Portal Motor Velocity and Internal Force Resisting Viral DNA Packaging in Bacteriophage ϕ29

Abstract

During the assembly of many viruses, a powerful molecular motor compacts the genome into a preassembled capsid. Here, we present measurements of viral DNA packaging in bacteriophage phi29 using an improved optical tweezers method that allows DNA translocation to be measured from initiation to completion. This method allowed us to study the previously uncharacterized early stages of packaging and facilitated more accurate measurement of the length of DNA packaged. We measured the motor velocity versus load at near-zero filling and developed a ramped DNA stretching technique that allowed us to measure the velocity versus capsid filling at near-zero load. These measurements reveal that the motor can generate significantly higher velocities and forces than detected previously. Toward the end of packaging, the internal force resisting DNA confinement rises steeply, consistent with the trend predicted by many theoretical models. However, the force rises to a higher magnitude, particularly during the early stages of packaging, than predicted by models that assume coaxial inverse spooling of the DNA. This finding suggests that the DNA is not arranged in that conformation during the early stages of packaging and indicates that internal force is available to drive complete genome ejection in vitro. The maximum force exceeds 100 pN, which is about one-half that predicted to rupture the capsid shell.

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

“…The terminase is qualitatively different with all packaging components present during translocation in eliminating all detectable YOYO-1 bound to even very short DNAs (70 bp or 280 bp) that are packaged. Resistance to packaging of short DNAs from DNA pressure within the prohead is expected to be low (17,18). It is thus unlikely that it is the packaged conformation of the DNA that quantitatively removes the dye but rather the translocation itself; indeed, the packaged DNA in the intact prohead can be readily stained with EthBr and also YOYO-1 (Figs.…”

Nonequilibrium dynamics and ultraslow relaxation of confined DNA during viral packaging

“…We note that our previous studies used the measured velocity vs. applied force relationship to infer an effective "internal force" resisting packaging (9,10,13). However, recent studies indicate that, although both prohead filling and applied force slow the motor, they have a different influence on the detailed motor stepping kinetics and thus cannot be directly equated (32).…”

“…This construct does not contain the gp3 DNA terminal protein needed for viral assembly in vivo. The DNA was tethered to 2.8-μm-diameter streptavidin-coated microspheres, and prohead-motor complexes were preassembled and attached via anti-phi29 antibodies to 2.1-μm protein G-coated microspheres, as described previously (10,13).…”

“…Packaging was initiated by bringing a microsphere carrying DNA into near contact with a microsphere carrying prohead-motor complexes in the presence of the standard packaging buffer containing 25 mM Tris·HCl, pH 7.5, 50 mM NaCl, 5 mM MgCl 2 , with 0.5 mM ATP, as described previously (10,13). Complexes were stalled by introducing the standard buffer with 0.4 mM γS-ATP via a capillary tube and were restarted by reintroducing standard packaging buffer.…”

“…DNA packaging in bacteriophages phi29, lambda, and T4 has been directly measured via single-molecule manipulation with optical tweezers and the packaging motors have been shown to generate forces of >60 pN, among the highest known for biomotors, while translocating DNA at rates ranging from ∼100 bp (for phage phi29, which packages a 19.3-kbp genome into a 42 × 54-nm prohead shell) up to as high as ∼2,000 bp/s (for phage T4, which packages a 171-kbp genome into a 120 × 86-nm prohead) (9)(10)(11)(12)(13)(14)(15). The force resisting packaging rises steeply with prohead filling and has been proposed to play an important role in driving viral DNA ejection (16).…”