Publications by authors named "Peter Tattersall"

44 Publications

Rapid development of a bromochloropyridine regioisomer purity method enabled by strategic LC screening.

J Pharm Biomed Anal 2020 Nov 28;191:113594. Epub 2020 Aug 28.

Chemical Process Development, Bristol Myers Squibb, 1 Squibb Dr., New Brunswick, NJ 08903, USA.

With the intent to provide aligned, impactful, and efficient strategies for liquid chromatography method development, tier-based stationary/mobile phase screening workflows have been implemented in the Chemical Process Development department at Bristol Myers Squibb. These workflows are utilized as tools that enable more rapid method generation for early to mid-stage clinical development programs. An illustrative example of applying this approach was the method development for 3-bromo-2-chloropyridine and six of its positional isomeric impurities. Several parameters (gradient time, flow rate, column geometry, particle size, temperature, and solvent effects) were evaluated to achieve a baseline resolved separation for this challenging mixture. The impact that the screening workflows have regarding timesavings, effort, and resourcing to develop and optimize this LC method will be discussed.
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http://dx.doi.org/10.1016/j.jpba.2020.113594DOI Listing
November 2020

ICTV Virus Taxonomy Profile: Parvoviridae.

J Gen Virol 2019 03 23;100(3):367-368. Epub 2019 Jan 23.

12​Centre de Recherche de Microbiologie et Biotechnologie, INRS-Institut Armand-Frappier Laval, QC H7V 1B7, Canada.

Members of the family Parvoviridae are small, resilient, non-enveloped viruses with linear, single-stranded DNA genomes of 4-6 kb. Viruses in two subfamilies, the Parvovirinae and Densovirinae, are distinguished primarily by their respective ability to infect vertebrates (including humans) versus invertebrates. Being genetically limited, most parvoviruses require actively dividing host cells and are host and/or tissue specific. Some cause diseases, which range from subclinical to lethal. A few require co-infection with helper viruses from other families. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the Parvoviridae, which is available at www.ictv.global/report/parvoviridae.
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http://dx.doi.org/10.1099/jgv.0.001212DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6537627PMC
March 2019

White paper on microbial anti-cancer therapy and prevention.

J Immunother Cancer 2018 08 6;6(1):78. Epub 2018 Aug 6.

0000 0001 2151 2636grid.215654.1Center for Immunotherapy, Vaccines and Virotherapy , Biodesign InstituteArizona State University 727 E Tyler Street, Room A330E 85281 Tempe AZ USA

In this White Paper, we discuss the current state of microbial cancer therapy. This paper resulted from a meeting ('Microbial Based Cancer Therapy') at the US National Cancer Institute in the summer of 2017. Here, we define 'Microbial Therapy' to include both oncolytic viral therapy and bacterial anticancer therapy. Both of these fields exploit tumor-specific infectious microbes to treat cancer, have similar mechanisms of action, and are facing similar challenges to commercialization. We designed this paper to nucleate this growing field of microbial therapeutics and increase interactions between researchers in it and related fields. The authors of this paper include many primary researchers in this field. In this paper, we discuss the potential, status and opportunities for microbial therapy as well as strategies attempted to date and important questions that need to be addressed. The main areas that we think will have the greatest impact are immune stimulation, control of efficacy, control of delivery, and safety. There is much excitement about the potential of this field to treat currently intractable cancer. Much of the potential exists because these therapies utilize unique mechanisms of action, difficult to achieve with other biological or small molecule drugs. By better understanding and controlling these mechanisms, we will create new therapies that will become integral components of cancer care.
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http://dx.doi.org/10.1186/s40425-018-0381-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6091193PMC
August 2018

Optimizing the Targeting of Mouse Parvovirus 1 to Murine Melanoma Selects for Recombinant Genomes and Novel Mutations in the Viral Capsid Gene.

Viruses 2018 01 30;10(2). Epub 2018 Jan 30.

Department of Laboratory Medicine, Yale University Medical School, New Haven, CT 06520, USA.

Combining virus-enhanced immunogenicity with direct delivery of immunomodulatory molecules would represent a novel treatment modality for melanoma, and would require development of new viral vectors capable of targeting melanoma cells preferentially. Here we explore the use of rodent protoparvoviruses targeting cells of the murine melanoma model B16F10. An uncloned stock of mouse parvovirus 1 (MPV1) showed some efficacy, which was substantially enhanced following serial passage in the target cell. Molecular cloning of the genes of both starter and selected virus pools revealed considerable sequence diversity. Chimera analysis mapped the majority of the improved infectivity to the product of the major coat protein gene, , in which linked blocks of amino acid changes and one or other of two apparently spontaneous mutations were selected. Intragenic chimeras showed that these represented separable components, both contributing to enhanced infection. Comparison of biochemical parameters of infection by clonal viruses indicated that the enhancement due to changes in VP2 operates after the virus has bound to the cell surface and penetrated into the cell. Construction of an in silico homology model for MPV1 allowed placement of these changes within the capsid shell, and revealed aspects of the capsid involved in infection initiation that had not been previously recognized.
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http://dx.doi.org/10.3390/v10020054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5850361PMC
January 2018

Atomic Resolution Structure of the Oncolytic Parvovirus LuIII by Electron Microscopy and 3D Image Reconstruction.

Viruses 2017 10 30;9(11). Epub 2017 Oct 30.

Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 32611, USA.

