Genetic elements in the VP region of porcine parvovirus are critical to replication efficiency in cell culture

Abstract

Factors controlling porcine parvovirus (PPV) replication efficiency are poorly characterized. Two prototype strains of PPV, NADL-2 and Kresse, differ greatly in pathogenic capacity both in vivo and in vitro, yet their genomic sequence is nearly identical (13 single-nucleotide substitutions and a 127-nucleotide noncoding repeated sequence). We have created a series of chimeras of these strains to identify the genetic elements involved in replication efficiency in the host porcine cell line. While the capsid proteins ultimately determine viral replication fitness, interaction between the NS1 protein and the VP gene occurs and involves interaction with the noncoding repeated sequence.

FIG. 1.

Construction of chimeras from the NADL-2 and Kresse PPV strains. (A) The genomes of the two wild-type strains differ by 13 nt in their coding regions (CR1 to CR3) (gray boxes) and by the repeated sequence downstream of the VP gene (white boxes). No differences are located within the hairpin termini (black boxes). The 127-nt (Rk) or the 254-nt (Rn) repeated sequence and the terminal 524 nt of the coding region (CR3) were amplified by PCR and swapped between backbones by seamless cloning (10). The CR1 and CR2 fragments were swapped in the infectious clones by classical methods. The CR1 fragment (PstI-HindIII, nt 290 to 3322) contained the entire NS coding region, including five synonymous mutations from NADL-2 to Kresse (a405g, c537t, a1533g, a1668c, a1971c) and the first of the nonsynonymous substitutions in the VP coding region (T45S in VP2 and L42V in SAT from NADL-2 to Kresse) (3, 28). The CR2 fragment (HindIII-SacI, nt 3322 to 4025) contained both silent mutations in the VP gene (nt g3163a, c3403t) and three of the nonsynonymous substitutions (VP2 I215T, D378G, H383Q from NADL-2 to Kresse). The C-terminal portion of the VP coding region contained the final two substitutions between the strains (VP2 residues S436P and R565K). Residues D378G and H383Q in CR2 and S436P in CR3 are in the BglII fragment that was previously identified as the allotropic determinant of the PPV strains in primary bovine testis cells (3). (B) A total of 14 chimeras were constructed by swapping CR1, CR2, CR3, and Rn or Rk between the NADL-2 (N2, dark gray) and Kresse (Kr, light gray) strains. The naming scheme is X-YnRxy, with X being the backbone, Yn the inset from the other strain, and Rxy the repeat.

FIG. 2.

Replication of the chimeras and wild-type strains in single-round infections assays. Early (A) and late (B) viral genome replication. Viral genome copy numbers were measured by qPCR and normalized to cell numbers using the c-myc gene to account for differences in efficiency in DNA extractions and between independent experiments (4, 26). Results are expressed as the increase in the log of genome copy equivalents (GCE) between 8 and 10 h p.i. (A) or between 8 and 20 h p.i. (B) (mean + standard deviation [SD] from at least three representative independent experiments). (C) Infectious virus production at 24 h p.i. Supernatant from cells infected with either chimera was titrated by immunofluorescence (IF) (4) and expressed as log FFU/ml (mean + standard error of the mean [SEM]) from at least three representative independent experiments. (D) Infectivity of the virus released at 20 h p.i. Supernatants from infected cells were titrated by IF, and viral genome copy numbers were determined by qPCR. Results are expressed as log GCE/FFU (mean + SD). Means were compared by unpaired two-tailed t test for NADL-2 (*, P < 0.05; **, P < 0.01) and Kresse (°, P < 0.01).

FIG. 3.

Relative replication fitness of the chimeras. (A) The chimeras were cotransfected in pairs in PT cells, and the supernatants were used to reinfect cells for one or two viral passages. PCR of final supernatants (Sn) were analyzed by sequencing (A) to determine dominance. As an example, shown is the amplification of CR2 (c3958a) after cotransfection of chimeras N-K3 and N-K2Rk (P0, sequencing of input DNA for transfection; P1, sequencing of Sn from 1st viral passage; P2, sequencing of Sn from 2nd viral passage). (B) Dominance was also verified by comparing the amount of DNA in Sn at different times posttransfection (P1) and after the first viral passage (P2), in cotransfection with chimeras differing in the origin of Rn (top band) and Rk (bottom band). P1, PCR of Sn from cotransfections with N2 and N-Rk, N2 and K-N3, or K-N3Rn and N-Rk. Lane 1, PCR of input DNA; lane 2, PCR of Sn harvested at 24 h; lane 3, PCR of Sn harvested at 48 h; lane 4, PCR of Sn harvested at 72 h. P2, Sn harvested at 24 h from cotransfections was used to infected fresh cells. Lane 1, PCR of input virus (same as P1, lane 2); lane 2, PCR of Sn harvested at 48 h p.i. from 1st viral passage. Based on the results shown, N2 is dominant over N-Rk, N2 is dominant over K-N3, and N-Rk is dominant over K-N3Rn. (C) Relative replication fitness of each chimera was determined from dominance of each clone in cotransfection and reinfection experiments (120 combinations in total, two rounds of reinfection, in three independent experiments) determined as shown in panels A and B.