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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 duringMVMinfection 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 MVMinfection was likely due to our observation that Rad9 failed to associate with chromatin atMVMAPAR bodies. Additionally, early in infection, prior to the onset of the virus-induced DNA damage response (DDR), stalling of the replication ofMVM genomes with hydroxyurea (HU) resulted in Chk1 phosphorylation in a virus dose-dependent manner. However, upon establishment of full viral replication,MVMinfection prevented activation of Chk1 in response to HU and various other drug treatments. Finally, ATR phosphorylation became undetectable uponMVMinfection, 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 thatMVMinfection disables the ATR signaling pathway. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: Translation of goose parvovirus (GPV) 72kDa 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 52kDa 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 &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Adeno-associated virus type 5 is unique among adenoassociated 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. [ABSTRACT FROM AUTHOR]

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 (pA)p to allow accumulation of RNAs that extend into the capsid gene, and it enhances splicing of the upstream adjacent 3rd 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 3rd intron, as well as regions outside of the classical motifs that were 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 G2/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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 eIF2 α. 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 eIF2 α. The expression of both the closely related goat-derived AAV and the prototype AAV2 capsid gene transcription units also induced the phosphorylation of eIF2 α, suggesting that the induction of the PKR/eIF2 α cellular response may be a previously unrecognized general feature of at least the Dependovirus genus of the Parvovirinae. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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)pl 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. [ABSTRACT FROM AUTHOR]

Adeno-associated virus type 5 (AAV5) has a linear, single-stranded DNA genome of ca. 5 kb and an overlapping transcription profile featuring multiple promoters and a single intron in the center of the genome. Unlike the situation for the prototype AAV2, AAV5 RNAs transcribed from upstream promoters at map units 7 (P7) and 19 (P19), which encode the viral Rep proteins, are predominantly polyadenylated at a site within the intron [(pA)p]. RNAs generated from the AAV5 capsid gene promoter P41, which is only 78 nucleotides (nt) upstream of the intron donor, and 281 nt upstream of (pA)p, primarily readthrough (pA)p, are polyadenylated at a more distal site at the 3' end of the genome [(pA)d] and ultimately spliced. The intron contains the core sequences sufficient for polyadenylation at (pA)p, which is governed by a G/U-rich downstream element that overlaps with the intron 3' A2 splice acceptor. In addition, polyadenylation of P7- and P19-generated RNAs at (pA)p is influenced by an upstream element that lies 5' to the start of the P41 transcript. Our results also suggest that splicing and polyadenylation of P41-generated RNA can compete for the same pool of precursor pre-mRNA molecules. The cis-acting signals within the A2 3' splice site that govern polyadenylation and splicing of AAV5 RNAs must be optimized to program both (i) the levels of polyadenylation of P7- and P19-generated RNA at (pA)p required to generate the proper levels of the essential Rep proteins and (ii) the splicing of P41-generated RNAs to generate the proper ratio of capsid proteins during AAV5 infection. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. Author summary: Parvoviruses are among the simplest of viruses, depending almost exclusively on host cell factors to successfully replicate. We have previously shown that the parvovirus Minute Virus of Mice (MVM) establishes replication centers at sites that are associated with cellular regions of DNA damage. These sites are primed to contain factors necessary to efficiently initiate vigorous virus lytic infection. The process by which viral proteins and viral DNA specifically localize to these sites has previously remained unknown. In this study we show that the essential viral protein NS1 possesses the intrinsic ability to localize to cellular sites of DNA damage. Additionally, wild-type NS1, but not its DNA binding mutant, could localize to sites of DNA damage both the MVM genome, or a heterologous DNA molecule engineered to contain NS1 binding sites. This work provides the first evidence that NS1 may function as a bridging molecule to localize the MVM genome to cellular sites of DNA damage to facilitate ongoing replication. [ABSTRACT FROM AUTHOR]

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 premRNA. 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 celldependent 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

An abstract of the article "Transfection of Mammalian Cells Using Linear Polyethylenimine Is a Simple and Effective Means of Producing Recombinant AAV," by Sharon E. Reed, Elizabeth M. Staley, David J. Pintel and Gregory E. Tullis is presented.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 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 β-galactosidase in order to assay the amount of virus produced. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]