Human Parainfluenza Virus Type 3 HN-Receptor Interaction: Effect of 4-Guanidino-Neu5Ac2en on a Neuraminidase-Deficient Variant

Abstract

The envelope of human parainfluenza virus type 3 (HPF3) contains two viral glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion protein (F). HN, which is responsible for receptor attachment and for promoting F-mediated fusion, also possesses neuraminidase (receptor-destroying) activity. We reported previously that 4-guanidino-neu5Ac2en (4-GU-DANA) and related sialic acid-based inhibitors of HPF3 neuraminidase activity also inhibit HN-mediated receptor binding and fusion processes not involving neuraminidase activity. We have now examined this mechanism, as well as neuraminidase's role in the viral life cycle, using a neuraminidase-deficient HPF3 variant (C28a) and stable cell lines expressing C28a or wild-type (wt) HN. C28a, which has a wt F sequence and two point mutations in the HN gene corresponding to two amino acid changes in the HN protein, is the first HPF3 variant with insignificant neuraminidase activity. Cells expressing C28a HN did not bind erythrocytes at 4°C unless pretreated with neuraminidase, but no such pretreatment was required for hemadsorption activity (HAD) at 22 or 37°C. HAD was blocked by 4-GU-DANA, attesting to the ability of this compound to inhibit HN's receptor-binding activity. C28a or wt plaque enlargement, a process that involves cell-cell fusion and does not depend on virion release, is diminished by the presence of 4-GU-DANA, confirming the inhibitory effect of 4-GU-DANA on the fusogenic function of C28a HN. In C28a-infected cell monolayers, virion release and thus multicycle replication are severely restricted. This defect was corrected by supplementation of exogenous neuraminidase and also by the addition of 4-GU-DANA; neuraminidase destroys the receptors whereby newly formed C28a virions would remain attached to the cell surface, whereas 4-GU-DANA prevents the attachment itself, obviating the need for receptor cleavage. In accord with the ability of 4-GU-DANA to prevent attachment, the neuraminidase inhibitory effect of 4-GU-DANA on wt HPF3 did not diminish virion release into the medium. Thus, it is by inhibition of viral entry and syncytium formation that sialic acid analogs like 4-GU-DANA may counteract wt HPF3 infection.

Premature Activation of the Paramyxovirus Fusion Protein before Target Cell Attachment with Corruption of the Viral Fusion Machinery

“…Measurement of Neuraminidase Activity-Assays were performed in transiently transfected 293T cell monolayers, as described previously (26,30). Briefly, 293T cells expressing WT HN were added to 96-well plates in CO 2 -independent medium at pH 5.0.…”

“…Hemadsorption Assays-Hemadsorption was performed and quantified as previously described (30). Briefly, growth medium from HN/F-cotransfected 293T cell monolayers in 48-well Biocoat plates (BD Biosciences Labware) was aspirated and replaced with 150 l of CO 2 -independent medium (pH 7.3, Invitrogen) containing different concentrations of compounds and of 1% sialic acid receptor-bearing erythrocytes (RBCs) in serum-free, CO 2 -independent medium, and placed at 4°C for 30 min.…”

Caracterización biológica de tres aislamientos naturales del Rubulavirus porcino (México)

“…Esto sugiere la existencia de una relación directa entre las actividades de la proteína HN y la virulencia. Tal relación se ha discutido en estudios con otros virus de la familia, como los virus de parainfluenza Peluso 1991, 1992;Huberman et al 1995, Porotto et al 2001, donde se sugiere que virus deficientes en actividad NA inducen mayor fusión celular, mientras que virus con actividades altas de NA son menos fusogéni-cos, porque la actividad enzimática elimina el receptor de las células vecinas y esto reduce la infección viral célula-célula. También se ha postulado que las dos actividades de la proteí-na HN, hemaglutinación y neuraminidasa, se encuentran equilibradas en las variantes naturales, tanto en paramixovirus como en ortomixovirus; es decir, que si por alguna mutación se causa una ganancia en actividad HA, también se eleva la actividad de NA y, viceversa; ambas actividades pueden disminuir de manera coordinada (Solórzano et al 2000).…”

Entry of parainfluenza virus into cells as a target for interrupting childhood respiratory disease

“…Since the HN protein has 3 functions (receptor binding, neuraminidase, and F protein activation) that each impact the fusion potential of the virus, the field had been hindered by the inability to study just 1 function - triggering - independently of the influence of other HN protein activities. A strategy for quantitating F protein triggering was developed using a panel of mutant HN proteins (17,18,24,30) in order to map the 3 functions of the HN protein to specific regions of the protein. One of the mutant HN proteins displayed a defect in triggering the F protein, which demonstrated for the first time that F protein triggering is a distinct function of the HN protein (30).…”

“…The matrix protein binds to the nucleocapsid and also interacts with the cytoplasmic tails of the HN and F proteins, in this way mediating the alignment of the nucleocapsid with the areas of the plasma membrane containing viral glycoproteins in order to set the scenario for budding (16). The neuraminidase or receptor-cleaving activity of the HN molecule cleaves sialic acid-containing receptor moieties that would attach the viral HN protein to the cell surface and allows the release of newly budded particles from the cell to begin a new round of infection (17,18).…”