The adeno-associated virus 2 genome and Rep 68/78 proteins interact with cellular sites of DNA damage.

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.

Binding of CCCTC-Binding Factor (CTCF) to the Minute Virus of Mice Genome Is Important for Proper Processing of Viral P4-Generated Pre-mRNAs.

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 transcription profile of Aleutian mink disease virus in CRFK cells is generated by alternative processing of pre-mRNAs produced from a single promoter.

A reevaluation of the transcription profile of Aleutian mink disease parvovirus (AMDV)-infected CRFK cells at either 32 degrees C or 37 degrees C has determined that strain AMDV-G encodes six species of mRNAs produced by alternative splicing and alternative polyadenylation of a pre-mRNA generated by a single promoter at the left end of the genome. Three different splicing patterns are used, and each type is found polyadenylated at either the 3' end of the genome (the distal site) or at a site in the center of the genome (the proximal site). All spliced species accumulate similarly over the course of infection, with the R2 RNA predominant throughout. The R2 RNA, which contains and can express the NS2 coding region, encodes the viral capsid proteins VP1 and VP2.

Minute virus of mice small non-structural protein NS2 localizes within, but is not required for the formation of, Smn-associated autonomous parvovirus-associated replication bodies.

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.

Minute virus of mice NS1 interacts with the SMN protein, and they colocalize in novel nuclear bodies induced by parvovirus infection.

The human survival motor neuron (SMN) gene is the spinal muscular atrophy-determining gene, and a knockout of the murine Smn gene results in preembryonic lethality. Here we show that SMN can directly interact in vitro and in vivo with the large nonstructural protein NS1 of the autonomous parvovirus minute virus of mice (MVM), a protein essential for viral replication and a potent transcriptional activator. Typically, SMN localizes within nuclear Cajal bodies and diffusely in the cytoplasm. Following transient NS1expression, SMN and NS1 colocalize within Cajal bodies. At early time points following parvovirus infection, NS1 fails to colocalize with SMN within Cajal bodies; however, during the course of MVM infection, dramatic nuclear alterations occur. Formerly distinct nuclear bodies such as Cajal bodies, promyelocytic leukemia gene product (PML) oncogenic domains (PODs), speckles, and autonomous parvovirus-associated replication (APAR) bodies are seen aggregating at later points in infection. These newly formed large nuclear bodies (termed SMN-associated APAR bodies) are active sites of viral replication and viral capsid assembly. These results highlight the transient nature of nuclear bodies and their contents and identify a novel nuclear body formed during infection. Furthermore, simple transient expression of the viral nonstructural proteins is insufficient to induce this nuclear reorganization, suggesting that this event is induced specifically by a step in the viral infection process.

Minute virus of mice small nonstructural protein NS2 interacts and colocalizes with the Smn protein.

The small nonstructural protein NS2 of the minute virus of mice (MVM) is required for efficient viral replication, although its mode of action is unclear. Here we demonstrate that NS2 and survival motor neuron protein (Smn) interact in vitro and in vivo. NS2 and Smn also colocalize in infected nuclei at late times following MVM infection.