LuIII, a protoparvovirus pathogenic to rodents, replicates in human mitotic cells, making it applicable for use to kill cancer cells. This virus group includes H-1 parvovirus (H-1PV) and minute virus of mice (MVM). However, LuIII displays enhanced oncolysis compared to H-1PV and MVM, a phenotype mapped to the major capsid viral protein 2 (VP2). This suggests that within LuIII VP2 are determinants for improved tumor lysis. To investigate this, the structure of the LuIII virus-like-particle was determined using single particle cryo-electron microscopy and image reconstruction to 3.17 Å resolution, and compared to the H-1PV and MVM structures. The LuIII VP2 structure, ordered from residue 37 to 587 (C-terminal), had the conserved VP topology and capsid morphology previously reported for other protoparvoviruses. This includes a core β-barrel and α-helix A, a depression at the icosahedral 2-fold and surrounding the 5-fold axes, and a single protrusion at the 3-fold axes. Comparative analysis identified surface loop differences among LuIII, H-1PV, and MVM at or close to the capsid 2- and 5-fold symmetry axes, and the shoulder of the 3-fold protrusions. The 2-fold differences cluster near the previously identified MVM sialic acid receptor binding pocket, and revealed potential determinants of protoparvovirus tumor tropism.
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http://dx.doi.org/10.3390/v9110321DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5707528PMC
October 2017

Cryo-EM maps reveal five-fold channel structures and their modification by gatekeeper mutations in the parvovirus minute virus of mice (MVM) capsid.

Virology 2017 10 24;510:216-223. Epub 2017 Jul 24.

Department of Medicine, Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033, USA. Electronic address:

In minute virus of mice (MVM) capsids, icosahedral five-fold channels serve as portals mediating genome packaging, genome release, and the phased extrusion of viral peptides. Previous studies suggest that residues L172 and V40 are essential for channel function. The structures of MVMi wildtype, and mutant L172T and V40A virus-like particles (VLPs) were solved from cryo-EM data. Two constriction points, termed the mid-gate and inner-gate, were observed in the channels of wildtype particles, involving residues L172 and V40 respectively. While the mid-gate of V40A VLPs appeared normal, in L172T adjacent channel walls were altered, and in both mutants there was major disruption of the inner-gate, demonstrating that direct L172:V40 bonding is essential for its structural integrity. In wildtype particles, residues from the N-termini of VP2 map into claw-like densities positioned below the channel opening, which become disordered in the mutants, implicating both L172 and V40 in the organization of VP2 N-termini.
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http://dx.doi.org/10.1016/j.virol.2017.07.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5601314PMC
October 2017

The MVMp P4 promoter is a host cell-type range determinant in vivo.

Virology 2017 06 6;506:141-151. Epub 2017 Apr 6.

Department of Microbiology, Immunology and Genetics, Faculty of Health Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel. Electronic address:

The protoparvovirus early promoters, e.g. P4 of Minute Virus of Mice (MVM), play a critical role during infection. Initial P4 activity depends on the host transcription machinery only. Since this is cell-type dependent, it is hypothesized that P4 is a host cell-type range determinant. Yet host range determinants have mapped mostly to capsid, never P4. Here we test the hypothesis using the mouse embryo as a model system. Disruption of the CRE element of P4 drastically decreased infection levels without altering range. However, when we swapped promoter elements of MVM P4 with those from equivalent regions of the closely related H1 virus, we observed elimination of infection in fibroblasts and chondrocytes and the acquisition of infection in skeletal muscle. We conclude that P4 is a host range determinant and a target for modifying the productive infection potential of the virus - an important consideration in adapting these viruses for oncotherapy.
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http://dx.doi.org/10.1016/j.virol.2017.03.012DOI Listing
June 2017

In Vivo Examination of Mouse APOBEC3- and Human APOBEC3A- and APOBEC3G-Mediated Restriction of Parvovirus and Herpesvirus Infection in Mouse Models.

J Virol 2016 09 12;90(17):8005-12. Epub 2016 Aug 12.

Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA

Unlabelled: APOBEC3 knockout and human APOBEC3A and -3G transgenic mice were tested for their ability to be infected by the herpesviruses herpes simplex virus 1 and murine herpesvirus 68 and the parvovirus minute virus of mice (MVM). Knockout, APOBEC3A and APOBEC3G transgenic, and wild-type mice were equally infected by the herpesviruses, while APOBEC3A but not mouse APOBEC3 conferred resistance to MVM. No viruses showed evidence of cytidine deamination by mouse or human APOBEC3s. These data suggest that in vitro studies implicating APOBEC3 proteins in virus resistance may not reflect their role in vivo

Importance: It is well established that APOBEC3 proteins in different species are a critical component of the host antiretroviral defense. Whether these proteins also function to inhibit other viruses is not clear. There have been a number of in vitro studies suggesting that different APOBEC3 proteins restrict herpesviruses and parvoviruses, among others, but whether they also work in vivo has not been demonstrated. Our studies looking at the role of mouse and human APOBEC3 proteins in transgenic and knockout mouse models of viral infection suggest that these restriction factors are not broadly antiviral and demonstrate the importance of testing their activity in vivo.
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http://dx.doi.org/10.1128/JVI.00973-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4988146PMC
September 2016

Structures of minute virus of mice replication initiator protein N-terminal domain: Insights into DNA nicking and origin binding.

Virology 2015 Feb 18;476:61-71. Epub 2014 Dec 18.

Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045, USA. Electronic address:

Members of the Parvoviridae family all encode a non-structural protein 1 (NS1) that directs replication of single-stranded viral DNA, packages viral DNA into capsid, and serves as a potent transcriptional activator. Here we report the X-ray structure of the minute virus of mice (MVM) NS1 N-terminal domain at 1.45Å resolution, showing that sites for dsDNA binding, ssDNA binding and cleavage, nuclear localization, and other functions are integrated on a canonical fold of the histidine-hydrophobic-histidine superfamily of nucleases, including elements specific for this Protoparvovirus but distinct from its Bocaparvovirus or Dependoparvovirus orthologs. High resolution structural analysis reveals a nickase active site with an architecture that allows highly versatile metal ligand binding. The structures support a unified mechanism of replication origin recognition for homotelomeric and heterotelomeric parvoviruses, mediated by a basic-residue-rich hairpin and an adjacent helix in the initiator proteins and by tandem tetranucleotide motifs in the replication origins.
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http://dx.doi.org/10.1016/j.virol.2014.11.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4699654PMC
February 2015

Parvoviruses: Small Does Not Mean Simple.

Annu Rev Virol 2014 Nov 9;1(1):517-37. Epub 2014 Jul 9.

Departments of 1Laboratory Medicine and.

Parvoviruses are small, rugged, nonenveloped protein particles containing a linear, nonpermuted, single-stranded DNA genome of ∼5 kb. Their limited coding potential requires optimal adaptation to the environment of particular host cells, where entry is mediated by a variable program of capsid dynamics, ultimately leading to genome ejection from intact particles within the host nucleus. Genomes are amplified by a continuous unidirectional strand-displacement mechanism, a linear adaptation of rolling circle replication that relies on the repeated folding and unfolding of small hairpin telomeres to reorient the advancing fork. Progeny genomes are propelled by the viral helicase into the preformed capsid via a pore at one of its icosahedral fivefold axes. Here we explore how the fine-tuning of this unique replication system and the mechanics that regulate opening and closing of the capsid fivefold portals have evolved in different viral lineages to create a remarkably complex spectrum of phenotypes.
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http://dx.doi.org/10.1146/annurev-virology-031413-085444DOI Listing
November 2014

Complementation for an essential ancillary non-structural protein function across parvovirus genera.

Virology 2014 Nov 6;468-470:226-237. Epub 2014 Sep 6.

Department of Laboratory Medicine, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510, USA; Department of Genetics, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510, USA. Electronic address:

Parvoviruses encode a small number of ancillary proteins that differ substantially between genera. Within the genus Protoparvovirus, minute virus of mice (MVM) encodes three isoforms of its ancillary protein NS2, while human bocavirus 1 (HBoV1), in the genus Bocaparvovirus, encodes an NP1 protein that is unrelated in primary sequence to MVM NS2. To search for functional overlap between NS2 and NP1, we generated murine A9 cell populations that inducibly express HBoV1 NP1. These were used to test whether NP1 expression could complement specific defects resulting from depletion of MVM NS2 isoforms. NP1 induction had little impact on cell viability or cell cycle progression in uninfected cells, and was unable to complement late defects in MVM virion production associated with low NS2 levels. However, NP1 did relocate to MVM replication centers, and supports both the normal expansion of these foci and overcomes the early paralysis of DNA replication in NS2-null infections.
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http://dx.doi.org/10.1016/j.virol.2014.07.043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4254310PMC
November 2014

Genome sequence of tumor virus x, a member of the genus protoparvovirus in the family parvoviridae.

Genome Announc 2014 Jul 31;2(4). Epub 2014 Jul 31.

The orphan parvovirus tumor virus X (TVX) has potent oncolytic activity. Compared to other viruses from the species Rodent protoparvovirus 1, TVX has a 111 nucleotide deletion in its nonstructural (NS) gene, a 24 nucleotide insertion in VP1, and a 93 nucleotide repeat initiating from the C-terminus of the capsid gene.
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http://dx.doi.org/10.1128/genomeA.00758-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4118071PMC
July 2014

Profiling of glycan receptors for minute virus of mice in permissive cell lines towards understanding the mechanism of cell recognition.

PLoS One 2014 27;9(1):e86909. Epub 2014 Jan 27.

Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida, United States of America.

The recognition of sialic acids by two strains of minute virus of mice (MVM), MVMp (prototype) and MVMi (immunosuppressive), is an essential requirement for successful infection. To understand the potential for recognition of different modifications of sialic acid by MVM, three types of capsids, virus-like particles, wild type empty (no DNA) capsids, and DNA packaged virions, were screened on a sialylated glycan microarray (SGM). Both viruses demonstrated a preference for binding to 9-O-methylated sialic acid derivatives, while MVMp showed additional binding to 9-O-acetylated and 9-O-lactoylated sialic acid derivatives, indicating recognition differences. The glycans recognized contained a type-2 Galβ1-4GlcNAc motif (Neu5Acα2-3Galβ1-4GlcNAc or 3'SIA-LN) and were biantennary complex-type N-glycans with the exception of one. To correlate the recognition of the 3'SIA-LN glycan motif as well as the biantennary structures to their natural expression in cell lines permissive for MVMp, MVMi, or both strains, the N- and O-glycans, and polar glycolipids present in three cell lines used for in vitro studies, A9 fibroblasts, EL4 T lymphocytes, and the SV40 transformed NB324K cells, were analyzed by MALDI-TOF/TOF mass spectrometry. The cells showed an abundance of the sialylated glycan motifs recognized by the viruses in the SGM and previous glycan microarrays supporting their role in cellular recognition by MVM. Significantly, the NB324K showed fucosylation at the non-reducing end of their biantennary glycans, suggesting that recognition of these cells is possibly mediated by the Lewis X motif as in 3'SIA-Le(X) identified in a previous glycan microarray screen.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0086909PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3903596PMC
September 2014

The family Parvoviridae.

