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.

FIG. 1.

Sequence alignment and NS1 model. (A) Organization of functional domains in CPV NS1, including the N-terminal origin of replication (amino acids 16 to 275), the SF3 helicase domain (amino acids 299 to 486), and the C-terminal transactivation domain (amino acids 600 to 667). (B) Alignment of CPV NS1 and AAV2 REP40 helicase domain protein sequences. Numbers above the alignment refer to the CPV NS1 sequence, and those below refer to the AAV2 REP40 sequence. Positions of conserved domains (A, B, B′, and C) are indicated with blue boxes above the alignment. Secondary structures according to REP40 are shown below the alignment (34). α-Helices are shown in purple, and β-sheets are shown in orange. (C) Comparative model of CPV NS1 helicase domain. α-Helices (orange) and β-sheets (purple) are shown according to the alignment. (Back) Close-up of the area denoted in green, as seen from behind the structure. R508 and R510 are shown as space-filling models, and adjacent positively charged amino acids are shown in a stick model. (Front) Close-up of the ATP-binding pocket, denoted as a red square in the model. K406 from the A motif and E444 and E445 from the B motif are shown as space-filling models.

FIG. 2.

NS1 fluorescent fusion constructs. (A) Schematic presentation of NS1-EYFP and NS1-deYFP constructs. CPV NS1 with an N-terminal EYFP fusion is under the control of the CMV immediate-early (IE) promoter. NS1-deYFP constructs have mutations in the P38 promoter area and in the start codon of EYFP. Western blots are shown for whole-cell lysates of NLFK cells transfected with NS1-deYFP constructs and detected with anti-GFP (B) and anti-NS1 (C) antibodies.

FIG. 3.

Functions of mutated NS1 constructs. (A) Secondary infections with concentrated medium collected at 2, 3, and 4 days p.t. are shown with white, gray, and black bars, respectively. Infectivity is the percentage of NS1-positive nuclei in the sample (n > 5,100). Error bars indicate standard deviations. (B) Percentages of transfected cells with signs of infection (n = 60). Chromatin marginalization is indicated in white, nuclear capsid antibody staining in black, and cells with both markers in gray. (C) NS1-deYFP-expressing infected cell with marginalized chromatin and nuclear capsid accumulation, K406M-deYFP-expressing infected cell with nuclear capsid accumulation but normal chromatin distribution, and CPV-infected cell, fixed at 48 h p.i., with marginalized chromatin and cytoplasmic capsid localization. Bars, 5 μm. DIC, differential interference contrast. (D) P38 transactivation activities of mutated NS1 proteins. Average transactivation values from EYFP (white boxes) and pIC (black circles) experiments are shown (n = 5). Transactivation values are average values for the product under the P38 promoter (EYFP or VP2) divided by the value for the corresponding NS1 protein (NS1-EYFP or NS1) and normalized to the value for nonmutated NS1. Proteins were detected in Western blots with anti-GFP (NS1-EYFP and EYFP), anti-NS1 (NS1), or anti-capsid (VP2) antibody. Error bars indicate standard errors of the means.

FIG. 4.

Nuclear distribution of NS1-deYFP constructs and H2B-EYFP under different imaging conditions. (Live) NS1-deYFP constructs under live-cell imaging conditions. (Digitonin) Permeabilization experiments are divided into three columns depending on the imaging conditions: ATP−, imaged in buffer without ATP; ATP+, imaged with 1 mM ATP; and AMP-PNP, imaged with 1 mM AMP-PNP. Bars, 5 μm. Images are shown with inverted gray-scale coloring.

FIG. 5.

In vitro FRAP experiments with NS1-deYFP constructs. (A) NS1-deYFP. (B) K406M-deYFP. (C) E445Q-deYFP. (D) E444Q-deYFP. (E) R508A-deYFP. (F) R510A-deYFP. Average recovery in buffer without ATP is shown in blue, that in buffer with 1 mM ATP is shown in red, that in buffer with 1 mM AMP-PNP is shown in black, and that in buffer with 1 mM ATP-γS is shown in purple (only in panels A and D). The y axis is normalized average fluorescence, and the x axis is the time after bleaching, in seconds. Error bars show standard errors (SE). Twenty or more cells were analyzed within each condition. (G) Inverted gray-scale images of NS1-deYFP recovery in ivFRAP experiments under different imaging conditions. The bleached area is highlighted with a black line. Bar, 5 μm.

FIG. 6.

Half-nucleus FRAP experiments and virtual cell simulations. The blue line denotes normalized half-nucleus recovery of the construct. Error bars are SE. The red line shows the virtual cell simulation reconstruction of recovery. (A) NS1-deYFP. (B) K406M-deYFP. (C) E444Q-deYFP. (D) E445Q-deYFP. (E) R508A-deYFP. (F) R510A-deYFP. The y axis is normalized average fluorescence, and the x axis is time, in seconds. (G) Average binding times for both binding sites (Bound 1 and Bound 2) and average free diffusion times (free), in seconds. Binding reaction pseudo-on rates (k*_{1, on} and k*_{2, on}) and off rates (k_{1, off} and k_{2, off}) were obtained from simulations.