Structure determination of bacteriophage PP7 from<i>Pseudomonas aeruginosa</i>: from poor data to a good map

Tars, K.; Fridborg, K.; Bundule, M. F.; Liljas, Lars

doi:10.1107/s0907444900001232

Cited by 16 publications

(12 citation statements)

References 23 publications

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“…To improve the R factor, it may be necessary to release the strict NCS constraints and impose them as restraints at high resolutions (e.g., 2.3 Å), particularly when more than one particle is present in the crystallographic asymmetric unit. This phenomenon is even more pronounced in a high-symmetry space group relative to low-symmetry space group P1 as seen in a few cases (Khayat et al, 2011; Tars et al, 2000a, 2000b). Even though NCS restraints were used in the refinement of simple (T = 1) viruses, where 60-fold NCS was present (Larson et al, 2014), it is difficult to implement the same with 120-fold NCS because of a greater number of chains (120 × 2 = 240) involved.…”

Section: Methodsmentioning

confidence: 86%

Crystal Structure and Proteomics Analysis of Empty Virus-like Particles of Cowpea Mosaic Virus

et al. 2016

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SUMMARY Empty virus-like particles (eVLPs) of Cowpea mosaic virus (CPMV) are currently being utilized as reagents in various biomedical and nanotechnology applications. Here, we report the crystal structure of CPMV eVLPs determined using X-ray crystallography at 2.3 Å resolution and compare it with previously reported cryo-electron microscopy (cryo-EM) of eVLPs and virion crystal structures. Although the X-ray and cryo-EM structures of eVLPs are mostly similar, there exist significant differences at the C terminus of the small (S) subunit. The intact C terminus of the S subunit plays a critical role in enabling the efficient assembly of CPMV virions and eVLPs, but undergoes proteolysis after particle formation. In addition, we report the results of mass spectrometry-based proteomics analysis of coat protein subunits from CPMV eVLPs and virions that identify the C termini of S subunits undergo proteolytic cleavages at multiple sites instead of a single cleavage site as previously observed.

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Section: Methodsmentioning

confidence: 86%

Crystal Structure and Proteomics Analysis of Empty Virus-like Particles of Cowpea Mosaic Virus

et al. 2016

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“…They shut off viral replicase synthesis by binding an RNA hairpin that contains the replicase ribosome binding site. Recent x-ray structure determination of RNA phages shows that homologies evident from comparisons of coat protein amino acid sequences are reflected in their tertiary structures (1)(2)(3)(4)(5)(6)(7). The coat protein dimer, which is both the repressor and the basic building block of the virus particle, consists of two intertwined monomers that together form a large ␤-sheet surface upon which the RNA is bound.…”

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confidence: 99%

Translational Repression and Specific RNA Binding by the Coat Protein of the Pseudomonas Phage PP7

