Mutations in Human Parainfluenza Virus Type 3 Hemagglutinin-Neuraminidase Causing Increased Receptor Binding Activity and Resistance to the Transition State Sialic Acid Analog 4-GU-DANA (Zanamivir)

Abstract

Entry and fusion of human parainfluenza virus type 3 (HPF3) require the interaction of the viral hemagglutinin-neuraminidase (HN) glycoprotein with its sialic acid receptor. 4-GU-DANA, a potent inhibitor of influenza virus neuraminidase, inhibits not only HPF3 neuraminidase but also the receptor binding activity of HPF3 HN and thus its ability to promote attachment and fusion. We previously generated a 4-GU-DANAresistant HPF3 virus variant (ZM1) with a markedly fusogenic plaque morphology that harbored two HN gene mutations resulting in amino acid alterations. The present study using cells that express the individual mutations of ZM1 HN shows that one of these mutations is responsible for the increases in receptor binding and neuraminidase activities as well as the diminished sensitivity of both activities to the inhibitory effect of 4-GU-DANA. To examine the hypothesis that increased receptor binding avidity underlies 4-GU-DANA resistance, parallel studies were carried out on the high-affinity HN variant virus C22 and cells expressing the C22 variant HN. This variant also exhibited reduced sensitivity to 4-GU-DANA in terms of receptor binding and infectivity but without concomitant changes in the neuraminidase activity of HN. Another high-affinity HN variant, C0, was not resistant in terms of infectivity; however, a small increase in the receptor binding activity of C0 HN and a partial resistance of this activity to 4-GU-DANA were revealed by sensitive methods that we developed. In each virus variant, one mutation in HN accounted for both increased receptor binding avidity and 4-GU-DANA resistance; the higher affinity for the receptor overcomes the inhibitory effect of 4-GU-DANA. Thus, in contrast to influenza viruses for which 4-GU-DANA escape variants include hemagglutinin mutants with decreased receptor binding avidity that promotes virion release, for HPF3, HN mutants with increased receptor binding avidity are those that can escape the growth inhibitory effect of 4-GU-DANA.

Roles of the highly conserved amino acids in the globular head and stalk region of the Newcastle disease virus HN protein in the membrane fusion process

“…In Nipah and Hendra viruses, attachment proteins can be used interchangeably, which suggests slightly different F and HN interaction mechanisms among these paramyxoviruses (3). However, no final conclusion has yet been reached on the pattern of interaction between HN and F proteins (4,11,12,14).…”

“…When NDV infects the host cells, the attachment protein first binds to the sialic acid on the cell surface, and then triggers the fusion (4,5). It has been reported that behavior of the receptor-binding promotes dramatic conformational changes of the F protein, which facilitate the membrane fusion process.…”

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

“…While 4-GU-DANA inhibits HPIV neuraminidase activity, it does not prevent release of virus from the infected cell surface (45) as it does in the case of influenza viruses; instead, it blocks interaction between the parainfluenza HN protein and its receptor and thus - surprisingly - aids in the release of newly assembled virions from the infected cell (18). However, by interfering with HN-receptor interaction, 4-GU-DANA blocks receptor binding and thereby blocks fusion and viral entry (18,45). These findings have stimulated interest in designing binding/entry inhibitors for treatment of paramyxovirus infection.…”

“…The structure of the globular head region is shown in Figure 4A complexed with sialic acid. A single alteration in the HN protein - T193I - leads to an HPIV3 variant with phenotypic resistance to the effects of 4-GU-DANA in terms of both neuraminidase activity and receptor binding (45). Increased receptor-binding avidity alone can confer drug resistance and indeed accounts for part of the variant virus's 4-GU-DANA-resistant properties (45).…”

“…A single alteration in the HN protein - T193I - leads to an HPIV3 variant with phenotypic resistance to the effects of 4-GU-DANA in terms of both neuraminidase activity and receptor binding (45). Increased receptor-binding avidity alone can confer drug resistance and indeed accounts for part of the variant virus's 4-GU-DANA-resistant properties (45). However, the T193I substitution does not confer resistance to a smaller molecule, DANA (identical to 4-GU-DANA except for a smaller substituent group at C4).…”