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During infection, the autonomous parvovirus minute virus of mice (MVM) induces cellular DNA breaks and localizes to such sites, which presumably affords an environment beneficial for genome replication. MVM replication also benefits from the DNA damage response (DDR) mediated by the ataxia-telangiectasia mutated (ATM) kinase, while the ataxia telangiectasia and Rad-3 related (ATR) arm of the DDR is disabled, which prevents activation of its primary target, checkpoint kinase 1 (Chk1). We find here that Chk1 inactivation strongly correlates with dephosphorylation of one of its targets, RAD51, known to play a pivotal role in homologous recombination repair (HRR), thus leading to substantial inhibition of DNA repair in infected cells. We demonstrate colocalization of replicating MVM DNA with cellular double-strand breaks (DSBs) during infection, and show that an agent that exogenously induces cellular DSBs significantly increases viral DNA replication levels, establishing a role for cellular genome damage in facilitating virus DNA replication. Additionally, overexpression of active Chk1 during MVM infection was found to re-establish the activating phosphorylation of RAD51 Thr 309, significantly suppress infection-induced reduction of HRR efficiency with a concomitant increase in cellular genome DSBs, and reduce viral DNA replication levels. Thus, we conclude that during infection, MVM inhibition of Chk1 activation enhances viral replication, at least in part, by inhibiting cellular HRR.IMPORTANCEThe autonomous parvovirus minute virus of mice (MVM) has a compact DNA genome encoding a minimum number of proteins. During infection, it induces cellular DNA damage and both utilizes and modifies the subsequent cellular DNA damage response (DDR) in various ways to facilitate its replication. One of MVM's activities in this regard is to inhibit one of the primary arms of the DDR, the ataxia telangiectasia and Rad-3 related (ATR) pathway, which prevents activation of checkpoint kinase 1 (Chk1), a key protein involved in controlling the cellular DDR and preserving genome integrity. We show that prevention by MVM of Chk1 activation leads to inhibition of homologous recombination repair (HRR) of cellular DNA, which helps sustain viral replication. This work illuminates another way in which autonomous parvoviruses adjust the cellular environment for their replicative advantage.

During infection the autonomous parvovirus minute virus of mice (MVM) generates extensive DNA damage which facilitates virus replication and induces a cellular DNA damage response (DDR) driven by the ataxia telangiectasia mutated (ATM) kinase. Atypically, the ataxia telangiectasia and Rad-3-related (ATR) DDR pathway remains inactive. Upon DNA damage ATR is normally recruited to single-stranded DNA sequences formed at genomic DNA damage sites, and while within a multiprotein complex activates, via phosphorylation, the key DDR regulator checkpoint kinase 1 (Chk1). Inactivation of ATR during MVM infection leads to the accumulation of damaged DNA and enhancement of virus replication. Although ATR is inactivated, we show that during infection, the Chk1 activation pathway downstream of the initial ATR activating events remained functional. Activation of ATR, and consequently of Chk1, requires interaction with TopBP1, which itself is maintained in proximity to ATR by interaction with the phosphorylated S387 residue of Rad9, part of the Rad9-Hus1-Rad1 (911) complex. Both MVM infection and MVM NS1 overexpression inhibited Rad9 S387 phosphorylation and subsequent ATR activation. ATR inactivation during infection was suppressed by expression of Rad9 bearing a phosphomimetic 387 residue, indicating that this site, and the function it served, was the target of NS1 inhibition. NS1 interaction with CK2α and CK2α enzymatic activity was both required to prevent ATR activation, indicating MVM retargeted this kinase's activity during infection. Inhibition of the protein phosphatase 2C (PP2C) prevented Rad9 S387 dephosphorylation and Chk1 inactivation during MVM infection and NS1 overexpression revealing its role in the pathway's suppression., Importance: Infection by the parvovirus minute virus of mice (MVM) causes significant DNA damage and induces a potent DNA damage response (DDR) which the virus exploits to further its replication. The cell responds to infection with an ATM-regulated DDR; however, atypically, the ATR-regulated DDR pathway is disabled during infection. This prevents Chk1 activation, thus allowing the accumulation of damaged DNA which facilitates virus replication. We describe here how MVM, and specifically the main viral replication protein NS1, inhibits ATR activation. Activation of ATR, and consequently Chk1, requires TopBP1 localization into the activating complex via its interaction with a phosphorylated residue of Rad9. We show that NS1 redirects casein kinase 2 to activate a phosphatase in the PP2C family which causes dephosphorylation of this critical residue, thus inhibiting ATR activation. This work provides mechanistic insight into one of the ways by which parvoviruses modify the host DDR response to facilitate their replication.

It is unclear how the activation of HIV-1 transcription affects chromatin structure. We interrogated chromatin organization both genome-wide and nearby HIV-1 integration sites using Hi-C and ATAC-seq. In conjunction, we analyzed the transcription of the HIV-1 genome and neighboring genes. We found that long-range chromatin contacts did not differ significantly between uninfected cells and those harboring an integrated HIV-1 genome, whether the HIV-1 genome was actively transcribed or inactive. Instead, the activation of HIV-1 transcription changes chromatin accessibility immediately downstream of the provirus, demonstrating that HIV-1 can alter local cellular chromatin structure. Finally, we examined HIV-1 and neighboring host gene transcripts with long-read sequencing and found populations of chimeric RNAs both virus-to-host and host-to-virus. Thus, multiomics profiling revealed that the activation of HIV-1 transcription led to local changes in chromatin organization and altered the expression of neighboring host genes., Competing Interests: The authors declare no competing interests., (© 2022.)

