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. 2017 Jul 27;91(16):e00770-17.
doi: 10.1128/JVI.00770-17. Print 2017 Aug 15.

Newcastle Disease Virus Establishes Persistent Infection in Tumor Cells In Vitro: Contribution of the Cleavage Site of Fusion Protein and Second Sialic Acid Binding Site of Hemagglutinin-Neuraminidase

Udaya S Rangaswamy  1 , Weijia Wang  1 , Xing Cheng  1 , Patrick McTamney  2 , Danielle Carroll  3 , Hong Jin  4
Affiliations

Newcastle Disease Virus Establishes Persistent Infection in Tumor Cells In Vitro: Contribution of the Cleavage Site of Fusion Protein and Second Sialic Acid Binding Site of Hemagglutinin-Neuraminidase

Udaya S Rangaswamy et al. J Virol. .

Abstract

Newcastle disease virus (NDV) is an oncolytic virus being developed for the treatment of cancer. Following infection of a human ovarian cancer cell line (OVCAR3) with a recombinant low-pathogenic NDV, persistent infection was established in a subset of tumor cells. Persistently infected (PI) cells exhibited resistance to superinfection with NDV and established an antiviral state, as demonstrated by upregulation of interferon and interferon-induced genes such as myxoma resistance gene 1 (Mx1) and retinoic acid-inducing gene-I (RIG-I). Viruses released from PI cells induced higher cell-to-cell fusion than the parental virus following infection in two tumor cell lines tested, HT1080 and HeLa, and remained attenuated in chickens. Two mutations, one in the fusion (F) protein cleavage site, F117S (F117S), and another in hemagglutinin-neuraminidase (HN), G169R (HN169R), located in the second sialic acid binding region, were responsible for the hyperfusogenic phenotype. F117S improves F protein cleavage efficiency, facilitating cell-to-cell fusion, while HN169R possesses a multifaceted role in contributing to higher fusion, reduced receptor binding, and lower neuraminidase activity, which together result in increased fusion and reduced viral replication. Thus, establishment of persistent infection in vitro involves viral genetic changes that facilitate efficient viral spread from cell to cell as a potential mechanism to escape host antiviral responses. The results of our study also demonstrate a critical role in the viral life cycle for the second receptor binding region of the HN protein, which is conserved in several paramyxoviruses.IMPORTANCE Oncolytic Newcastle disease virus (NDV) could establish persistent infection in a tumor cell line, resulting in a steady antiviral state reflected by constitutively expressed interferon. Viruses isolated from persistently infected cells are highly fusogenic, and this phenotype has been mapped to two mutations, one each in the fusion (F) and hemagglutinin-neuraminidase (HN) proteins. The F117S mutation in the F protein cleavage site improved F protein cleavage efficiency while the HN169R mutation located at the second receptor binding site of the HN protein contributed to a complex phenotype consisting of a modest increase in fusion and cell killing, lower neuraminidase activity, and reduced viral growth. This study highlights the intricate nature of these two mutations in the glycoproteins of NDV in the establishment of persistent infection. The data also shed light on the critical balance between the F and HN proteins required for efficient NDV infection and their role in avian pathogenicity.

Keywords: NDV; Newcastle disease virus; fusion; paramyxovirus; persistent infection.