Arch Virol 2014 May 9;159(5):1239-47. Epub 2013 Nov 9.

Department of Laboratory Medicine, Yale University School of Medicine, New Haven, CT, USA,

A set of proposals to rationalize and extend the taxonomy of the family Parvoviridae is currently under review by the International Committee on Taxonomy of Viruses (ICTV). Viruses in this family infect a wide range of hosts, as reflected by the longstanding division into two subfamilies: the Parvovirinae, which contains viruses that infect vertebrate hosts, and the Densovirinae, encompassing viruses that infect arthropod hosts. Using a modified definition for classification into the family that no longer demands isolation as long as the biological context is strong, but does require a near-complete DNA sequence, 134 new viruses and virus variants were identified. The proposals introduce new species and genera into both subfamilies, resolve one misclassified species, and improve taxonomic clarity by employing a series of systematic changes. These include identifying a precise level of sequence similarity required for viruses to belong to the same genus and decreasing the level of sequence similarity required for viruses to belong to the same species. These steps will facilitate recognition of the major phylogenetic branches within genera and eliminate the confusion caused by the near-identity of species and viruses. Changes to taxon nomenclature will establish numbered, non-Latinized binomial names for species, indicating genus affiliation and host range rather than recapitulating virus names. Also, affixes will be included in the names of genera to clarify subfamily affiliation and reduce the ambiguity that results from the vernacular use of "parvovirus" and "densovirus" to denote multiple taxon levels.
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http://dx.doi.org/10.1007/s00705-013-1914-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4013247PMC
May 2014

Distinct host cell fates for human malignant melanoma targeted by oncolytic rodent parvoviruses.

Virology 2013 Nov 9;446(1-2):37-48. Epub 2013 Aug 9.

Medical Scientist Training Program, Yale University Medical School, 333 Cedar Street, New Haven, CT 06510, United States; Department of Genetics, Yale University Medical School, 333 Cedar Street, New Haven, CT 06510, United States.

The rodent parvoviruses are known to be oncoselective, and lytically infect many transformed human cells. Because current therapeutic regimens for metastatic melanoma have low response rates and have little effect on improving survival, this disease is a prime candidate for novel approaches to therapy, including oncolytic parvoviruses. Screening of low-passage, patient-derived melanoma cell lines for multiplicity-dependent killing by a panel of five rodent parvoviruses identified LuIII as the most melanoma-lytic. This property was mapped to the LuIII capsid gene, and an efficiently melanoma tropic chimeric virus shown to undergo three types of interaction with primary human melanoma cells: (1) complete lysis of cultures infected at very low multiplicities; (2) acute killing resulting from viral protein synthesis and DNA replication, without concomitant expansion of the infection, due to failure to export progeny virions efficiently; or (3) complete resistance that operates at an intracellular step following virion uptake, but preceding viral transcription.
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http://dx.doi.org/10.1016/j.virol.2013.07.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3811133PMC
November 2013

Parvoviral left-end hairpin ears are essential during infection for establishing a functional intranuclear transcription template and for efficient progeny genome encapsidation.

J Virol 2013 Oct 31;87(19):10501-14. Epub 2013 Jul 31.

Departments of Laboratory Medicine.

The 121-nucleotide left-end telomere of Minute Virus of Mice (MVM) can be folded into a Y-shaped hairpin with short axial ears that are highly conserved within genus Parvovirus. To explore their potential role(s) during infection, we constructed infectious plasmid clones that lacked one or other ear. Although these were nonviable when transfected into A9 cells, excision of the viral genome and DNA amplification appeared normal, and viral transcripts and proteins were expressed, but progeny virion production was minimal, supporting the idea of a potential role for the ears in genome packaging. To circumvent the absence of progeny that confounded further analysis of these mutants, plasmids were transfected into 293T cells both with and without an adenovirus helper construct, generating single bursts of progeny. These virions bound to A9 cells and were internalized but failed to initiate viral transcription, protein expression, or DNA replication. No defects in mutant virion stability or function could be detected in vitro. Significantly, mutant capsid gene expression and DNA replication could be rescued by coinfection with wild-type virions carrying a replication-competent, capsid-gene-replacement vector. To pinpoint where such complementation occurred, prior transfection of plasmids expressing only MVM nonstructural proteins was explored. NS1 alone, but not NS2, rescued transcription and protein expression from both P4 and P38 promoters, whereas NS1 molecules deleted for their C-terminal transactivation domain did not. These results suggest that the mutant virions reach the nucleus, uncoat, and are converted to duplex DNA but require an intact left-end hairpin structure to form the initiating transcription complex.
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http://dx.doi.org/10.1128/JVI.01393-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3807388PMC
October 2013

Parvovirus evades interferon-dependent viral control in primary mouse embryonic fibroblasts.

Virology 2013 Jul 12;442(1):20-7. Epub 2013 May 12.

Department of Immunobiology, Yale University, New Haven, CT 06520, USA.