Inverted terminal repeats (ITRs) are the only wild-type components retained in the genome of adeno-associated virus (AAV) vectors. To determine whether ITR modification is a viable approach for AAV vector engineering, we rationally deleted all CpG motifs in the ITR and examined whether CpG elimination compromises AAV-vector production and transduction. Modified ITRs were stable in the plasmid and maintained the CpG-free nature in purified vectors. Replacing the wild-type ITR with the CpG-free ITR did not affect vector genome encapsidation. However, the vector yield was decreased by approximately 3-fold due to reduced vector genome replication. To study the biological potency, we made micro-dystrophin (μDys) AAV vectors carrying either the wild-type ITR or the CpG-free ITR. We delivered the CpG-free μDys vector to one side of the tibialis anterior muscle of dystrophin-null mdx mice and the wild-type μDys vector to the contralateral side. Evaluation at four months after injection showed no difference in the vector genome copy number, microdystrophin expression, and muscle histology and force. Our results suggest that the complete elimination of the CpG motif in the ITR does not affect the biological activity of the AAV vector. CpG-free ITRs could be useful in engineering therapeutic AAV vectors., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Nuclear DNA viruses simultaneously access cellular factors that aid their life cycle while evading inhibitory factors by localizing to distinct nuclear sites. Adeno-associated viruses (AAVs), which are Dependoviruses in the family Parvovirinae, are non-enveloped icosahedral viruses, which have been developed as recombinant AAV vectors to express transgenes. AAV2 expression and replication occur in nuclear viral replication centers (VRCs), which relies on cellular replication machinery as well as coinfection by helper viruses such as adenoviruses or herpesviruses, or exogenous DNA damage to host cells. AAV2 infection induces a complex cellular DNA damage response (DDR), in response to either viral DNA or viral proteins expressed in the host nucleus during infection, where VRCs co-localized with DDR proteins. We have previously developed a modified iteration of a viral chromosome conformation capture (V3C-seq) assay to show that the autonomous parvovirus minute virus of mice localizes to cellular sites of DNA damage to establish and amplify its replication. Similar V3C-seq assays to map AAV2 show that the AAV2 genome co-localized with cellular sites of DNA damage under both non-replicating and replicating conditions. The AAV2 non-structural protein Rep 68/78, also localized to cellular DDR sites during both non-replicating and replicating infections, and also when ectopically expressed. Ectopically expressed Rep could be efficiently re-localized to DDR sites induced by micro-irradiation. Recombinant AAV2 gene therapy vector genomes derived from AAV2 localized to sites of cellular DNA damage to a lesser degree, suggesting that the inverted terminal repeat origins of replication were insufficient for targeting., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Pregnane X receptor (PXR) is activated by chemicals to transcriptionally regulate drug disposition and possibly decrease drug efficacy and increase resistance, suggesting therapeutic value for PXR antagonists. We previously reported the antagonist SPA70 and its analog SJB7, which unexpectedly is an agonist. Here, we describe another unexpected observation: mutating a single residue (W299A) within the PXR ligand-binding domain converts SPA70 to an agonist. After characterizing wild-type and W299A PXR activity profiles, we used molecular dynamics simulations to reveal that in wild-type PXR, agonists stabilize the activation function 2 (AF-2) helix in an "inward" position, but SPA70 displaces the AF-2. In W299A, however, SPA70 stabilizes the AF-2 "inward", like agonists. We validated our model by predicting the antagonist SJC2 to be a W299A agonist, which was confirmed experimentally. Our work correlates previously unobserved ligand-induced conformational changes to PXR cellular activity and, for the first time, reveals how PXR antagonists work.

Specific chromatin immunoprecipitation of salt-fractionated infected cell extracts has demonstrated that the CCCTC-binding factor (CTCF), a highly conserved, 11-zinc-finger DNA-binding protein with known roles in cellular and viral genome organization and gene expression, specifically binds the genome of Minute Virus of Mice (MVM). Mutations that diminish binding of CTCF to MVM affect processing of the P4-generated pre-mRNAs. These RNAs are spliced less efficiently to generate the R1 mRNA, and definition of the NS2-specific exon upstream of the small intron is reduced, leading to relatively less R2 and the generation of a novel exon-skipped product. These results suggest a model in which CTCF is required for proper engagement of the spliceosome at the MVM small intron and for the first steps of processing of the P4-generated pre-mRNA.

The autonomous parvovirus Minute Virus of Mice (MVM) localizes to cellular DNA damage sites to establish and sustain viral replication centers, which can be visualized by focal deposition of the essential MVM non-structural phosphoprotein NS1. How such foci are established remains unknown. Here, we show that NS1 localized to cellular sites of DNA damage independently of its ability to covalently bind the 5' end of the viral genome, or its consensus DNA binding sequence. Many of these sites were identical to those occupied by virus during infection. However, localization of the MVM genome to DNA damage sites occurred only when wild-type NS1, but not its DNA-binding mutant was expressed. Additionally, wild-type NS1, but not its DNA binding mutant, could localize a heterologous DNA molecule containing the NS1 binding sequence to DNA damage sites. These findings suggest that NS1 may function as a bridging molecule, helping the MVM genome localize to cellular DNA damage sites to facilitate ongoing virus replication., Competing Interests: Matthew S. Fuller is employed by Ultragenyx Gene Therapy. There are no other competing interests to declare.

The folding mechanisms of the mammalian genome package our genetic material into the nucleus, and in doing so, dictate its appropriate replication and expression. Chromosome conformation capture technology has enabled the dissection of the folding principles of the cellular genome. This has led to a better understanding of the role played by architectural proteins in forming and dissolving 3D-chromatin-structure. These assays are based on the principle of crosslinking distant cellular sites that are proximal to each other in 3D space using formaldehyde followed by digestion of formed hybrid DNA junctions. Invading viruses, such as the lytic parvovirus Minute Virus of Mice (MVM), establish distinct replication centers within the nuclear environment at cellular sites that preferentially undergo DNA damage, but do not integrate into the cellular DNA. We have adapted chromosome conformation capture technology to study the trans-interaction between MVM and the cellular genome, which we have dubbed V3C, which can be extended to a whole-genome analysis we term V3C-seq. This protocol describes the procedure for performing, as well as analyzing V3C-seq assays, and can be adapted for mapping the cellular interaction sites of any non-integrating DNA virus., Competing Interests: Competing interests The authors declare no competing financial interest.

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.

The NP1 protein of minute virus of canines (MVC) governs production of the viral capsid proteins via its role in pre-mRNA processing. NP1 suppresses polyadenylation and cleavage at its internal site, termed the proximal polyadenylation (pA)p site, to allow accumulation of RNAs that extend into the capsid gene, and it enhances splicing of the upstream adjacent third intron, which is necessary to properly enter the capsid protein open reading frame. We find the (pA)p region to be complex. It contains redundant classical cis -acting signals necessary for the cleavage and polyadenylation reaction and splicing of the adjacent upstream third intron, as well as regions outside the classical motifs that are necessary for responding to NP1. NP1, but not processing mutants of NP1, bound to MVC RNA directly. The cellular RNA processing factor CPSF6 interacted with NP1 in transfected cells and participated with NP1 to modulate its effects. These experiments further characterize the role of NP1 in parvovirus gene expression. IMPORTANCE The Parvovirinae are small nonenveloped icosahedral viruses that are important pathogens in many animal species, including humans. Unlike other parvoviruses, the bocavirus genus controls expression of its capsid proteins via alternative RNA processing, by both suppressing polyadenylation at an internal site, termed the proximal polyadenylation (pA)p site, and by facilitating splicing of an upstream adjacent intron. This regulation is mediated by a small genus-specific protein, NP1. Understanding the cis -acting targets of NP1, as well as the cellular factors with which it interacts, is necessary to more clearly understand this unique mode of parvovirus gene expression., (Copyright © 2019 American Society for Microbiology.)

