imaging | molecular motors A s part of a virus life cycle, genetic information needs to be incorporated into the newly produced virus particles. Tailed bacteriophages, which probably form the largest biomass of the planet (1), and many eukaryotic viruses such as herpes viruses use powerful ATPase motors to achieve this (2). These motors generate forces as high as 80-100 pN and translocate DNA into a preformed prohead until a DNA condensate of near crystalline density fills the interior (3).The viral packaging motors share a common architecture with the ASCE (additional strand, conserved E) superfamily of multimeric ring ATPases that perform diverse functions such as chromosome segregation (helicases), protein remodeling (chaperones and proteasomes), and cargo transport (dyneins) (4). Although much has been learned about the mechanochemistry of these motors, little is known about how a functional motor is assembled and its activity is initiated. The packaging motors have the difficult task of precisely inserting the end of a viral genome into the capsid at the time of initiation.In a general virus assembly pathway shared by dsDNA viruses, assembly starts at a unique fivefold vertex of the prohead called the portal vertex, which is formed from 12 molecules of the portal protein (5). A protein shell assembles around a protein scaffold and later becomes an empty prohead after the scaffold leaves, or is degraded (6). In most dsDNA bacteriophages as well as herpes viruses a complex of two proteins, known as small and large "terminase" proteins, recognize a specific sequence of DNA in the concatemeric viral genome (e.g., cos site in phage λ and pac site in phage P22) and make a cut to create a dsDNA end (7,8). The small terminase is responsible for binding to the cos or pac site, whereas the large terminase makes the cut. However, phage phi29 and adenoviruses do not require DNA cutting because the genome is a unit-length m...

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Function and horizontal transfer of the small terminase subunit of the tailed bacteriophage Sf6 DNA packaging nanomotor

Cited by 40 publications

References 98 publications

An RNA Domain Imparts Specificity and Selectivity to a Viral DNA Packaging Motor

An RNA Domain Imparts Specificity and Selectivity to a Viral DNA Packaging Motor

Exploring the Balance between DNA Pressure and Capsid Stability in Herpesviruses and Phages

Single-molecule packaging initiation in real time by a viral DNA packaging machine from bacteriophage T4

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