Engagement of innate viral sensors elicits a robust antiviral program via the induction of type I interferons (IFNs). Innate defense mechanisms against ssDNA viruses are not well defined. Here, we examine type I IFN induction and effectiveness in controlling a ssDNA virus. Using mouse embryonic fibroblasts (MEFs), we found that a murine parvovirus, minute virus of mice (MVMp), induced a delayed but significant IFN response. MEFs deficient in mitochondrial antiviral signaling protein (MAVS) mounted a wild-type IFN response to MVMp infection, indicating that RIG-I-dependent RNA intermediate recognition is not required for innate sensing of this virus. However, MVMp-induced IFNs, as well recombinant type I IFNs, were unable to inhibit viral replication. Finally, MVMp infected cells became unresponsive to Poly (I:C) stimulation. Together, these data suggest that the MVMp efficiently evades antiviral immune mechanisms imposed by type I IFNs, which may in part explain their efficient transmission between mice.
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http://dx.doi.org/10.1016/j.virol.2013.03.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3767977PMC
July 2013

Toll-like receptor 9 in plasmacytoid dendritic cells fails to detect parvoviruses.

J Virol 2013 Mar 9;87(6):3605-8. Epub 2013 Jan 9.

Department of Immunobiology, Yale University, New Haven, CT, USA.

Toll-like receptor 9 (TLR9) recognizes genomes of double-stranded DNA (dsDNA) viruses in the endosome to stimulate plasmacytoid dendritic cells (pDCs). However, how and if viruses with single-stranded DNA (ssDNA) genomes are detected by pDCs remain unclear. Here we have shown that despite the ability of purified genomic DNA to stimulate TLR9 and despite the ability to enter TLR9 endosomes, ssDNA viruses of the Parvoviridae family failed to elicit an interferon (IFN) response in pDCs.
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http://dx.doi.org/10.1128/JVI.03155-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3592163PMC
March 2013

Parvovirus diversity and DNA damage responses.

Cold Spring Harb Perspect Biol 2013 Feb 1;5(2). Epub 2013 Feb 1.

Department of Laboratory Medicine, Yale University School of Medicine, New Haven, CT 06510, USA.

Parvoviruses have a linear single-stranded DNA genome, around 5 kb in length, with short imperfect terminal palindromes that fold back on themselves to form duplex hairpin telomeres. These contain most of the cis-acting information required for viral "rolling hairpin" DNA replication, an evolutionary adaptation of rolling-circle synthesis in which the hairpins create duplex replication origins, prime complementary strand synthesis, and act as hinges to reverse the direction of the unidirectional cellular fork. Genomes are packaged vectorially into small, rugged protein capsids ~260 Å in diameter, which mediate their delivery directly into the cell nucleus, where they await their host cell's entry into S phase under its own cell cycle control. Here we focus on genus-specific variations in genome structure and replication, and review host cell responses that modulate the nuclear environment.
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http://dx.doi.org/10.1101/cshperspect.a012989DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3552509PMC
February 2013

Functional glycomic analysis of human milk glycans reveals the presence of virus receptors and embryonic stem cell biomarkers.

J Biol Chem 2012 Dec 31;287(53):44784-99. Epub 2012 Oct 31.

Department of Biochemistry and the Glycomics Center, Emory University School of Medicine, Atlanta, Georgia 30322, USA.

Human milk contains a large diversity of free glycans beyond lactose, but their functions are not well understood. To explore their functional recognition, here we describe a shotgun glycan microarray prepared from isolated human milk glycans (HMGs), and our studies on their recognition by viruses, antibodies, and glycan-binding proteins (GBPs), including lectins. The total neutral and sialylated HMGs were derivatized with a bifunctional fluorescent tag, separated by multidimensional HPLC, and archived in a tagged glycan library, which was then used to print a shotgun glycan microarray (SGM). This SGM was first interrogated with well defined GBPs and antibodies. These data demonstrated both the utility of the array and provided preliminary structural information (metadata) about this complex glycome. Anti-TRA-1 antibodies that recognize human pluripotent stem cells specifically recognized several HMGs that were then further structurally defined as novel epitopes for these antibodies. Human influenza viruses and Parvovirus Minute Viruses of Mice also specifically recognized several HMGs. For glycan sequencing, we used a novel approach termed metadata-assisted glycan sequencing (MAGS), in which we combine information from analyses of glycans by mass spectrometry with glycan interactions with defined GBPs and antibodies before and after exoglycosidase treatments on the microarray. Together, these results provide novel insights into diverse recognition functions of HMGs and show the utility of the SGM approach and MAGS as resources for defining novel glycan recognition by GBPs, antibodies, and pathogens.
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http://dx.doi.org/10.1074/jbc.M112.425819DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3531791PMC
December 2012

Maintenance of the flip sequence orientation of the ears in the parvoviral left-end hairpin is a nonessential consequence of the critical asymmetry in the hairpin stem.

J Virol 2012 Nov 29;86(22):12187-97. Epub 2012 Aug 29.

Department of Laboratory Medicine, Yale University Medical School, New Haven, Connecticut, USA.