Human bocavirus 1 (HBoV1) encodes a genus-specific protein, NP1, which regulates viral alternative pre-mRNA processing. Similar to NP1 of the related bocavirus minute virus of canine (MVC), HBoV1 NP1 suppressed cleavage and polyadenylation of RNAs at the viral internal polyadenylation site (pA)p. HBoV1 (pA)p is a complex region. It contains 5 significant cleavage and polyadenylation sites, and NP1 was found to regulate only the three of these sites that are governed by canonical AAUAAA hexamer signals. HBoV1 NP1 also facilitated splicing of the upstream intron adjacent to (pA)p. Alternative polyadenylation and splicing of the upstream intron were independent of each other, functioned efficiently within an isolated transcription unit, and were responsive independent of NP1. Characterization of HBoV1 NP1 generalizes its function within the genus Bocaparvovirus , uncovers important differences, and provides important comparisons with MVC NP1 for mechanistic and evolutionary considerations. IMPORTANCE The Parvovirinae are small nonenveloped icosahedral viruses that are important pathogens in many animal species, including humans. The NP1 protein of human bocavirus 1 (HBoV1), similar to NP1 of the bocavirus minute virus of canine (MVC), regulates viral alternative RNA processing by both suppressing polyadenylation at an internal site, (pA)p, and facilitating splicing of an upstream adjacent intron. These effects allow both extension into the capsid gene and splicing of the viral pre-mRNA that correctly registers the capsid gene open reading frame. Characterization of HBoV1 NP1 generalizes this central mode of parvovirus gene regulation to another member of the bocavirus genus and uncovers both important similarities and differences in function compared to MVC NP1 that will be important for future comparative studies., (Copyright © 2018 American Society for Microbiology.)

We have developed a generally adaptable, novel high-throughput Viral Chromosome Conformation Capture assay (V3C-seq) for use in trans that allows genome-wide identification of the direct interactions of a lytic virus genome with distinct regions of the cellular chromosome. Upon infection, we found that the parvovirus Minute Virus of Mice (MVM) genome initially associated with sites of cellular DNA damage that in mock-infected cells also exhibited DNA damage as cells progressed through S-phase. As infection proceeded, new DNA damage sites were induced, and virus subsequently also associated with these. Sites of association identified biochemically were confirmed microscopically and MVM could be targeted specifically to artificially induced sites of DNA damage. Thus, MVM established replication at cellular DNA damage sites, which provide replication and expression machinery, and as cellular DNA damage accrued, virus spread additionally to newly damaged sites to amplify infection. MVM-associated sites overlap significantly with previously identified topologically-associated domains (TADs)., Competing Interests: KM, JW, MB, JR, TJ, DP No competing interests declared, MF is employed by Ultragenyx Pharmaceutical. There are no other competing interests to declare., (© 2018, Majumder et al.)

Protoparvoviruses are simple single-stranded DNA viruses that infect many animal species. The protoparvovirus minute virus of mice (MVM) infects murine and transformed human cells provoking a sustained DNA damage response (DDR). This DDR is dependent on signaling by the ATM kinase and leads to a prolonged pre-mitotic cell cycle block that features the inactivation of ATR-kinase mediated signaling, proteasome-targeted degradation of p21, and inhibition of cyclin B1 expression. This review explores how protoparvoviruses, and specifically MVM, co-opt the common mechanisms regulating the DDR and cell cycle progression in order to prepare the host nuclear environment for productive infection., Competing Interests: The authors declare no conflict of interest.

Replication of minute virus of mice (MVM) induces a sustained cellular DNA damage response (DDR) which the virus then exploits to prepare the nuclear environment for effective parvovirus takeover. An essential aspect of the MVM-induced DDR is the establishment of a potent premitotic block, which we previously found to be independent of activated p21 and ATR/Chk1 signaling. This arrest, unlike others reported previously, depends upon a significant, specific depletion of cyclin B1 and its encoding RNA, which precludes cyclin B1/CDK1 complex function, thus preventing mitotic entry. We show here that while the stability of cyclin B1 RNA was not affected by MVM infection, the production of nascent cyclin B1 RNA was substantially diminished at late times postinfection. Ectopic expression of NS1 alone did not reduce cyclin B1 expression. MVM infection also reduced the levels of cyclin B1 protein, and RNA levels normally increased in response to DNA-damaging reagents. We demonstrated that at times of reduced cyclin B1 expression during infection, there was a significantly reduced occupancy of RNA polymerase II and the essential mitotic transcription factor FoxM1 on the cyclin B1 gene promoter. Additionally, while total FoxM1 levels remained constant, there was a significant decrease of the phosphorylated, likely active, forms of FoxM1. Targeting of a constitutively active FoxM1 construct or the activation domain of FoxM1 to the cyclin B1 gene promoter via clustered regularly interspaced short palindromic repeats (CRISPR)-enzymatically inactive Cas9 in MVM-infected cells increased both cyclin B1 protein and RNA levels, implicating FoxM1 as a critical target for cyclin B1 inhibition during MVM infection. IMPORTANCE Replication of the parvovirus minute virus of mice (MVM) induces a sustained cellular DNA damage response (DDR) which the virus exploits to prepare the nuclear environment for effective takeover. An essential aspect of the MVM-induced DDR is establishment of a potent premitotic block. This block depends upon a significant, specific depletion of cyclin B1 and its encoding RNA that precludes cyclin B1/CDK1 complex functions necessary for mitotic entry. We show that reduced cyclin B1 expression is controlled primarily at the level of transcription initiation. Additionally, the essential mitotic transcription factor FoxM1 and RNA polymerase II were found to occupy the cyclin B1 gene promoter at reduced levels during infection. Recruiting a constitutively active FoxM1 construct or the activation domain of FoxM1 to the cyclin B1 gene promoter via CRISPR-catalytically inactive Cas9 (dCas9) in MVM-infected cells increased expression of both cyclin B1 protein and RNA, implicating FoxM1 as a critical target mediating MVM-induced cyclin B1 inhibition., (Copyright © 2017 American Society for Microbiology.)

Parvoviruses use a variety of means to control the expression of their compact genomes. The bocaparvovirus minute virus of canines (MVC) encodes a small, genus-specific protein, NP1, which governs access to the viral capsid gene via its role in alternative polyadenylation and alternative splicing of the single MVC pre-mRNA. In addition to NP1, MVC encodes five additional nonstructural proteins (NS) that share an initiation codon at the left end of the genome and which are individually encoded by alternative multiply spliced mRNAs. We found that three of these proteins were encoded by mRNAs that excise the NP1-regulated MVC intron immediately upstream of the internal polyadenylation site, (pA)p, and that generation of these proteins was thus regulated by NP1. Splicing of their progenitor mRNAs joined the amino termini of these proteins to the NP1 open reading frame, and splice site mutations that prevented their expression inhibited virus replication in a host cell-dependent manner. Thus, in addition to controlling capsid gene access, NP1 also controls the expression of three of the five identified NS proteins via its role in governing MVC pre-mRNA splicing. IMPORTANCE The Parvovirinae are small nonenveloped icosahedral viruses that are important pathogens in many animal species, including humans. Minute virus of canine (MVC) is an autonomous parvovirus in the genus Bocaparvovirus It has a single promoter that generates a single pre-mRNA. NP1, a small genus-specific MVC protein, participates in the processing of this pre-mRNA and so controls capsid gene access via its role in alternative internal polyadenylation and splicing. We show that NP1 also controls the expression of three of the five identified NS proteins via its role in governing MVC pre-mRNA splicing. These NS proteins together are required for virus replication in a host cell-dependent manner., (Copyright © 2017 American Society for Microbiology.)

