Building a Virus from Scratch: Assembly of an Infectious Virus Using Purified Components in a Rigorously Defined Biochemical Assay System

Gaussier, Hélène; Yang, Qin; Catalano, Carlos Enrique

doi:10.1016/j.jmb.2006.01.013

Cited by 50 publications

(67 citation statements)

References 55 publications

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“…Our result suggests that either our assumption of linearity is not correct or the rate-liming step of virion assembly is not at the steps regulated by the late promoter, e.g., genomic DNA replication, etc., and thus not influenced by variation in the late promoter activity. However, a recent study of phage l assembly showed that, at least under the in vitro condition, the ratelimiting step of phage assembly, when compared to the genomic DNA packaging, is at the step of head-tail joining (Gaussier et al 2006), a step in which the component concentrations would certainly be influenced by the late promoter activity. To complicate matters further, in the same study, the authors also showed that not all the relationships between component concentrations and virion assembly are as straightforward as the Michelis-Menten kind.…”

Section: Discussionmentioning

confidence: 99%

Effect of Late Promoter Activity on Bacteriophage λ Fitness

Shao¹,

Wang

2009

Genetics

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For many bacteriophages (phages), the proteins responsible for host lysis and virion morphogenesis are expressed from the same polycistronic transcript. Such an expression pattern can potentially have a pleiotropic effect on the assembly rate and lysis time, thus affecting phage fitness. To study the effects of late promoter activity on phage life history traits and fitness, we constructed a series of isogenic phage l strains that differ only in their late promoter p R 9 sequences. The resulting late promoter activities ranged from 6 to 100% of the wild type's. The lysis times, burst sizes, and relative fitness were empirically determined for these strains. Our results showed that the lysis time is more sensitive than the assembly rate to variation in p R 9 activity. However, except for the strain with the lowest activity, the relative fitnesses of all the other strains are not significantly different from each other. Ad hoc models describing the effects of the late promoter activity on lysis time and assembly rate were constructed. The expected phage burst size and fitness curve were predicted from these models. Evolution of the late promoter activity was discussed in the context of phage life history trait evolution.

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Section: Discussionmentioning

confidence: 99%

Effect of Late Promoter Activity on Bacteriophage λ Fitness

Shao¹,

Wang

2009

Genetics

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“…36,37 These studies have demonstrated that the packaging reaction is magnesium dependent and that the optimal MgCl 2 concentration is ∼ 5 mM. This presents a problem when trying to package into the expanded capsids since they contract in the presence of N 1 mM Mg 2+ (Fig.…”

Section: Biological Activity Of the Expanded λ Capsidsmentioning

confidence: 99%

Thermodynamic Characterization of Viral Procapsid Expansion into a Functional Capsid Shell

Medina

Nakatani

Kruse

et al. 2012

Journal of Molecular Biology

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“…More recently, bulk studies using purified components have demonstrated that the assembly of infectious λ virus is reduced to 2% of maximum when gpD is omitted from the reaction mixture 44 . All of these authors concluded that gpD binds to the DNA-filled capsid, stabilizing the expanded structure to prevent DNA release.…”

Section: Rupture Of the Immature Procapsidmentioning

confidence: 99%

Measurements of Single DNA Molecule Packaging Dynamics in Bacteriophage λ Reveal High Forces, High Motor Processivity, and Capsid Transformations

Fuller

Raymer

Rickgauer

et al. 2007

Journal of Molecular Biology

158

204

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Molecular motors drive genome packaging into preformed procapsids in many dsDNA viruses. Here, we present optical tweezers measurements of single DNA molecule packaging in bacteriophage λ. DNA-gpA-gpNu1 complexes were assembled with recombinant gpA and gpNu1 proteins and tethered to microspheres, and procapsids were attached to separate microspheres. DNA binding and initiation of packaging were observed within a few seconds of bringing these microspheres into proximity in the presence of ATP. The motor was observed to generate greater than 50 picoNewtons (pN) of force, in the same range as observed with bacteriophage ϕ29, suggesting that high force generation is a common property of viral packaging motors. However, at low capsid filling the packaging rate averaged ~600 bp/s, which is 3.5-fold higher than ϕ29, and the motor processivity was also 3-fold higher, with less than one slip per genome length translocated. The packaging rate slowed significantly with increasing capsid filling, indicating a buildup of internal force reaching 14 pN at 86% packaging, in good agreement with the force driving DNA ejection measured in osmotic pressure experiments and calculated theoretically. Taken together, these experiments show that the internal force that builds during packaging is largely available to drive subsequent DNA ejection. In addition, we observed an 80 bp/s dip in the average packaging rate at 30% packaging, suggesting that procapsid expansion occurs at this point following the buildup of an average of 4 pN of internal force. In experiments with a DNA construct longer than the wild-type genome, a sudden acceleration in packaging rate was observed above 90% packaging in many cases, and greater than 100% of the genome length was translocated, suggesting that internal force can rupture the immature procapsid.

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Building a Virus from Scratch: Assembly of an Infectious Virus Using Purified Components in a Rigorously Defined Biochemical Assay System

Cited by 50 publications

References 55 publications

Effect of Late Promoter Activity on Bacteriophage λ Fitness

Effect of Late Promoter Activity on Bacteriophage λ Fitness

Thermodynamic Characterization of Viral Procapsid Expansion into a Functional Capsid Shell

Measurements of Single DNA Molecule Packaging Dynamics in Bacteriophage λ Reveal High Forces, High Motor Processivity, and Capsid Transformations

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