Parvoviral terminal hairpins are essential for viral DNA amplification but are also implicated in multiple additional steps in the viral life cycle. The palindromes at the two ends of the minute virus of mice (MVM) genome are dissimilar and are processed by different resolution mechanisms that selectively direct encapsidation of predominantly negative-sense progeny genomes and conserve a single Flip sequence orientation at the 3' (left) end of such progeny. The sequence and predicted structure of these 3' hairpins are highly conserved within the genus Parvovirus, exemplified by the 121-nucleotide left-end sequence of MVM, which folds into a Y-shaped hairpin containing small internal palindromes that form the "ears" of the Y. To explore the potential role(s) of this hairpin in the viral life cycle, we constructed infectious clones with the ear sequences either inverted, to give the antiparallel Flop orientation, or with multiple transversions, conserving their base composition but changing their sequence. These were compared with a "bubble" mutant, designed to activate the normally silent origin in the inboard arm of the hairpin, thus potentially rendering symmetric the otherwise asymmetric junction resolution mechanism that drives maintenance of Flip. This mutant exhibited a major defect in viral duplex and single-strand DNA replication, characterized by the accumulation of covalently closed turnaround forms of the left end, and was rapidly supplanted by revertants that restored asymmetry. In contrast, both sequence and orientation changes in the hairpin ears were tolerated, suggesting that maintaining the Flip orientation of these structures is a consequence of, but not the reason for, asymmetric left-end processing.
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http://dx.doi.org/10.1128/JVI.01450-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3486466PMC
November 2012

Mutations at the base of the icosahedral five-fold cylinders of minute virus of mice induce 3'-to-5' genome uncoating and critically impair entry functions.

J Virol 2012 Jan 19;86(1):69-80. Epub 2011 Oct 19.

Department of Laboratory Medicine, Yale University School of Medicine, New Haven, Connecticut, USA.

The linear single-stranded DNA genome of minute virus of mice can be ejected, in a 3'-to-5' direction, via a cation-linked uncoating reaction that leaves the 5' end of the DNA firmly complexed with its otherwise intact protein capsid. Here we compare the phenotypes of four mutants, L172T, V40A, N149A, and N170A, which perturb the base of cylinders surrounding the icosahedral 5-fold axes of the virus, and show that these structures are strongly implicated in 3'-to-5' release. Although noninfectious at 37°C, all mutants were viable at 32°C, showed a temperature-sensitive cell entry defect, and, after proteolysis of externalized VP2 N termini, were unable to protect the VP1 domain, which is essential for bilayer penetration. Mutant virus yields from multiple-round infections were low and were characterized by the accumulation of virions containing subgenomic DNAs of specific sizes. In V40A, these derived exclusively from the 5' end of the genome, indicative of 3'-to-5' uncoating, while L172T, the most impaired mutant, had long subgenomic DNAs originating from both termini, suggesting additional packaging portal defects. Compared to the wild type, genome release in vitro following cation depletion was enhanced for all mutants, while only L172T released DNA, in both directions, without cation depletion following proteolysis at 37°C. Analysis of progeny from single-round infections showed that uncoating did not occur during virion assembly, release, or extraction. However, unlike the wild type, the V40A mutant extensively uncoated during cell entry, indicating that the V40-L172 interaction restrains an uncoating trigger mechanism within the endosomal compartment.
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http://dx.doi.org/10.1128/JVI.06119-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3255873PMC
January 2012

The parvoviral capsid controls an intracellular phase of infection essential for efficient killing of stepwise-transformed human fibroblasts.

Virology 2011 Jul 20;416(1-2):32-41. Epub 2011 May 20.

Department of Laboratory Medicine, Yale University Medical School, New Haven, CT 06520, USA.

Members of the rodent subgroup of the genus Parvovirus exhibit lytic replication and spread in many human tumor cells and are therefore attractive candidates for oncolytic virotherapy. However, the significant variation in tumor tropism observed for these viruses remains largely unexplained. We report here that LuIII kills BJ-ELR 'stepwise-transformed' human fibroblasts efficiently, while MVM does not. Using viral chimeras, we mapped this property to the LuIII capsid gene, VP2, which is necessary and sufficient to confer the killer phenotype on MVM. LuIII VP2 facilitates a post-entry, pre-DNA-amplification step early in the life cycle, suggesting the existence of an intracellular moiety whose efficient interaction with the incoming capsid shell is critical to infection. Thus targeting of human cancers of different tissue-type origins will require use of parvoviruses with capsids that effectively make this critical interaction.
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http://dx.doi.org/10.1016/j.virol.2011.04.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3112476PMC
July 2011

Structure of a packaging-defective mutant of minute virus of mice indicates that the genome is packaged via a pore at a 5-fold axis.

J Virol 2011 May 2;85(10):4822-7. Epub 2011 Mar 2.

Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907, USA.

The parvovirus minute virus of mice (MVM) packages a single copy of its linear single-stranded DNA genome into preformed capsids, in a process that is probably driven by a virus-encoded helicase. Parvoviruses have a roughly cylindrically shaped pore that surrounds each of the 12 5-fold vertices. The pore, which penetrates the virion shell, is created by the juxtaposition of 10 antiparallel β-strands, two from each of the 5-fold-related capsid proteins. There is a bottleneck in the channel formed by the symmetry-related side chains of the leucines at position 172. We report here the X-ray crystal structure of the particles produced by a leucine-to-tryptophan mutation at position 172 and the analysis of its biochemical properties. The mutant capsid had its 5-fold channel blocked, and the particles were unable to package DNA, strongly suggesting that the 5-fold pore is the packaging portal for genome entry.
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http://dx.doi.org/10.1128/JVI.02598-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3126206PMC
May 2011

Recruitment of DNA replication and damage response proteins to viral replication centers during infection with NS2 mutants of Minute Virus of Mice (MVM).