Contamination by the parvovirus minute virus of mice (MVM) remains a challenge in Chinese hamster ovary (CHO) biopharmaceutical production processes. Although infrequent, infection of a bioreactor can be catastrophic for a manufacturer, can impact patient drug supply and safety, and can have regulatory implications. We evaluated engineering a CHO parental cell line (CHOZN ® GS -/- ) to create a new host cell line that is resistant to MVM infection by modifying the major receptors used by the virus to enter cells. Attachment to a cell surface receptor is a key first step in the infection cycle for many viruses. While the exact functional receptor for MVM binding to CHO cell surface is unknown, sialic acid on the cell surface has been implicated. In this work, we used the zinc finger nuclease gene editing technology to validate the role of sialic acid on the cell surface in the binding and internalization of the MVM virus. Our approach was to systematically mutate genes involved in cell surface sialylation and then challenge each cell line for their ability to resist viral entry and propagation. To test the importance of sialylation, the following genes were knocked out: the CMP-sialic acid transporter, solute carrier family 35A1 (Slc35a1), the core 1-β-1,3-galactosyltransferase-1 specific chaperone (Cosmc), and mannosyl (α-1,3-)-glycoprotein β-1,2-N-acetylglucosaminyltransferase (Mgat1) as well as members of the sialyltransferase family. Slc35a1 is responsible for transporting sialic acid into the Golgi. Knocking out function of this gene in a cell results in asialylated glycan structures, thus eliminating the ability of MVM to bind to and enter the cell. The complete absence of sialic acid on the Slc35a1 knockout cell line led to complete resistance to MVM infection. The Cosmc and Mgat1 knockouts also show significant inhibition of infection likely due to their effect on decreasing cell surface sialic acid. Previously in vitro glycan analysis has been used to elucidate the precise sialic acid structures required for MVM binding and internalization. In this work, we performed the sequential knockout of various sialyltransferases that add terminal sialic acid to glycans with different linkage specificities. Cell lines with modifications of the various genes included in this study resulted in varying effects on MVM infection expanding on the knowledge of MVM receptors. MVM resistant host cell lines were also tested for the production of model recombinant proteins. Our data demonstrate that resistance against the MVM virus can be incorporated into CHO production cell lines, adding another level of defense against the devastating financial consequences of MVM infection without compromising recombinant protein yield or quality. Biotechnol. Bioeng. 2017;114: 576-588. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Unlabelled: Minute virus of canines (MVC) is an autonomous parvovirus in the genus Bocaparvovirus. It has a single promoter that generates a single pre-mRNA processed via alternative splicing and alternative polyadenylation to produce at least 8 mRNA transcripts. MVC contains two polyadenylation sites, one at the right-hand end of the genome, (pA)d, and another complex site, (pA)p, within the capsid-coding region. During viral infection, the mRNAs must extend through (pA)p and undergo additional splicing of the immediately upstream 3D∕3A intron to access the capsid gene. MVC NP1 is a 22-kDa nuclear phosphoprotein unique to the genus Bocaparvovirus of the Parvovirinae which we have shown governs suppression of (pA)p independently of viral genome replication. We show here that in addition to suppression of (pA)p, NP1 is also required for the excision of the MVC 3D∕3A intron, independently of its effect on alternative polyadenylation. Mutations of the arginine∕serine (SR) di-repeats within the intrinsically disordered amino terminus of NP1 are required for splicing of the capsid transcript but not suppression of polyadenylation at (pA)p. 3'-end processing of MVC mRNAs at (pA)p is critical for viral genome replication and the optimal expression of NP1 and NS1. Thus, a finely tuned balance between (pA)p suppression and usage is necessary for efficient virus replication. NP1 is the first parvovirus protein implicated in RNA processing. Its characterization reveals another way that parvoviruses govern access to their capsid protein genes, namely, at the RNA level, by regulating the essential splicing of an intron and the suppression of an internal polyadenylation site., Importance: The Parvovirinae are small nonenveloped icosahedral viruses that are important pathogens in many animal species, including humans. Although parvoviruses have only subtle early-to-late expression shifts, they all regulate access to their capsid genes. Minute virus of canines (MVC) is an autonomous parvovirus in the genus Bocaparvovirus. It has a single promoter generating a single pre-mRNA which is processed via alternative splicing and alternative polyadenylation to generate at least 8 mRNA transcripts. MVC contains two polyadenylation sites, one at the right-hand end of the genome, (pA)d, and another, (pA)p, within the capsid-coding region. It had not been clear how the potent internal polyadenylation motif is suppressed to allow processing, export, and accumulation of the spliced capsid protein-encoding mRNAs. We show here that MVC NP1, the first parvovirus protein to be implicated in RNA processing, governs access to the MVC capsid gene by facilitating splicing and suppressing internal polyadenylation of MVC pre-mRNAs., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Unlabelled: The ATR kinase has essential functions in maintenance of genome integrity in response to replication stress. ATR is recruited to RPA-coated single-stranded DNA at DNA damage sites via its interacting partner, ATRIP, which binds to the large subunit of RPA. ATR activation typically leads to activation of the Chk1 kinase among other substrates. We show here that, together with a number of other DNA repair proteins, both ATR and its associated protein, ATRIP, were recruited to viral nuclear replication compartments (autonomous parvovirus-associated replication [APAR] bodies) during replication of the single-stranded parvovirus minute virus of mice (MVM). Chk1, however, was not activated during MVM infection even though viral genomes bearing bound RPA, normally a potent trigger of ATR activation, accumulate in APAR bodies. Failure to activate Chk1 in response to MVM infection was likely due to our observation that Rad9 failed to associate with chromatin at MVM APAR bodies. Additionally, early in infection, prior to the onset of the virus-induced DNA damage response (DDR), stalling of the replication of MVM genomes with hydroxyurea (HU) resulted in Chk1 phosphorylation in a virus dose-dependent manner. However, upon establishment of full viral replication, MVM infection prevented activation of Chk1 in response to HU and various other drug treatments. Finally, ATR phosphorylation became undetectable upon MVM infection, and although virus infection induced RPA32 phosphorylation on serine 33, an ATR-associated phosphorylation site, this phosphorylation event could not be prevented by ATR depletion or inhibition. Together our results suggest that MVM infection disables the ATR signaling pathway., Importance: Upon infection, the parvovirus MVM activates a cellular DNA damage response that governs virus-induced cell cycle arrest and is required for efficient virus replication. ATM and ATR are major cellular kinases that coordinate the DNA damage response to diverse DNA damage stimuli. Although a significant amount has been discovered about ATM activation during parvovirus infection, involvement of the ATR pathway has been less studied. During MVM infection, Chk1, a major downstream target of ATR, is not detectably phosphorylated even though viral genomes bearing the bound cellular single-strand binding protein RPA, normally a potent trigger of ATR activation, accumulate in viral replication centers. ATR phosphorylation also became undetectable. In addition, upon establishment of full viral replication, MVM infection prevented activation of Chk1 in response to hydroxyurea and various other drug treatments. Our results suggest that MVM infection disables this important cellular signaling pathway., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

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.

