Effect of ATP Binding and Hydrolysis on Dynamics of Canine Parvovirus NS1

Abstract

The replication protein NS1 is essential for genome replication and protein production in parvoviral infection. Many of its functions, including recognition and site-specific nicking of the viral genome, helicase activity, and transactivation of the viral capsid promoter, are dependent on ATP. An ATP-binding pocket resides in the middle of the modular NS1 protein in a superfamily 3 helicase domain. Here we have identified key ATP-binding amino acid residues in canine parvovirus (CPV) NS1 protein and mutated amino acids from the conserved A motif (K406), B motif (E444 and E445), and positively charged region (R508 and R510). All mutations prevented the formation of infectious viruses. When provided in trans, all except the R508A mutation reduced infectivity in a dominant-negative manner, possibly by hindering genome replication. These results suggest that the conserved R510 residue, but not R508, is the arginine finger sensory element of CPV NS1. Moreover, fluorescence recovery after photobleaching (FRAP), complemented by computer simulations, was used to assess the binding properties of mutated fluorescent fusion proteins. These experiments identified ATP-dependent and -independent binding modes for NS1 in living cells. Only the K406M mutant had a single binding site, which was concluded to indicate ATP-independent binding. Furthermore, our data suggest that DNA binding of NS1 is dependent on its ability to both bind and hydrolyze ATP.Canine parvovirus (CPV), an autonomous parvovirus, has two transcriptional units in its ϳ5.3-kb single-stranded DNA genome (51). The right-side unit produces VP1 and VP2 proteins, from which the ϳ26-nm icosahedral capsid is formed (70). The nonstructural proteins NS1 and NS2 are expressed from the left-side transcriptional unit. NS1 is a 76.7-kDa multifunctional nuclear phosphoprotein and is the only essential nonstructural protein in CPV (66). Many of its functions are necessary for parvoviral replication. NS1 initiates genomic replication by binding site specifically to the viral right-hand origin, together with endogenous high-mobility-group proteins (18), and to the left-hand origin, along with glucocorticoid modulatory element-binding proteins (11). Recognition at both origins leads to strand-and site-specific nicking of viral DNA (12, 45). These processes require ATP for tight binding and subsequent nicking (12,13,18). NS1 remains covalently linked to the 5Ј end of nicked DNA, and the remaining 3Ј-hydroxyl group can be used for synthesis of the nascent strand (17, 45). Parvoviral replication is most likely mediated by polymerase ␦, in a process that depends on the sliding clamp protein proliferating cell nuclear antigen, the single strandbinding protein replication protein A, and NS1 (15). In this process, NS1 is needed as an ATP-powered helicase to resolve terminal hairpin structures of the viral genome (68). Transcription from a viral capsid promoter, P38, is also enhanced by the NS1 protein, in an ATP-dependent manner (10, 41, 52). In addition to these functions, NS1 is re...

Mutations in DNA Binding and Transactivation Domains Affect the Dynamics of Parvovirus NS1 Protein

“…All mutations were made to NS1-deYFP, which contains NS1 cloned to the multiple cloning site of the pEYFP-N3 plasmid (Clontech), conserved mutations at the P38 promoter area of the NS1 gene, and an ATG-to-ACG mutation at the start codon of the enhanced yellow fluorescent protein (EYFP) gene. This construct has been characterized (15,35). To study P38 promoter activity and replication efficacy, some of the mutations were also made to NS1-EYFP, in which the P38 promoter is intact (15), and to infectious clone pIC (pBI265 in reference36).…”

“…This construct has been characterized (15,35). To study P38 promoter activity and replication efficacy, some of the mutations were also made to NS1-EYFP, in which the P38 promoter is intact (15), and to infectious clone pIC (pBI265 in reference36). Singlemutation-containing constructs were named after the amino acid change (E121A and Y212F).…”

“…The comparative model of the CPV NS1 helicase domain has been described before (15). In brief, the monomeric model was built with Modeler (31), and it is based on alignment of amino acid residues 277 to 490 of the AAV2 Rep40 protein (PDB accession numbers 1S9H and 1U0J) (18,32) and residues 338 to 556 of the CPV NS1 protein.…”

“…Constructs containing mutations at multiple sites were named by combining the names indicated above (e.g., K2A-K470/2A). ATP binding mutant K406M has been characterized previously (15). Mutations were made with PCR-based sitedirected mutagenesis or the two-step PCR method.…”

“…ORI recognition, nicking, and helicase activity are dependent on ATP. However, a requirement for energy input is evident only for the helicase function, and ATP binding is likely to promote NS1 oligomerization for the other activities (8,9,12,14,15).…”