Virology 2011 Feb 30;410(2):375-84. Epub 2010 Dec 30.

Department of Laboratory Medicine, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510, USA.

MVM NS2 is essential for viral DNA amplification, but its mechanism of action is unknown. A classification scheme for autonomous parvovirus-associated replication (APAR) center development, based on NS1 distribution, was used to characterize abnormal APAR body maturation in NS2null mutant infections, and their organization examined for defects in host protein recruitment. Since acquisition of known replication factors appeared normal, we looked for differences in invoked DNA damage responses. We observed widespread association of H2AX/MDC1 damage response foci with viral replication centers, and sequestration and complex hyperphosphorylation of RPA(32), which occurred in wildtype and mutant infections. Quantifying these responses by western transfer indicated that both wildtype and NS2 mutant MVM elicited ATM activation, while phosphorylation of ATR, already basally activated in asynchronous A9 cells, was downregulated. We conclude that MVM infection invokes multiple damage responses that influence the APAR environment, but that NS2 does not modify the recruitment of cellular proteins.
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http://dx.doi.org/10.1016/j.virol.2010.12.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3072075PMC
February 2011

Depletion of virion-associated divalent cations induces parvovirus minute virus of mice to eject its genome in a 3'-to-5' direction from an otherwise intact viral particle.

J Virol 2010 Feb 2;84(4):1945-56. Epub 2009 Dec 2.

Department of Laboratory Medicine, Yale University Medical School, 333 Cedar Street, New Haven, CT 06510, USA.

We describe a structural rearrangement that can occur in parvovirus minute virus of mice (MVMp) virions following prolonged exposure to buffers containing 0.5 mM EDTA. Such particles remain stable at 4 degrees C but undergo a conformational shift upon heating to 37 degrees C at pH 7.2 that leads to the ejection of much of the viral genome in a 3'-to-5' direction, leaving the DNA tightly associated with the otherwise intact capsid. This rearrangement can be prevented by the addition of 1 mM CaCl(2) or MgCl(2) prior to incubation at 37 degrees C, suggesting that readily accessible divalent cation binding sites in the particle are critical for genome retention. Uncoating was not seen following the incubation of virions at pH 5.5 and 37 degrees C or at pH 7.2 and 37 degrees C in particles with subgenomic DNA, suggesting that pressure exerted by the full-length genome may influence this process. Uncoated genomes support complementary-strand synthesis by T7 DNA polymerase, but synthesis aborts upstream of the right-hand end, which remains capsid associated. We conclude that viral genomes are positioned so that their 3' termini and coding sequences can be released from intact particles at physiological temperatures by a limited conformational rearrangement. In the presence of divalent cations, incremental heating between 45 degrees C and 65 degrees C induces structural transitions that first lead to the extrusion of VP1 N termini, followed by genome exposure. However, in cation-depleted virions, the sequence of these shifts is blurred. Moreover, cation-depleted particles that have been induced to eject their genomes at 37 degrees C continue to sequester their VP1 N termini within the intact capsid, suggesting that these two extrusion events represent separable processes.
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http://dx.doi.org/10.1128/JVI.01563-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2812381PMC
February 2010

Seroepidemiology of human bocavirus defined using recombinant virus-like particles.

J Infect Dis 2008 Jul;198(1):41-50

Dept. of Pediatrics, Div. of Infectious Diseases, Yale University School of Medicine, PO Box 208064, New Haven, CT 06520-8064,

Background: Human bocavirus (HBoV) is a newly identified human parvovirus for which seroepidemiology and antigenic properties remain undefined.

Methods: The HBoV VP2 gene, expressed from a baculovirus vector, produced virus-like particles (VLPs), which were used to raise rabbit anti-HBoV antisera and to develop an enzyme-linked immunosorbent assay (ELISA). The VLP-based ELISA was used to screen for HBoV-specific immunoglobulin G antibodies in a convenience sample of 270 serum specimens, mostly from children, obtained at Yale-New Haven Hospital; 208 specimens were also screened for erythrovirus B19-specific antibodies by a B19 VLP-based ELISA.

Results: Immunofluorescence and ELISA showed that human parvoviruses HBoV and B19 are antigenically distinct. By the HBoV VLP-based ELISA, 91.8% and 63.6% of serum specimens from infants in the first and second months of life, respectively, were found to be seropositive, as were 45.4% from 3-month-old infants and 25.0% from 4-month-old infants. The percentages of HBoV-seropositive children increased to 40.7%-60.0% for children 5-47 months of age and to >85% for individuals >or=48 months old. However, the overall percentage of B19-seropositive individuals was <40.5% for all age groups screened.

Conclusions: HBoV infection is common during childhood, but a minority of children and young adults screened have evidence of B19 infection.
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http://dx.doi.org/10.1086/588674DOI Listing
July 2008

Evolution to pathogenicity of the parvovirus minute virus of mice in immunodeficient mice involves genetic heterogeneity at the capsid domain that determines tropism.

J Virol 2008 Feb 28;82(3):1195-203. Epub 2007 Nov 28.

Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid), 28049 Cantoblanco, Madrid, Spain.