Infection by the autonomous parvovirus minute virus of mice (MVM) induces a vigorous DNA damage response in host cells which it utilizes for its efficient replication. Although p53 remains activated, p21 protein levels remain low throughout the course of infection. We show here that efficient MVM replication required the targeting for degradation of p21 during this time by the CRL4Cdt2 E3-ubiquitin ligase which became re-localized to MVM replication centers. PCNA provides a molecular platform for substrate recognition by the CRL4Cdt2 E3-ubiquitin ligase and p21 targeting during MVM infection required its interaction both with Cdt2 and PCNA. PCNA is also an important co-factor for MVM replication which can be antagonized by p21 in vitro. Expression of a stable p21 mutant that retained interaction with PCNA inhibited MVM replication, while a stable p21 mutant which lacked this interaction did not. Thus, while interaction with PCNA was important for targeting p21 to the CRL4Cdt2 ligase re-localized to MVM replication centers, efficient viral replication required subsequent depletion of p21 to abrogate its inhibition of PCNA.

Parvoviruses halt cell cycle progression following initiation of their replication during S-phase and continue to replicate their genomes for extended periods of time in arrested cells. The parvovirus minute virus of mice (MVM) induces a DNA damage response that is required for viral replication and induction of the S/G2 cell cycle block. However, p21 and Chk1, major effectors typically associated with S-phase and G2-phase cell cycle arrest in response to diverse DNA damage stimuli, are either down-regulated, or inactivated, respectively, during MVM infection. This suggested that parvoviruses can modulate cell cycle progression by another mechanism. In this work we show that the MVM-induced, p21- and Chk1-independent, cell cycle block proceeds via a two-step process unlike that seen in response to other DNA-damaging agents or virus infections. MVM infection induced Chk2 activation early in infection which led to a transient S-phase block associated with proteasome-mediated CDC25A degradation. This step was necessary for efficient viral replication; however, Chk2 activation and CDC25A loss were not sufficient to keep infected cells in the sustained G2-arrested state which characterizes this infection. Rather, although the phosphorylation of CDK1 that normally inhibits entry into mitosis was lost, the MVM induced DDR resulted first in a targeted mis-localization and then significant depletion of cyclin B1, thus directly inhibiting cyclin B1-CDK1 complex function and preventing mitotic entry. MVM infection thus uses a novel strategy to ensure a pseudo S-phase, pre-mitotic, nuclear environment for sustained viral replication.

Although precluded from using splicing to produce multiple small Rep proteins, adeno-associated virus type 5 (AAV5) generates a Rep40-like protein by alternative translation initiation at an internal AUG. A defined region upstream of the internal AUG was both required and sufficient to program internal initiation within AAV5 and may act similarly in heterologous contexts. The internally initiated AAV5 Rep40-like protein was functional and had helicase activity similar to that of AAV2 Rep40. Surprisingly, both the AAV5 Rep40-like protein and Rep52 were able to be translated from the AAV5 upstream P7-generated RNAs; however, the relative level of small to large Rep proteins was reduced compared to that of the wild type. A P19 mutant AAV5 infectious clone generated near-wild-type levels of the double-stranded monomer replicative form (mRF) replicative intermediate but reduced levels of virus, consistent with the previously defined role of Rep40-like proteins in genome encapsidation. Levels of mutant virus were dramatically reduced upon amplification.

We present a detailed characterization of a single-cycle infection of the bocavirus minute virus of canines (MVC) in canine WRD cells. This has allowed identification of an additional smaller NS protein that derives from an mRNA spliced within the NS gene that had not been previously reported. In addition, we have identified a role for the viral NP1 protein during infection. NP1 is required for read-through of the MVC internal polyadenylation site and, thus, access of the capsid gene by MVC mRNAs. Although the mechanism of NP1's action has not yet been fully elucidated, it represents the first parvovirus protein to be implicated directly in viral RNA processing.

The DNA damage response to infection with minute virus of mice (MVM) leads to activated p53; however, p21 levels are reduced via a proteasome-mediated mechanism. This loss was sustained, as virus replicated in infected cells held at the G(2)/M border. Addition of the cyclin-dependent kinase (CDK) inhibitor roscovitine after S-phase entry reduced MVM replication, suggesting that CDK activity was critical for continued viral replication and virus-induced reduction of p21 may thus be necessary to prevent inhibition of CDK.

Translation of goose parvovirus (GPV) 72 kDa Rep 1 is initiated from unspliced P9-generated mRNAs in ORF1 from the first in-frame AUG (537 AUG); however, this AUG is bypassed in spliced P9-generated RNA: translation of the 52 kDa Rep 2 protein from spliced RNA is initiated in ORF2 at the next AUG downstream (650 AUG). Usage of the 537 AUG was restored in spliced RNA when the GPV intron was replaced with a chimeric SV40 intron, or following specific mutations of the GPV intron which did not appear in the final spliced mRNA. Additionally, 650 AUG usage was gained in unspliced RNA when the GPV intron splice sites were debilitated. Splicing-dependent regulation of translation initiation was mediated in cis by GPV RNA surrounding the target AUGs. Thus, nuclear RNA processing of GPV P9-generated pre-mRNAs has a complex, but significant, effect on alternative translation initiation of the GPV Rep proteins., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The ribonuclease protection assay (RPA) has emerged as an important methodology for the detection, mapping, and quantification of RNAs. In this assay, total or cytoplasmic RNAs are hybridized to a high-specific activity antisense radioactive RNA probe synthesized by in vitro transcription from the SP6 or T7 promoter of an appropriate linearized plasmid template by the bacteriophage SP6 or T7 polymerase, respectively. The RNA hybrids are subjected to RNAse digestion and the protected products are resolved by denaturing polyacrylamide gel electrophoresis to allow detection of specific RNA fragments by subsequent autoradiography. RPAs are highly sensitive, the probes can be specifically targeted, and, when performed in probe excess, are quantitative, making them the method of choice for many analyses of RNA processing events.

