Entry of Newcastle Disease Virus into the host cell: Role of acidic pH and endocytosis

Abstract

Most paramyxoviruses enter the cell by direct fusion of the viral envelope with the plasma membrane. Our previous studies have shown the colocalization of Newcastle Disease Virus (NDV) with the early endosome marker EEA1 and the inhibition of NDV fusion by the caveolin-phosphorylating drug phorbol 12-myristate 13-acetate (PMA) prompted us to propose that NDV enters the cells via endocytosis. Here we show that the virus-cell fusion and cell-cell fusion promoted by NDV-F are increased by about 30% after brief exposure to low pH in HeLa and ELL-0 cells but not in NDV receptor- deficient cell lines such as GM95 or Lec1. After a brief low-pH exposure, the percentage of NDV fusion at 29 °C was similar to that at 37 °C without acid-pH stimulation, meaning that acid pH would decrease the energetic barrier to enhance fusion. Furthermore, preincubation of cells with the protein kinase C inhibitor bisindolylmaleimide led to the inhibition of about 30% of NDV infectivity, suggesting that a population of virus enters cells through receptor-mediated endocytosis. Moreover, the involvement of the GTPase dynamin in NDV entry is shown as its specific inhibitor, dynasore, also impaired NDV fusion and infectivity. Optimal infection of the host cells was significantly affected by drugs that inhibit endosomal acidification such as concanamycin A, monensin and chloroquine. These results support our hypothesis that entry of NDV into ELL-0 and HeLa cells occurs through the plasma membrane as well as by dynamin- low pH- and receptor- dependent endocytosis.

Human metapneumovirus uses endocytosis pathway for host cell entry

“…For example, Newcastle Disease Virus enters HeLa and ELL-0 cells via low pH-dependent endocytosis [25]. The role of pH in hMPV entry is controversial.…”

Unity in Diversity

“…Past work showed that HMPV, NiV, and RSV can use the endosomal pathway to enter cells 106–108 . Low pH exposure increases NDV fusion and subsequent syncytia formation while, reciprocally, fusion decreases in the presence of pH-acidification inhibitors 109 . Consistent with these results, the stability of NDV F (assessed through fusion assays) decreases after exposure to low pH; F is more easily activated under these conditions 109 .…”

“…Low pH exposure increases NDV fusion and subsequent syncytia formation while, reciprocally, fusion decreases in the presence of pH-acidification inhibitors 109 . Consistent with these results, the stability of NDV F (assessed through fusion assays) decreases after exposure to low pH; F is more easily activated under these conditions 109 . There may be an accessory pH-dependent pathway through the caveolin-mediated endocytosis for NDV.…”

Mutations in the Leucine Zipper-Like Motif of the Human Parainfluenza Virus 3 Fusion Protein Impair Fusion Activity

“…Pore formation follows hemifusion and results in mixing of both outer and inner leaflet membrane lipids with concomitant mixing of aqueous contents; it is measured by a reporter gene assay [26,27]. Pore expansion is the final stage, it leads to the formation of multinucleated giant cells, and it is measured by syncytium formation assay [24,38]. Six of the mutated F proteins (L257A, L271A, I299A, L271A-L278A, L271A-L285A, and L278A-L285A), which nearly lost their ability to form syncytia, directed less than 16.0% of the levels of pore formation of the wt F protein and exhibited a hemifusion activity almost similar to the negative control level, with the rate and extent of initial fusion below 9.5% of the wt F level.…”

“…Quantification of syncytia was accomplished by measuring the syncytia-covered area in 3 random fields and comparing this with the total area of the field. Areas were quantified using the analysis tool in Adobe Photoshop CS6 [24]. …”