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

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 (F 117S ), and another in hemagglutinin-neuraminidase (HN), G169R (HN 169R ), located in the second sialic acid binding region, were responsible for the hyperfusogenic phenotype. F 117S improves F protein cleavage efficiency, facilitating cell-to-cell fusion, while HN 169R 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 F 117S mutation in the F protein cleavage site improved F protein cleavage efficiency while the HN 169R 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

Potential of genotype VII Newcastle disease viruses to cause differential infections in chickens and ducks

“…The differential pathogenicities of these isolates suggested that NDV evolves in its natural host (duck) to increase its survival rate. Similar results describing NDV persistent infection due to a modification in the genome have been shown in an ovarian cell line (Rangaswamy et al., ), but persistent infection in ducks, geese or chickens has yet to be studied. Our chicken‐origin NDV isolate did not evolve in ducks, and thus, it caused high mortality.…”

“…A mutation in the stalk region of the HN protein modulates the fusion process and expression of the HN‐F complex on the cellular surface (Iorio, Melanson, & Mahon, ; Mirza & Iorio, ). Previous studies have revealed that the amino acid residues at 169, 174, 175, 192, 198, 236, 258, 299, 317, 401, 416, 498, 516, 517, 519, 526, 552, 553 and 557 are important for HN protein function (presented in red in Figure b) (Connaris et al., ; Estevez, King, Luo, & Yu, ; McGinnes & Morrison, ; Rangaswamy et al., ; Takimoto, Taylor, Connaris, Crennell, & Portner, ; Zaitsev et al., ). In the present study, we identified amino acid substitutions at positions 9, 323, 331 and 514 when we compared the Ch/CH/SD/2008/128 and Du/CH/SD/2009/134 isolates.…”

Parainfluenza Virus Infection Sensitizes Cancer Cells to DNA-Damaging Agents: Implications for Oncolytic Virus Therapy

“…Consistent with this, a persistent NDV infection was successfully established in one ovarian cancer cell line, OVCAR3, while other cell lines, such as OAW28, CAL27, FaDu, and PE/CA PJ15, were unable to establish a persistent infection (47). PI cells can have an altered antiviral state induced by low levels of IFN production and the presence of IFN-stimulated genes, as seen in the case of NDV infection (47,48). The P/V-CPI Ϫ virus is a potent inducer of IFN (18), and work is in progress to understand the role that this cytokine plays in the differential ability to establish P/V-CPI Ϫ PI cells in different cancer cell lines.…”

“…Host cell type can also be key in developing a persistent paramyxovirus infection (46). Consistent with this, a persistent NDV infection was successfully established in one ovarian cancer cell line, OVCAR3, while other cell lines, such as OAW28, CAL27, FaDu, and PE/CA PJ15, were unable to establish a persistent infection (47). PI cells can have an altered antiviral state induced by low levels of IFN production and the presence of IFN-stimulated genes, as seen in the case of NDV infection (47,48).…”

Residues 315 and 369 in HN Protein Contribute to the Thermostability of Newcastle Disease Virus

“…The NDV genome contains six genes encoding the nucleoprotein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase protein (HN) and large RNA polymerase (L) (Dimitrov et al, 2019). Infection of host cells by NDV is accomplished by the interaction of two surface glycoproteins, F and HN (Gravel and Morrison, 2003;Rangaswamy et al, 2017). The F protein directs the viral fusion activity and is supposed to play a major role in determining NDV virulence (Samal et al, 2013).…”