Infection by DNA viruses can elicit DNA damage responses (DDRs) in host cells. In some cases the DDR presents a block to viral replication that must be overcome, and in other cases the infecting agent exploits the DDR to facilitate replication. We find that low multiplicity infection with the autonomous parvovirus minute virus of mice (MVM) results in the activation of a DDR, characterized by the phosphorylation of H2AX, Nbs1, RPA32, Chk2 and p53. These proteins are recruited to MVM replication centers, where they co-localize with the main viral replication protein, NS1. The response is seen in both human and murine cell lines following infection with either the MVMp or MVMi strains. Replication of the virus is required for DNA damage signaling. Damage response proteins, including the ATM kinase, accumulate in viral-induced replication centers. Using mutant cell lines and specific kinase inhibitors, we show that ATM is the main transducer of the signaling events in the normal murine host. ATM inhibitors restrict MVM replication and ameliorate virus-induced cell cycle arrest, suggesting that DNA damage signaling facilitates virus replication, perhaps in part by promoting cell cycle arrest. Thus it appears that MVM exploits the cellular DNA damage response machinery early in infection to enhance its replication in host cells.

Adeno-associated virus (AAV) type 2 and 5 proteins Rep52 and Rep40 were polyubiquitinated during AAV-adenovirus type 5 (Ad5) coinfection and during transient transfection in either the presence or absence of Ad5 E4orf6 and E1b-55k. Polyubiquitination of small Rep proteins via lysine 48 (K48) linkages, normally associated with targeting of proteins for proteasomal degradation, was detected only in the presence of E4orf6. The small Rep proteins were ubiquitinated via lysine 63 (K63) following transfection in either the presence or absence of E4orf6 or following coinfection with Ad5. E4orf6/E1b-55k-dependent K48-specific polyubiquitination of small Rep proteins could be inhibited using small interfering RNA (siRNA) to cullin 5.

Alternative splicing of adeno-associated virus type 2 (AAV2) P19-generated pre-mRNAs generates the small Rep proteins Rep52 and Rep40, which differ in their carboxyl termini. Both proteins are required for optimal packaging of AAV2 genomes. AAV5 Rep-encoding P19-generated transcripts are primarily polyadenylated within the central intron and not efficiently spliced; however, surprisingly, AAV5 was found to generate high levels of a Rep40-like protein. The AAV5 Rep40-like protein was generated by internal initiation and has the same C terminus as Rep52. Although precluded from using alternative splicing to generate multiple Rep isoforms, AAV5 ensures the production of a Rep40-like protein by utilizing a novel internal translation initiation event.

The goose parvovirus (GPV) Rep 1 and Rep 2 proteins are encoded by P9-generated mRNAs that are either unspliced or spliced within the rep gene region, respectively. These mRNAs are present in an approximately equal ratio. The translation of Rep 1 was initiated from the first AUG in unspliced P9-generated mRNA; however, this AUG was bypassed in spliced P9-generated RNA and Rep 2 translation initiated predominately at the next initiating AUG downstream. We show that the choice of the site of initiation of translation of GPV Rep-encoding mRNAs is governed both by the splicing process itself and by the nature of the excised intron.

The APOBEC3 proteins form a multigene family of cytidine deaminases with inhibitory activity against viruses and retrotransposons. In contrast to APOBEC3G (A3G), APOBEC3A (A3A) has no effect on lentiviruses but dramatically inhibits replication of the parvovirus adeno-associated virus (AAV). To study the contribution of deaminase activity to the antiviral activity of A3A, we performed a comprehensive mutational analysis of A3A. By mutation of non-conserved residues, we found that regions outside of the catalytic active site contribute to both deaminase and antiviral activities. Using A3A point mutants and A3A/A3G chimeras, we show that deaminase activity is not required for inhibition of recombinant AAV production. We also found that deaminase-deficient A3A mutants block replication of both wild-type AAV and the autonomous parvovirus minute virus of mice (MVM). In addition, we identify specific residues of A3A that confer activity against AAV when substituted into A3G. In summary, our results demonstrate that deaminase activity is not necessary for the antiviral activity of A3A against parvoviruses.

Adeno-associated viral (AAV) capsid proteins, thought to be a rate-limiting step in the production of recombinant AAV (rAAV), are translated from spliced mRNAs. Improvement of the native AAV nonconsensus donor sequence increases splicing yet leaves the relative levels of VP1- and VP2/3-encoding mRNAs unchanged, and thus provides a means to increase delivery of correct ratios of AAV capsid proteins. This effect is independent of the AAV serotype used, and occurs whether the rep and cap genes supplied in trans are on the same or separate expression vectors. In the split-vector system, replacement of the more traditionally used cytomegalovirus promoter with that of the AAV5 P41 promoter allowed for even greater levels of splicing, and together with an improved intron donor, led to a 10- to 15-fold increase in the levels of splicing, rAAV production, and transduction compared with levels achieved by traditional cotransfection methods. Thus, the enhancement of splicing presents a useful method to enhance rAAV production via transient transfection.

The pre-mRNA processing strategy of the B19 virus is unique among parvoviruses. B19 virus-generated pre-mRNAs are transcribed from a single promoter and are extensively processed by alternative splicing and alternative polyadenylation to generate 12 transcripts. Blockage of the production of full-length B19 virus transcripts at the internal polyadenylation site [(pA)p] was previously reported to be a limiting step in B19 virus permissiveness. We show here that in the absence of genome replication, internal polyadenylation of B19 virus RNAs at (pA)p is favored in cells which are both permissive and nonpermissive for B19 viral replication. Replication of the B19 virus genome, however, introduced either by viral infection or by transfection of an infectious clone into permissive cells or forced by heterologous replication systems in nonpermissive cells, enhanced readthrough of (pA)p and the polyadenylation of B19 virus transcripts at the distal site [(pA)d]. Therefore, replication of the genome facilitates the generation of sufficient full-length transcripts that encode the viral capsid proteins and the essential 11-kDa nonstructural protein. Furthermore, we show that polyadenylation of B19 viral RNA at (pA)p likely competes with splicing at the second intron. Thus, we conclude that replication of the B19 virus genome is the primary limiting step governing B19 virus tropism.

Degradation of de novo-generated adeno-associated virus type 5 (AAV5) Rep52 and capsid proteins is part of the limited target specificity displayed by adenovirus type 5 E4Orf6-E1B-55k as part of a cullin 5-containing E3 ligase complex. Both Rep and capsid proteins can be found in the ligase complex, and their presence is dependent on interaction between E4Orf6 and elongins B and C. Degradation of AAV5 proteins can be inhibited by a dominant-negative ubiquitin that prevents chain elongation or by small interfering RNA directed against cullin 5.

The parvoviruses bovine adeno-associated virus (B-AAV) and adeno-associated virus type 5 (AAV5) have similar transcription maps. However, while the AAV5 capsid gene promoter P41 possesses a high basal level in 293 cells, and is further activated only poorly by Rep during adenovirus type 5 (Ad5) infection, the B-AAV P41 promoter has a low basal activity within RepCap constructs in these cells and can be strongly activated by its Rep protein in the presence of Ad5 when a Rep-binding element (RBE) is included in cis at either end of the molecule. These differences are not due to differences in the intrinsic activating capability of the individual Rep proteins. Both viral promoters contain AP1 and CRE elements that contribute to their basal activity; however, the nature of the B-AAV P41 promoter itself and the surrounding sequences contribute to its relatively lower basal activity. In addition, the B-AAV upstream transcription units themselves also are activated in the presence of Ad5 and Rep. Thus, although the transcription map of B-AAV is much more closely related to AAV5, activation of its promoters is functionally more like the prototype AAV2.

