Deaminase-Independent Inhibition of Parvoviruses by the APOBEC3A Cytidine Deaminase

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

Parvovirus Minute Virus of Mice Induces a DNA Damage Response That Facilitates Viral Replication

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

The minute virus of mice P39 transcription unit can encode both capsid proteins

The right-hand 80% of the genome of minute virus of mice (MVM) was cloned into the bovine papillomavirus type I shuttle vector and used to transfect mouse C127 cells. Transformed lines were isolated that efficiently produce both authentic MVM capsid proteins at a ratio similar to that seen in a normal viral infection, and these proteins assemble into intact empty virions. The only transcription of MVM sequences detected in these lines was representative of the viral P39 transcription unit, which therefore contains sufficient information to encode both authentic capsid proteins at the same regulated ratio seen in an infected cell.</jats:p

Generation and characterization of a temperature-sensitive mutation in the NS-1 gene of the autonomous parvovirus minute virus of mice

In-phase single-codon insertion mutations were constructed in the open reading frames of the NS-1 and NS-2 genes of the autonomous parvovirus minute virus of mice. A viral mutant containing an isoleucine insertion exclusively within NS-1 between residues 229 and 230 was isolated that produced approximately 3 orders of magnitude fewer plaques at 39 degrees C than at 32 degrees C. Preliminary characterization of the mutant demonstrated that the NS-1 gene product is independently required for both genome amplification and the regulation of the temporal expression between the two viral transcription units during lytic infection.</jats:p

Characterization of the minute virus of mice P38 core promoter elements

While the minute virus of mice (MVM) P4 promoter, which drives the viral nonstructural genes, is highly active in the absence of viral proteins, P38, the capsid gene promoter, is strictly dependent on the viral nonstructural protein NS1. Once fully transactivated, however, P38 mediates twice the steady-state level of expression achieved by P4. In this report, we address the discrepancy between the ability of P38 to mediate very high levels of activated transcription yet only low levels of basal expression, and we investigate the determinants that govern P38 basal expression. The isolated P38 core promoter elements (the P38 Sp1-binding site and TATA element) are at least as transcriptionally competent as the analogous P4 promoter elements. Proximally positioning P4 enhancer factor-binding sequences (nucleotides [nt] 57 to 157) upstream of isolated P38 core transcription regulatory elements or upstream of a native, though abbreviated, P38 cassette (MVM nt 1938 to 2072) confers significant levels of expression to P38 in the absence of NS1, while the full left-end hairpin sequences (nt 1 to 133) elevate basal P38 activity to levels equivalent to P4 basal levels. In the context of the complete viral genome, however, proximally positioned enhancer sequences are unable to confer significant levels of expression to P38, suggesting that low P38 basal levels are a consequence not only of a lack of proximal enhancer elements but also of additional positional regulatory constraints which can be overcome by NS1.</jats:p

The two transcription units of the autonomous parvovirus minute virus of mice are transcribed in a temporal order

Using quantitative RNase protection assays, we have monitored the appearance of mRNAs generated during lytic infection of tightly synchronized murine cells by the autonomous parvovirus minute virus of mice. Our results demonstrate that transcripts from the P4 promoter can be detected prior to those from the P39 promoter, providing direct evidence for a temporal order of expression between the two parvovirus promoters.</jats:p

Inclusion of the NS2-Specific Exon in Minute Virus of Mice mRNA Is Facilitated by an Intronic Splicing Enhancer That Affects Definition of the Downstream Small Intron

AbstractAlternative splicing of pre-mRNAs plays a critical role in maximizing the coding capacity of the small parvovirus genome. The small-intron region of minute virus of mice (MVM) pre-mRNAs undergoes an unusual pattern of overlapping alternative splicing, using two donors, D1 and D2, and two acceptors, A1 and A2, within a region of 120 nucleotides, that governs the steady-state ratios of the various viral mRNAs. In a previous report we demonstrated that a complex interaction between both donor and acceptor sequences, as well as the constraints of size, defines the small intron and governs its alternative splicing. We also identified a G-rich intronic splicing enhancer sequence (IES) that appeared to function as both an intron- and an exon-defining element. In this report we further examined the components that govern MVM small-intron splicing. In fully processed wild-type mRNAs, A1 is used preferentially over A2. In this report, we show that in the context of the wild-type small intron the position of the downstream acceptor A2 was preferred, and the primary sequence of A1 must be stronger for it to be utilized at wild-type efficiency. Use of A2 in generation of the minor spliced forms D2/A2 required the IES because of a weak A2 polypyrimidine tract and because of the relative strength of A1. The small size of the intron and the relative position of the IES were also shown to play a critical role in donor and acceptor site selection. Finally, we have further characterized how the IES functions as an intronic enhancer of upstream exon definition. When the small intron was expanded, upstream exon inclusion was dependent upon the position of the IES. Within the context of the small intron, alterations of the small intron that overcame the requirement for the IES for splicing to A2 also permitted wild-type levels of upstream exon inclusion in the absence of the IES, suggesting that, in its natural context, the IES facilitates upstream exon inclusion by affecting small-intron definition

Intron Definition Is Required for Excision of the Minute Virus of Mice Small Intron and Definition of the Upstream Exon

ABSTRACT Alternative splicing of pre-mRNAs plays a critical role in maximizing the coding capacity of the small parvovirus genome. The small-intron region of minute virus of mice (MVM) pre-mRNAs undergoes an unusual pattern of overlapping alternative splicing—using two donors (D1 and D2) and two acceptors (A1 and A2) within a region of 120 nucleotides—that determines the steady-state ratios of the various viral mRNAs. In this report, we show that the determinants that govern excision of the small intron are complex and are also required for efficient definition of the upstream exon. For the MVM small intron in its natural context, the two donors appear to compete for the splicing machinery: the position of D1 favors its usage, while the primary sequence of D2 must be more like the consensus sequence than is D1 to be used efficiently. We have genetically defined the branch points that are used for generation of the major and minor spliced forms and show that recognition of components of the small-intron acceptors is likely to be the dominant determinant in alternative small-intron excision. We have also identified a G-rich intronic enhancer sequence within the small intron that is essential for splicing of the minor form (D2 to A2) but not the major form (D1 to A1) of MVM mRNAs and is required for efficient definition of the upstream NS2-specific exon. In its natural context, the small intron appears to be excised by a mechanism consistent with intron definition. When the MVM small intron is expanded, various parameters of its excision are altered, indicating that critical cis -acting signals are context dependent. Relative use of the donors and acceptors is altered, and the upstream NS2-specific exon is no longer efficiently defined. The fact that definition of the upstream NS2-specific exon can be achieved by the MVM small intron in its natural context, but not when it is expanded, suggests that the multiple determinants that govern definition and excision of the small intron are required, in concert, for upstream exon definition. Our data are consistent with a model in which alternative splicing of the MVM P4-generated pre-mRNAs is governed by a hybrid of intron- and exon-defining mechanisms. </jats:p