Lim

Downey

Peabody

2001

Journal of Biological Chemistry

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PP7 is a single-strand RNA bacteriophage of Pseudomonas aeroginosa and a distant relative to coliphages like MS2 and Q␤. Here we show that PP7 coat protein is a specific RNA-binding protein, capable of repressing the translation of sequences fused to the translation initiation region of PP7 replicase. Its RNA binding activity is specific since it represses the translational operator of PP7, but does not repress the operators of the MS2 or Q␤ phages. Conditions for the purification of coat protein and for the reconstitution of its RNA binding activity from disaggregated virus-like particles were established. Its dissociation constant for PP7 operator RNA in vitro was determined to be about 1 nM. Using a genetic system in which coat protein represses translation of a replicase-␤-galactosidase fusion protein, amino acid residues important for binding of PP7 RNA were identified.The coat proteins of several single-strand RNA bacteriophages are known translational repressors. They shut off viral replicase synthesis by binding an RNA hairpin that contains the replicase ribosome binding site. Recent x-ray structure determination of RNA phages shows that homologies evident from comparisons of coat protein amino acid sequences are reflected in their tertiary structures (1-7). The coat protein dimer, which is both the repressor and the basic building block of the virus particle, consists of two intertwined monomers that together form a large ␤-sheet surface upon which the RNA is bound. Each of the coat proteins uses a common structural framework to bind different RNAs, thereby presenting an opportunity to investigate the basis of specific RNA-protein recognition. In previous work we characterized the RNA binding sites of MS2, GA, and Q␤ coat proteins (8 -10). Here we describe the RNA binding properties of the coat protein of PP7, an RNA bacteriophage of Pseudomonas aeroginosa whose coat protein shows only 13% amino acid sequence identity to that of MS2. We present the following findings. 1) The coat protein of PP7 is a translational repressor. 2) An RNA hairpin containing the PP7 replicase translation initiation site is specifically bound by PP7 coat protein both in vivo and in vitro, indicating that this structure represents the translational operator.3) The RNA binding site resides on the coat protein ␤-sheet. A map of this site is presented. EXPERIMENTAL PROCEDURESPlasmid Constructions-The PP7 coat sequence cloned on a plasmid was kindly provided to us by Gordon Garde. We amplified the coat sequence using polymerase chain reaction and a 5Ј-primer (5Ј-GGGTCTAGACGTTACAGCGACTACTGAAACGTAAG-3Ј) that introduced a XbaI site about 40 nucleotides upstream of the coat initiation codon, and a 3Ј primer (5Ј-GGGGGATCCATACACACGGGTACACCG-CAGGGCC-3Ј) that created a BamHI site a few nucleotides downstream of the stop codon. This and subsequent amplifications were conducted using Pfu DNA polymerase. After digestion with XbaI and BamHI, the fragment was cloned between the corresponding restriction sites within the polylinker of pUC119 (11...

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“…1a–c). The icosahedral capsid of the Pseudomonas aeruginosa RNA bacteriophage PP7 self-assembles from 180 coat protein monomers 31, 32 , whose structural arrangement is equivalent to 90 coat protein dimers 31 . Recombinantly-expressed PP7 coat protein self-assembles into stable VLPs consistent in size with the bacteriophage PP7 icosahedral capsid 28, 31, 33 .…”

Section: Resultsmentioning

confidence: 99%

VLP-based vaccine induces immune control of Staphylococcus aureus virulence regulation

Daly

Joyner

Triplett

et al. 2017

Sci Rep

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Staphylococcus aureus is the leading cause of skin and soft tissue infections (SSTIs) and mounting antibiotic resistance requires innovative treatment strategies. S. aureus uses secreted cyclic autoinducing peptides (AIPs) and the accessory gene regulator (agr) operon to coordinate expression of virulence factors required for invasive infection. Of the four agr alleles (agr types I-IV and corresponding AIPs1-4), agr type I isolates are most frequently associated with invasive infection. Cyclization via a thiolactone bond is essential for AIP function; therefore, recognition of the cyclic form of AIP1 may be necessary for antibody-mediated neutralization. However, the small sizes of AIPs and labile thiolactone bond have hindered vaccine development. To overcome this, we used a virus-like particle (VLP) vaccine platform (PP7) for conformationally-restricted presentation of a modified AIP1 amino acid sequence (AIP1S). Vaccination with PP7-AIP1S elicited AIP1-specific antibodies and limited agr-activation in vivo. Importantly, in a murine SSTI challenge model with a highly virulent agr type I S. aureus isolate, PP7-AIP1S vaccination reduced pathogenesis and increased bacterial clearance compared to controls, demonstrating vaccine efficacy. Given the contribution of MRSA agr type I isolates to human disease, vaccine targeting of AIP1-regulated virulence could have a major clinical impact in the fight against antibiotic resistance.

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Structure determination of bacteriophage PP7 fromPseudomonas aeruginosa: from poor data to a good map

Cited by 16 publications

References 23 publications

Crystal Structure and Proteomics Analysis of Empty Virus-like Particles of Cowpea Mosaic Virus

Crystal Structure and Proteomics Analysis of Empty Virus-like Particles of Cowpea Mosaic Virus

Translational Repression and Specific RNA Binding by the Coat Protein of the Pseudomonas Phage PP7

VLP-based vaccine induces immune control of Staphylococcus aureus virulence regulation

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