Mutants of adenovirus type 5 (Ad5) virus-associated RNA I deficient in inhibiting the activation and subsequent phosphorylation of protein kinase R (PKR) could neither function as helpers for adeno-associated virus type 5 (AAV5) replication nor enhance AAV5 protein accumulation in either the presence or absence of Ad5 E4Orf6 and E2a. Furthermore, a short region of the AAV5 capsid gene RNA leader sequence surrounding the AUG of VP1 could induce the phosphorylation of eIF2alpha. Both short interfering RNA directed against PKR and the addition of the herpes simplex virus ICP34.5 protein enhanced the accumulation of AAV5 capsid protein in the presence of the AAV5 capsid gene PKR-inducing element, suggesting that VA RNA acted to overcome direct AAV5-induced activation of PKR that led to the phosphorylation of eIF2alpha. The expression of both the closely related goat-derived AAV and the prototype AAV2 capsid gene transcription units also induced the phosphorylation of eIF2alpha, suggesting that the induction of the PKR/eIF2alpha cellular response may be a previously unrecognized general feature of at least the Dependovirus genus of the Parvovirinae.

Full replication of adeno-associated virus type 5 (AAV5) is sustained by adenovirus type 5 (Ad5) helper functions E1a, E1b, E2a, E4Orf6, and virus-associated (VA) RNA; however, their combined net enhancement of AAV5 replication was comprised of both positive and negative individual effects. Although Ad5 E4Orf6 was required for AAV5 genomic DNA replication, it also functioned together with E1b to degrade de novo-expressed, preassembled AAV5 capsid proteins and Rep52 in a proteosome-dependent manner. VA RNA enhanced accumulation of AAV5 protein, overcoming the degradative effects of E4Orf6, and was thus required to restore adequate amounts of AAV5 proteins necessary to achieve efficient virus production.

In contrast to the prototype adeno-associated virus type 2 (AAV2), the capsid gene P41 promoter of AAV5, within viral constructs that lack inverted terminal repeat sequences, displays a high basal level of expression in 293 cells in the absence of coinfecting adenovirus. Here we demonstrate that this was due to differences in the relative strengths of the core promoter elements and to the presence of active binding sites for the transcription factors CREB and AP1 within the upstream region of P41 that are absent from the AAV2 capsid gene promoter P40. These differences also governed the relative basal activity of the AAV capsid gene promoters within near-full-length viral genomes.

We have developed a simple protocol to transfect mammalian cells using linear polyethylenimine (PEI). Our linear PEI protocol is as effective as commercial reagents in the transfection of HeLa cells and XDC293 cells, a derivative of HEK293 cells, but at a fraction of the cost. Greater than 90% of XDC293 cells and 98% of HeLa cells transfected using our method were positive for EGFP expression as determined by flow cytometery. Our protocol should be useful for many different applications such as large-scale production of recombinant protein and viruses, which requires transient transfection of mammalian cells in large batches. We have used this protocol to produce recombinant adeno-associated virus (AAV) in XDC293 cells and in HeLa cells. This requires transient expression of three adenovirus gene-products (E2A, E4orf6, and VA RNAs) as well as the AAV replication (Rep78, Rep68, Rep52, and Rep40) and capsid (VP1, VP2, and VP3) proteins. Production of a recombinant AAV that expresses green fluorescent protein was assessed by quantitative PCR and by transduction of HeLa cells. Linear PEI is a better transfection reagent than calcium phosphate for the production of recombinant AAV in both HEK293 and HeLa cells. In addition, when both HeLa and XDC293 cells were by our method, HeLa cells in the absence of E1A generated three-fold more recombinant AAV than XDC293 cells, which constitutively express E1A.

Recombinant AAV vectors are produced by transient transfection of mammalian cells. The virus is usually purified from a combination of lysed cells and spent culture medium by HPLC. We have developed a quantitative, real-time PCR assay for quantifying encapsidated single-stranded viral DNA (i.e. DNA-containing virions) in cell lysates and the spent culture medium. This requires extensive DNaseI digestion to reduce the amount of AAV replicative DNA, as well as plasmid and cellular DNA, to negligible amounts. To demonstrate the utility of this assay, we produced recombinant AAV in HeLa cells and five different types of 293 cells. We used primers to the EGFP transgene to detect the production of a recombinant AAV. We assayed the cell lysates and media by both our quantitative PCR assay and a functional transduction assay. The quantitative PCR assay data correlated well with the transduction assay data. Because this assay only requires standard PCR primers and SYBR Green I dye to detect the amplification of the PCR template, it will readily adapt to any target DNA sequence within the recombinant AAV genome. The recombinant AAV vector does not need to express a reporter gene, such as EGFP or beta-galactosidase in order to assay the amount of virus produced.

Efficient expression of the adeno-associated virus type 5 (AAV5) P41 capsid gene promoter required adenovirus E1A and/or E1B; however, in contrast to what was observed for expression of the AAV2 capsid gene promoter (P40), neither adenovirus infection nor the large Rep protein was required. Although both the AAV2 and the AAV5 large Rep proteins efficiently bound the (GAGY)(3) Rep-binding element, the AAV5 large Rep protein transactivated transcription of the inducible AAV2 P40 promoter much less well than AAV2 large Rep. Differences in their activation potentials were mapped to the amino-terminal region of the proteins, and the poorly transactivating AAV5 Rep protein could competitively inhibit AAV2 Rep transactivation.

We present the first detailed expression profiles of nonprimate-derived adeno-associated viruses, namely, bovine adeno-associated virus (B-AAV) and avian adeno-associated virus (A-AAV), which were obtained after the infection of cell lines derived from their natural hosts. In general, the profiles of B-AAV and A-AAV were quite similar to that of AAV5; however, both exhibited features found for AAV2 as well. Like adeno-associated virus type 5 (AAV5), B-AAV and A-AAV utilized an internal polyadenylation site [(pA)p]; however, it was used to greater relative levels by B-AAV than by A-AAV. Similar to AAV5, >99% of B-AAV RNAs generated from upstream promoters were polyadenylated at (pA)p and hence not spliced. In contrast, ca. 50% of the A-AAV RNAs generated from upstream promoters read through (pA)p, as seen for AAV2. However, A-AAV generated lower levels of spliced P5 and P19 products than does AAV2, suggesting that A-AAV generates lower relative levels of Rep 68 and Rep 40. An additional difference in the expression profile of these viruses was that B-AAV generated a greater level of ITR-initiated RNAs than did A-AAV or AAV5. In addition, we demonstrate that, like AAV2, transactivation of transcription of the capsid-gene promoter of B-AAV required both adenovirus and targeting of its Rep protein to the transcription template; however, expression of the capsid-gene promoter of A-AAV was, like AAV5, largely independent of both adenovirus and its Rep proteins.

