Interaction between the hemagglutinin-neuraminidase and fusion glycoproteins of human parainfluenza virus type III regulates viral growth in vivo

Abstract

Paramyxoviruses, enveloped RNA viruses that include human parainfluenza virus type 3 (HPIV3), cause the majority of childhood viral pneumonia. HPIV3 infection starts when the viral receptor-binding protein engages sialic acid receptors in the lung and the viral envelope fuses with the target cell membrane. Fusion/entry requires interaction between two viral surface glycoproteins: tetrameric hemagglutinin-neuraminidase (HN) and fusion protein (F). In this report, we define structural correlates of the HN features that permit infection in vivo. We have shown that viruses with an HN-F that promotes growth in cultured immortalized cells are impaired in differentiated human airway epithelial cell cultures (HAE) and in vivo and evolve in HAE into viable viruses with less fusogenic HN-F. In this report, we identify specific structural features of the HN dimer interface that modulate HN-F interaction and fusion triggering and directly impact infection. Crystal structures of HN, which promotes viral growth in vivo, show a diminished interface in the HN dimer compared to the reference strain's HN, consistent with biochemical and biological data indicating decreased dimerization and decreased interaction with F protein. The crystallographic data suggest a structural explanation for the HN's altered ability to activate F and reveal properties that are critical for infection in vivo.

Importance: Human parainfluenza viruses cause the majority of childhood cases of croup, bronchiolitis, and pneumonia worldwide. Enveloped viruses must fuse their membranes with the target cell membranes in order to initiate infection. Parainfluenza fusion proceeds via a multistep reaction orchestrated by the two glycoproteins that make up its fusion machine. In vivo, viruses adapt for survival by evolving to acquire a set of fusion machinery features that provide key clues about requirements for infection in human beings. Infection of the lung by parainfluenzavirus is determined by specific interactions between the receptor binding molecule (hemagglutinin-neuraminidase [HN]) and the fusion protein (F). Here we identify specific structural features of the HN dimer interface that modulate HN-F interaction and fusion and directly impact infection. The crystallographic and biochemical data point to a structural explanation for the HN's altered ability to activate F for fusion and reveal properties that are critical for infection by this important lung virus in vivo.

FIG 1

The strength of HN-F interaction for the lung-fit virus HPIV3-HN_Q552/R559/F_D396 is reduced relative to that of the parental virus (HPIV3-HN_Q552/F_G396) in 293T cells. (a) Cells were cotransfected with constructs encoding the indicated F fused to C-CFP and HN fused to N-Venus (or HA fused to N-Venus) and treated overnight with 10 mM zanamivir. One hour prior to confocal microscopy analysis, fresh 10 mM zanamivir and cycloheximide were added. Fluorescence (the mean fluorometric ratio) resulting from HN-F interaction was quantified. Values are means and standard errors of the means (SEM) from 4 to 7 separate experiments. ***, P < 0.001 (one-way analysis of variance). (b) In a competition assay, HN_Q552/R559 N-Venus and F C-CFP were cotransfected with pCAGGS (mock), HN_H552 pCAGGS (cold HN_H552), HN_Q552 (cold HN_Q552), or HN_Q552/R559 (cold HN_Q552/R559) and treated overnight with 10 mM zanamivir. The expression levels of HN and F (in the uncleaved precursor form [F0] and the proteolytically cleaved form [F1]) are similar for each HN-F pair, as shown by Western blotting using tubulin (55 kDa) as a loading control. (c) Fluorescence (the mean fluorometric ratio) resulting from HN-F interaction in the competition assay was quantified as described for panel a. Representative fluorescence images of cotransfected 293T cells 20 h after transfection show HN-F interaction on the cell surface. Cells were also transfected with mCherry (red).

FIG 2

Hypofusion-triggering HN (Q552/R559) shows decreased HN dimerization. (a) Mean fluorometric ratios of BiFC resulting from the oligomerization of HN. Constructs containing R559 (HN_Q552/R559 and HN_R559) show lower mean fluorometric ratios and weaker association than the reference strain HN with H552 and HN_Q552. Values are means and SEM of results from five experiments. ***, P < 0.001 (one-way analysis of variance). (b) 293T cells were cotransfected with HN_Q552/R559 N-Venus, HN_Q552/R559 C-Venus, and a nontagged (cold) HN construct. BiFC from the oligomerization of HN_Q552/R559 N-Venus and HN_Q552/R559 C-Venus can be disrupted by cold HN, which results in a decreased mean fluorometric ratio. Cold HN_Q552 is more effective than cold HN_H552 and cold HN_Q552/R559 to compete with tagged HN_Q552/R559 for homo-oligomerization. Values are means and SEM of results from five experiments. *, P < 0.05 (Student’s t test). (c) Nonreducing SDS-PAGE autoradiography of the indicated untagged HNs. (d) Blue native gel autoradiography of the indicated untagged HNs.

FIG 3

Introduction of a Q559R mutation weakens the HN-HN dimer interaction. (A) Structural comparison between HPIV3-HN_H552 (reference strain) and HN_Q552/R559. The HPIV3 H552 (reference strain) is shown in gray, and the two HN_Q552/R559 protomers that form a noncrystallographic dimer are superimposed on the reference strain and are in green and cyan, respectively. The mutations induce local conformational changes on the dimer interface. Most notably, HN_Q552/R559 displays a slightly wider separation across the dimer interface near the 550 loop region. (B) Dimer interface around the 550 loop in HN_H552 and HN_Q552/R559. The two HN_Q552/R559 protomers in the noncrystallographic dimer are in green and cyan, respectively. H552 (from the reference strain) is shown in gray for comparison.

FIG 4

Footprints of dimer interface in HN_H552 (reference strain) (A) and HN_Q552/R559 (B). HN residues in contact in the dimer interface are shown in red. Introduction of the double mutation results in the loss of about 800 of 3,700 Å² buried interface area in the dimer, mostly near the 550 loops.

FIG 5

HPIV3-HN_Q552/R559/F_D396 has enhanced fitness for growth in HAE compared to HPIV3-HN_H552 (reference strain) and HPIV3-HN_Q552. HAE cultures were coinfected with 4,000 PFU of the HPIV3 reference strain and HPIV3-HN_Q552/R559/F_D396 (a) or of HPIV3-HN_Q552 and HPIV3-HN_Q552/R559/F_D396 (b) at the ratios indicated. Viral titers (left) were determined at 1, 2, and 3 days postinfection. The ratios of viral genomes released (right) containing the codon CAA (glutamine) or CGA (arginine) corresponding to residue 559 on HN were quantified by qRT-PCR by SNP genotyping. The HPIV3 reference strain and HPIV3-HN_Q552 genomes carried Q559, while HPIV3 small plaques (HPIV3-HN_Q552/R559/F_D396) carried R559. Titers on days 1 to 3 after HAE infection were determined by plaque assay in CV1 cells based on plaque size. Viral genome was quantified using SNP genotyping specific to Q or R at position 559 in HN. All data points are means and standard deviations (SD) from triplicate experiments.

FIG 6

HPIV3-HN_Q552/R559/F_D396 is fit in vivo. Cotton rats (5 per group) were infected with HPIV3-HN_H552 (reference strain), HPIV3-HN_Q552, or HPIV3-HN_Q552/R559/F_D396. At 3 days postinfection, the viral titer (PFU/g lung tissue) was determined by plaque assay.