DNA Binding and Nicking Capabilities by the Human Parvovirus B19 NS1 Nuclease Domain

Abstract

The human parvovirus B19 (B19V) is a small nonenveloped single-stranded DNA virus that infects the majority of the population. Illnesses from B19V infection include erythema infectiosum (Fifth disease), hydrops fetalis, arthropathy, cardiomyopathy, and aplastic crisis. B19V infections have also seen associated with the development of various autoimmune diseases that may endure weeks to years. The viral genomic DNA from B19V has been identified to persist in many cellular tissues with unknown function, since B19V is able to infect many cell types but B19V is only able to replicate in precursor erythroid cells. Although the causative agent behind the triggering of autoimmune disease post B19V infections is unknown, several hypotheses have stated that the main replicative protein, nonstructural 1 (NS1), from B19V is involved. NS1 is crucial for viral replication as it contains predicted nuclease, helicase, and gene transactivation capabilities. These putative activities are thought to initiate autoimmune diseases through several mechanisms. As B19V can infect many cell types not necessarily important for viral replication, the presence of the viral genome will express the NS1 protein which is then able to transactivate host inflammatory genes and possibly lead to the development of autoimmune diseases. Similarly, infection of cell types not required for replication will lead to cellular apoptosis from DNA damage induced by NS1’s functions to bind and cleave DNA. Cellular apoptosis causes the release of apoptotic bodies that contain NS1 covalently attached to the host genomic DNA, which are recognized by the immune system and may lead to the production of antibodies against cellular genomic DNA. As NS1 is required for viral replication and speculated to be involved in triggering autoimmune diseases, biochemical characterization of the endonuclease domain of NS1 (NS1-nuc) is of importance. NS1-nuc was determined to bind cooperatively to B19V’s origin of replication. The NS1 binding element (NSBE) region within the viral origin of replication is bound by 5-7 copies of NS1-nuc in a cooperative manner. Previous reports of NS1 transactivation of host genes were tested to determine if the NS1-nuc is also able to directly bind DNA. Binding to the tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) promoters yielded very weak binding to no specific binding, respectively. When the NS1-nuc binding to random sequences consisting of different percentages of GC content DNA was tested, it yielded similar affinities as IL-6, indicating that the NS1-nuc is not able to bind in a sequence specific manner. Binding to TNF- was ten times stronger than that observed for the random sequences with differing percentages of GC content. On the other hand, the NS1-nuc was able to bind the p21 promoter responsible for cell cycle progression in a cooperative manner with 3 copies of the NS1-nuc binding to the promoter. The other part of NS1-nuc’s function is to cleave DNA. The ability to cleave DNA is required by NS1 to facilitate viral genomic replication and is also involved in causing host genomic DNA damage via cleavage. The cleavage at the viral origin of replication was mapped to the expected viral cleavage site termed, the terminal resolution site (TRS). NS1-nuc cleavage analysis indicated that divalent cations (in order of efficiency: Mn2+, Co2+, Ni2+, and Mg2+) are required to facilitate the cleavage reaction. The specificity of the TRS was found to be focused directly one to two nucleotides from the TRS and to sequences adjacent to the NSBE region. Interestingly, off-target DNA cleavage was also observed that could have important implications for cellular apoptosis from DNA damage. The NS1-nuc cleavage reaction was also found to produce a covalent adduct to the 5’ of the TRS. Due to nicking and covalent adduct formation by the NS1-nuc upon DNA cleavage, a fluorescence polarization assay was developed to analyze this ability. The assay may be used in high throughput manner to screen drug libraries to identify compounds that inhibit the DNA cleaving capacities of the NS1-nuc. Preliminary structural data on the NS1-nuc and full length NS1 protein are currently being performed. 1H-15N HSQC (Heteronuclear Single Quantum Coherence) NMR (Nuclear Magnetic Resonance) spectra of the NS1-nuc indicates it is good candidate for further structural studies at higher protein concentrations. The full-length NS1 protein was negatively stained and viewed through transmission electron microscopy, which indicated NS1 forms a ring like structure. Further analysis of NS1 by electron microscopy is required before any findings can be established. The initiated structural studies on the NS1-nuc and NS1 will undoubtedly provide a more complete picture of the mechanisms behind viral replication and the observed associations with autoimmune disease.