Polyadenylation of B19 pre-mRNAs at the major internal site, (pA)p1, is programmed by the nonconsensus core cleavage and polyadenylation specificity factor-binding hexanucleotide AUUAAA. Efficient use of this element requires both downstream and upstream cis-acting elements and is further influenced by an adjacent AAUAAC motif. The primary hexanucleotide element must be nonconsensus to allow efficient readthrough of P6-generated pre-mRNAs into the capsid-coding region. An additional cleavage and polyadenylation site, (pA)p2, 296 nucleotides downstream of (pA)p1 was shown to be used following both B19 infection and transfection of a genomic clone. RNAs polyadenylated at (pA)p2 comprise approximately 10% of B19 RNAs that are polyadenylated internally.

Two newly recognized parvovirus species, rat parvovirus 1 (RPV-1) and rat minute virus 1 (RMV-1), were recently identified in naturally infected rats. In this study, two polymerase chain reaction (PCR) assays were developed to specifically detect RPV-1 and RMV-1. The RPV-1 PCR assay amplified the expected 487-bp deoxyribonucleic acid (DNA) fragment only in the presence of RPV-1 DNA; the RMV-1 PCR assay amplified the expected 843-bp product only from RMV-1 DNA, not from other rodent parvoviruses. The RPV-1 and the RMV-1 PCR assays detected approximately 18 and 70 copies of DNA template, respectively. These two PCR assays were shown to be sensitive, specific and rapid methods for detecting RPV-1 and RMV-1 infections in rats. These assays may also be valuable for evaluation of biological specimens for parvovirus contamination.

The expression profile of the circovirus TTV has not yet been fully characterized. In this paper, we show that following transfection of a full-length viral clone of TTV genotype 6, each of the three virally encoded mRNAs is translated from two initiating AUGs, and therefore, the TTV genome generates at least six proteins. Localization studies of hemagglutinin-tagged versions of these proteins in fixed cells, and green fluorescent protein-tagged versions of these proteins in living cells, expressed following transfection, demonstrated that two were primarily nuclear, two were primarily cytoplasmic, and two were found throughout the cell.

The non-structural proteins NS1 and NS2 of the parvovirus minute virus of mice (MVM) are required for efficient virus replication. It has previously been shown that NS1 and NS2 interact and colocalize with the survival motor neuron (Smn) gene product in novel nuclear structures that are formed late in infection, termed Smn-associated APAR (autonomous parvovirus-associated replication) bodies (SAABs). It is not clear what molecular viral intermediate(s) contribute to SAAB formation. The current results address the role of NS2 in SAAB formation. In highly synchronized wild-type MVM infection of murine A9(2L) cells, NS2 colocalizes with Smn and other SAAB constituents. An MVM mutant that does not produce NS2 still generates SAABS, albeit with a temporal delay. The lag in SAAB formation seen in the absence of NS2 is probably related to the temporal delay in virus replication, suggesting that, whilst NS2 is required for efficient viral infection, it is dispensable for SAAB formation.

Simian parvovirus (SPV) is a member of the genus Erythrovirus and is closely related to the human parvovirus B19. Natural and experimental infection of monkeys with SPV resembles B19 infection of human. We report a detailed characterization of the viral RNAs and proteins generated following transfection of cloned SPV into COS cells and SPV infection of the human erythroid progenitor line UT-7/Epo-S1. SPV and B19 are 50% identical at the nucleotide level, and although their basic transcription and protein expression profiles were generally similar, there were also significant differences. SPV pre-mRNAs contain three introns, compared to two found for B19: an additional intron was found within the capsid-coding region. RNAs in which this intron was spliced were abundant and encoded the SPV 14-kDa protein (analogous to the B19 11-kDa protein), which initiated at an AUG in the exon preceding the third intron. Unlike B19, SPV RNAs were also spliced between the donor of the first intron and the acceptor of the second intron. The third intron was additionally spliced from a portion of these molecules; these mRNAs encoded the 14-kDa protein. A portion was not spliced further and encoded VP2. Like B19, SPV has a polyadenylation signal [AAUAAA (pA)p] in the middle of the genome, which directed efficient polyadenylation of both spliced and unspliced mRNAs (encoding a putative 10-kDa protein, analogous to the B19 7.5-kDa protein, and SPV NS1, respectively). The 14-kDa protein was localized to both in the nucleus and cytoplasm.

Therapeutic application of recombinant adeno-associated virus (AAV) has been limited by its small carrying capacity. To overcome this limitation trans-splicing vectors were developed recently. However, the transduction efficiency of trans-splicing vectors is considerably lower than that of a single intact vector in skeletal muscle. To improve trans-splicing vectors for skeletal muscle gene therapy, we examined whether coinfection efficiency is a rate-limiting factor in the mdx mouse, a model for Duchenne muscular dystrophy. Two different AAV viruses were delivered to the mdx muscle. Similar to previous reports in normal muscle, coinfection efficiency reached approximately 90% in the diseased muscle. This result suggests that coinfection is not a hurdle in dystrophic muscle. Another critical step in the trans-splicing method is the transcription and splicing across the inverted terminal repeat (ITR) junction in the reconstituted genome. To test whether this represented a significant obstacle, we systematically evaluated the transcription, pre-mRNA stability and splicing, and translation in a synthetic lacZ construct that mimicked the reconstituted genome. Although inserting an intron in the lacZ gene had no effect on its expression, inclusion of the ITR junction in the intron reduced expression by 50%. In construct containing the ITR junction, the mRNA transcript level was significantly reduced. This mRNA level reduction was associated with decreased pre-mRNA stability. These data suggest that the accumulation of mRNA is a rate-limiting factor in trans-splicing vector-mediated gene therapy.

Adeno-associated virus type 5 is unique among adeno-associated virus serotypes in that it uses a polyadenylation site in the center of the genome. The great majority of transcripts generated from the upstream P7 and P19 promoters are polyadenylated at a site in the central intron ((pA)p); however, most of the viral transcripts generated by the proximal P41 promoter are polyadenylated at the distal polyadenylation site at the 3' end of the genome (pA)d and subsequently spliced. Polyadenylation at (pA)p increases as the distance between the RNA initiation site and the intron and (pA)p site is increased. The steady-state level of RNAs polyadenylated at (pA)p is independent of the promoter used or of the intervening sequence but is dependent upon competition with splicing, inhibition by U1 snRNP binding to the intron donor, and the intrinsic efficiency of the cleavage/polyadenylation reaction. Each of these determinants shows a marked dependence on the distance between the RNA initiation site and the intron and (pA)p. Finally, unlike other reported systems, inhibition of (pA)p by U1 snRNP binding to the intron donor is decreased as the distance between the donor and (pA)p is increased.