Newcastle disease virus V protein is associated with viral pathogenesis and functions as an alpha interferon antagonist

Abstract

Newcastle disease virus (NDV) edits its P gene by inserting one or two G residues at the conserved editing site (UUUUUCCC, genome sense) and transcribes the P mRNA (unedited), the V mRNA (with a +1 frameshift), and the W mRNA (with a +2 frameshift). All three proteins are amino coterminal but vary at their carboxyl terminus in length and amino acid composition. Little is known about the role of the V and W proteins in NDV replication and pathogenesis. We have constructed and recovered two recombinant viruses in which the expression of the V or both the V and W proteins has been abolished. Compared to the parental virus, the mutant viruses showed impaired growth in cell cultures, except in Vero cells. However, transient expression of the carboxyl-terminal portion of the V protein enhanced the growth of the mutant viruses. In embryonated chicken eggs, the parental virus grew to high titers in embryos of different gestational ages, whereas the mutant viruses showed an age-dependent phenomenon, growing to lower titer in more-developed embryos. An interferon (IFN) sensitivity assay showed that the parental virus was more resistant to the antiviral effect of IFN than the mutant viruses. Moreover, infection with the parental virus resulted in STAT1 protein degradation, but not with the mutant viruses. These findings indicate that the V protein of NDV possesses the ability to inhibit alpha IFN and that the IFN inhibitory function lies in the carboxyl-terminal domain. Pathogenicity studies showed that the V protein of NDV significantly contributes to the virus virulence.

FIG. 1.

Construction and generation of the recombinant NDV mutants. (A) Schematic diagram of the genomic organization of the parental rBC NDV. NDV contains six genes. All genes are monocistronic except the P gene, which allows for potential expression of three proteins, P, V, and W, by RNA editing. (B) Nucleotide sequences around the editing site and patterns of P gene expression. Sequences are shown in positive sense, and mutated nucleotides are in lower case. Expression of the carboxyl-terminus-truncated V protein was obtained by introducing a stop codon TAG (underlined) in the V frame, which was silent to the P frame. Disruption of the P gene mRNA editing by introducing two silent nucleotide mutations (A to G and G to A) resulted in the inhibition of both V and W protein expression.

FIG. 2.

Western blot analysis performed with either purified viruses (A) or infected Vero cell extracts (B). Viral proteins were resolved in an SDS-12% polyacrylamide gel and transferred to polyvinylidene difluoride membranes in duplicate. The membranes were incubated with a cocktail of monoclonal HN antibodies or antipeptide serum specific for the carboxyl-terminal 18 amino acids of the V protein. The membrane was subsequently incubated with horseradish peroxidase-conjugated goat anti-mouse antibody or goat anti-rabbit IgG antibody, respectively. Reactive proteins were visualized in a visualization buffer.

FIG. 3.

Growth kinetics and plaque formation in tissue culture. Graphs show multiple-step growth curves of the parental rBC and mutant viruses on DF1 (A) or Vero (B) cells. Values are from two independent experiments, each performed in triplicate. Bars show standard deviations. (C) Plaque morphology on DF1 cells by the parental rBC and mutant viruses at 3 days postinfection.

FIG. 4.

Growth properties of rBC, rBC/V-Stop, and rBC/Edit viruses in SPF chicken embryonated eggs. Aliquots of 2 × 10³ PFU of parental rBC or mutant viruses in a volume of 100 μl were injected into the allantoic cavity of different gestational ages of embryonated eggs. Sixty hours after incubation at 37°C, allantoic fluids were harvested for virus titration on DF1 cells by plaque assay. Titers were determined from three independent experiments, each with pooled allantoic fluids from six inoculated eggs, with bars indicating standard deviations.

FIG. 5.

Viruses with editing-site mutation are more sensitive to IFN-α. DF1 cells were pretreated with an increasing concentration of chIFN-α for 24 h and were infected with rBC, rBC/V-Stop, or rBC/Edit at an MOI of 0.01. VSV was included as a control. Forty-eight hours postinfection, supernatant was harvested and used for virus titration. Results are mean values of three independent experiments, and bars show standard deviations.

FIG. 6.

Transient expression of the carboxyl-terminal portion of the NDV V protein enhanced the growth of editing-mutant viruses. DF1 cells were transfected with expression plasmid and 3 μg of pV, 1 μg of pW, or pV and pW combined. Blank plasmid pVAX1 was used as a negative control. Twenty-four hours posttransfection, the cells were infected with rBC, rBC/V-Stop, or rBC/Edit at an MOI of 0.01. Supernatant was collected 48 h after infection for assessment of the virus titers by plaque assay. Titers reflect two independent experiments, with bars indicating standard deviations.

FIG. 7.

NDV infection preferentially degraded STAT1 protein. 2fTGH cells (A) or Vero cells (B) were mock infected or infected with rBC, rBC/V-stop, rBC/Edit, SV5, or hPIV2. At 20 h postinfection, whole-cell extracts were subjected to electrophoresis and probed for STAT1 and STAT2 as well as viral protein HN by immunoblotting. Actin was used as a loading control.

FIG. 8.

Expression of the C terminus of the NDV V protein degraded STAT1 protein. 2fTGH cells (A) or Vero cells (B) were mock transfected or transfected with 3 μg of empty plasmid pVAX1, pV, or pW. Cells were lysed 20 h posttransfection. Lysates were separated by SDS-PAGE, and polypeptides were transferred to a nitrocellulose membrane and immunoblotted using antibodies specific for V, STAT1, or STAT2 proteins. Actin was used as a loading control.