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Figures

FIG 1
FIG 1
PI cells were resistant to superinfection with NDV. (A) Control cells or PI cells were either mock infected or infected with rNDV-GFP at an MOI of 3. At 24 h p.i., GFP expression was monitored by GFP fluorescence. (B) Control cells or PI cells were infected at an MOI of 0.001 in triplicates, their supernatants were collected from day 0 to day 4, and titers were determined by plaque assay. (C) Control cells or PI cells were either mock infected or infected at an increasing MOI in triplicates for 3 days. The percentage of dead cells relative to mock cells is plotted as means ± standard errors of the means. Data shown are from one study representative of two independent experiments.
FIG 2
FIG 2
Neutralization of IFN-β in PI cells. Control cells or PI cells were pretreated with antibody against IFN-β at 24 h prior to infection with rNDV-GFP. Supernatants were harvested at 48 h p.i. and tested for their levels of IFN-β by ELISA (top). Titers of these supernatants were determined by plaque assay (bottom). The data shown are means of triplicates. Error bars represent standard errors of the means.
FIG 3
FIG 3
PI cells were at an antiviral state. (A) PI cells or control cells were either mock infected or infected with rNDV-GFP at an MOI of 1. At 24 h p.i., cell lysates were harvested and tested for expression of ISGs as indicated. (B) Viral gene expression to detect F, HN, NP, and M proteins. The actin, NP, and M blots contain 20% of the input amount of lysate used for the F and HN blots. The data and images shown are representative of at least two independent experiments. The HN-to-NP ratio of each virus is indicated.
FIG 4
FIG 4
NDVpi was fusogenic compared to rNDV-GFP. (A) NDVpi and the parental virus rNDV-GFP were used to infect fresh HT1080 cells at an MOI of 0.001. The supernatants from HT1080 cells were harvested daily for 3 days. Viral titers were measured by plaque assay. (B) GFP fluorescence images of HT1080 cells infected with either NDVpi or rNDV-GFP at an MOI of 0.001 at 24 h p.i.
FIG 5
FIG 5
The HN169R mutation caused higher fusion than HN. (A) A fusion assay was performed on 293T cells transfected with the indicated plasmids that coexpress enhanced GFP. (B) GFP images were taken at 24 h posttransfection. (C) An NA assay was performed on 293T cells transfected using the indicated HN version of the plasmids. Data are representative of two or three independent experiments.
FIG 6
FIG 6
Characterization of recombinant viruses with F117S and HN1169R mutations. Recombinant viruses that contained either F117S or HN169R or both were used to infect HT1080 cells or HeLa cells. (A) Bright-field images were acquired with an EVOS microscope at 14 h p.i. in cells infected at an MOI of 1. (B) Cell killing was measured using a CellTiter-Glo assay on day 2 postinfection at an MOI of 0.1. ns, not significant.
FIG 7
FIG 7
Growth characteristics of recombinant viruses with F117S and HN1169R mutations. Recombinant viruses that contained either F117S or HN169R or both were used to infect HT1080 or HeLa cells. Viral supernatants were harvested at day 0 to day 3, and titers were determined by plaque assay. Data shown represent means ± standard errors from three replicate samples.
FIG 8
FIG 8
Characterization of recombinant viruses with F117S and HN169R mutations. Recombinant viruses that contained either F117S or HN1169R or both were used to infect HT1080 cells or HeLa cells. (A) Cell lysates were harvested from cells infected at an MOI of 1 at 9 h postinfection. Viral gene expression was detected by Western blotting. The data shown are representative of at least three experiments. (B) An NA assay was performed by harvesting lysates from infected cells at 24 h p.i.
FIG 9
FIG 9
The HN G169R mutation disrupts site II ligand binding. (A) Surface representation of HN shows residue 169 (yellow) located in site II (pink) at the dimer interface and distal to site I (orange). Residues of site I and site II binding pockets within 6 Å of the respective ligands are colored similarly. A comparison of site II binding pocket polar interactions (B, dashed black lines) to ligand and surface charge properties (C) between the wild type and the model of the G169R mutation is shown. HN monomeric units are colored individually, and residue 169 is shown in yellow in panel B. Site I and site II ligands (gray) as well as site II sialic acid polar contact residues in panel B are shown as sticks with oxygen and nitrogen atoms shown in red and blue, respectively. Waters directly involved in coordinating ligand residue contacts in panel B are shown as red spheres. Negative and positive charges in panel C are shown in blue and red, respectively. PDB accession number 1USR (38) was used for the HN structure and to model the structure of the G169R mutation, which was generated in PyMol.
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References

    1. Everts B, van der Poel HG. 2005. Replication-selective oncolytic viruses in the treatment of cancer. Cancer Gene Ther 12:141–161. doi:10.1038/sj.cgt.7700771. - DOI - PubMed
    1. Schirrmacher V, Fournier P. 2009. Newcastle disease virus: a promising vector for viral therapy, immune therapy, and gene therapy of cancer. Methods Mol Biol 542:565–605. doi:10.1007/978-1-59745-561-9_30. - DOI - PMC - PubMed
    1. Sinkovics JG, Horvath JC. 2000. Newcastle disease virus (NDV): brief history of its oncolytic strains. J Clin Virol 16:1–15. doi:10.1016/S1386-6532(99)00072-4. - DOI - PubMed
    1. Stojdl DF, Lichty B, Knowles S, Marius R, Atkins H, Sonenberg N, Bell JC. 2000. Exploiting tumor-specific defects in the interferon pathway with a previously unknown oncolytic virus. Nat Med 6:821–825. doi:10.1038/77558. - DOI - PubMed
    1. Stojdl DF, Lichty BD, ten Oever BR, Paterson JM, Power AT, Knowles S, Marius R, Reynard J, Poliquin L, Atkins H, Brown EG, Durbin RK, Durbin JE, Hiscott J, Bell JC. 2003. VSV strains with defects in their ability to shutdown innate immunity are potent systemic anti-cancer agents. Cancer Cell 4:263–275. doi:10.1016/S1535-6108(03)00241-1. - DOI - PubMed