Very little is known about the role that evolutionary dynamics plays in diseases caused by mammalian DNA viruses. To address this issue in a natural host model, we compared the pathogenesis and genetics of the attenuated fibrotropic and the virulent lymphohematotropic strains of the parvovirus minute virus of mice (MVM), and of two invasive fibrotropic MVM (MVMp) variants carrying the I362S or K368R change in the VP2 major capsid protein, in the infection of severe combined immunodeficient (SCID) mice. By 14 to 18 weeks after oronasal inoculation, the I362S and K368R viruses caused lethal leukopenia characterized by tissue damage and inclusion bodies in hemopoietic organs, a pattern of disease found by 7 weeks postinfection with the lymphohematotropic MVM (MVMi) strain. The MVMp populations emerging in leukopenic mice showed consensus sequence changes in the MVMi genotype at residues G321E and A551V of VP2 in the I362S virus infections or A551V and V575A changes in the K368R virus infections, as well as a high level of genetic heterogeneity within a capsid domain at the twofold depression where these residues lay. Amino acids forming this capsid domain are important MVM tropism determinants, as exemplified by the switch in MVMi host range toward mouse fibroblasts conferred by coordinated changes of some of these residues and by the essential character of glutamate at residue 321 for maintaining MVMi tropism toward primary hemopoietic precursors. The few viruses within the spectrum of mutants from mice that maintained the respective parental 321G and 575V residues were infectious in a plaque assay, whereas the viruses with the main consensus sequences exhibited low levels of fitness in culture. Consistent with this finding, a recombinant MVMp virus carrying the consensus sequence mutations arising in the K368R virus background in mice failed to initiate infection in cell lines of different tissue origins, even though it caused rapid-course lethal leukopenia in SCID mice. The parental consensus genotype prevailed during leukopenia development, but plaque-forming viruses with the reversion of the 575A residue to valine emerged in affected organs. The disease caused by the DNA virus in mice, therefore, involves the generation of heterogeneous viral populations that may cooperatively interact for the hemopoietic syndrome. The evolutionary changes delineate a sector of the surface of the capsid that determines tropism and that surrounds the sialic acid receptor binding domain.
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http://dx.doi.org/10.1128/JVI.01692-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2224436PMC
February 2008

Replication initiator protein NS1 of the parvovirus minute virus of mice binds to modular divergent sites distributed throughout duplex viral DNA.

J Virol 2007 Dec 26;81(23):13015-27. Epub 2007 Sep 26.

Department of Laboratory Medicine, Yale University Medical School, 333 Cedar Street, New Haven, CT 06510, USA.

To initiate DNA synthesis, the NS1 protein of minute virus of mice (MVM) first binds to a simple cognate recognition sequence in the viral origins, comprising two to three tandem copies of the tetranucleotide TGGT. However, this motif is also widely dispersed throughout the viral genome. Using an immunoselection procedure, we show that NS1 specifically binds to many internal sites, so that all viral fragments of more than approximately 170 nucleotides effectively compete for NS1, often binding with higher affinity to these internal sites than to sites in the origins. We explore the diversity of the internal sites using competitive binding and DNase I protection assays and show that they vary between two extreme forms. Simple sites with three somewhat degenerate, tandem TGGT reiterations bind effectively but are minimally responsive to ATP, while complex sites, containing multiple variably spaced TGGT elements arranged as opposing clusters, bind NS1 with an affinity that can be enhanced approximately 10-fold by ATP. Using immuno-selection procedures with randomized sequences embedded within specific regions of the genome, we explore possible binding configurations in these two types of site. We conclude that binding is modular, combinatorial, and highly flexible. NS1 recognizes two to six variably spaced, more-or-less degenerate forms of the 5'-TGGT-3' motif, so that it binds efficiently to a wide variety of sequences. Thus, despite complex coding constraints, binding sites are configured at frequent intervals throughout duplex forms of viral DNA, suggesting that NS1 may serve as a form of chromatin to protect and tailor the environment of replicating genomes.
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http://dx.doi.org/10.1128/JVI.01703-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2169109PMC
December 2007

Parvoviral host range and cell entry mechanisms.

Adv Virus Res 2007 ;70:183-232

Department of Laboratory Medicine, Yale University Medical School, New Haven, Connecticut 06510, USA.

Parvoviruses elaborate rugged nonenveloped icosahedral capsids of approximately 260 A in diameter that comprise just 60 copies of a common core structural polypeptide. While serving as exceptionally durable shells, capable of protecting the single-stranded DNA genome from environmental extremes, the capsid also undergoes sequential conformational changes that allow it to translocate the genome from its initial host cell nucleus all the way into the nucleus of its subsequent host. Lacking a duplex transcription template, the virus must then wait for its host to enter S-phase before it can initiate transcription and usurp the cell's synthetic pathways. Here we review cell entry mechanisms used by parvoviruses. We explore two apparently distinct modes of host cell specificity, first that used by Minute virus of mice, where subtle glycan-specific interactions between host receptors and residues surrounding twofold symmetry axes on the virion surface mediate differentiated cell type target specificity, while the second involves novel protein interactions with the canine transferrin receptor that allow a mutant of the feline leukopenia serotype, Canine parvovirus, to bind to and infect dog cells. We then discuss conformational shifts in the virion that accompany cell entry, causing exposure of a capsid-tethered phospholipase A2 enzymatic core that acts as an endosomolytic agent to mediate virion translocation across the lipid bilayer into the cell cytoplasm. Finally, we discuss virion delivery into the nucleus, and consider the nature of transcriptionally silent DNA species that, escaping detection by the cell, might allow unhampered progress into S-phase and hence unleash the parvoviral Trojan horse.
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http://dx.doi.org/10.1016/S0065-3527(07)70005-2DOI Listing
October 2007