Identification of Domains on the Fusion (F) Protein Trimer That Influence the Hemagglutinin-Neuraminidase Specificity of the F Protein in Mediating Cell-Cell Fusion

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Virus-specific interaction between the attachment protein (HN) and the fusion protein (F) is prerequisite for the induction of membrane fusion by parainfluenza viruses. This HN-F interaction presumably is mediated by particular amino acids in the HN stalk domain and those in the F head domain. We found in the present study, however, that a simian virus 41 (SV41) F-specific chimeric HPIV2 HN protein, SCA, whose cytoplasmic, transmembrane, and stalk domains were derived from the SV41 HN protein, could not induce cell-cell fusion of BHK-21 cells when coexpressed with an SV41 HN-specific chimeric PIV5 F protein, no. 36. Similarly, a headless form of the SV41 HN protein failed to induce fusion with chimera no. 36, whereas it was able to induce fusion with the SV41 F protein. Interestingly, replacement of 13 amino acids of the SCA head domain, which are located at or around the dimer interface of the head domain, with SV41 HN counterparts resulted in a chimeric HN protein, SCA-RII, which induced fusion with chimera no. 36 but not with the SV41 F protein. More interestingly, retroreplacement of 11 out of the 13 amino acids of SCA-RII with the SCA counterparts resulted in another chimeric HN protein, IM18, which induced fusion either with chimera no. 36 or with the SV41 F protein, similar to the SV41 HN protein. Thus, we conclude that the F protein specificity of the HN protein that is observed in the fusion event is not solely defined by the primary structure of the HN stalk domain. IMPORTANCEIt is appreciated that the HN head domain initially conceals the HN stalk domain but exposes it after the head domain has bound to the receptors, which allows particular amino acids in the stalk domain to interact with the F protein and trigger it to induce fusion. However, other regulatory roles of the HN head domain in the fusion event have been ill defined. We have shown in the current study that removal of the head domain or amino acid substitutions in a particular region of the head domain drastically change the F protein specificity of the HN protein, suggesting that the ability of a given HN protein to interact with an F protein is defined not only by the primary structure of the HN stalk domain but also by its conformation. This notion seems to account for the unidirectional substitutability among rubulavirus HN proteins in triggering noncognate F proteins.T he parainfluenza viruses are classified into the genera Rubulavirus, Avulavirus, and Respirovirus in the family Paramyxoviridae (1, 2). Human parainfluenza virus 1 (HPIV1) and HPIV3 are members of the genus Respirovirus, while HPIV2, HPIV4A, HPIV4B, Simian virus 41 (SV41), and Parainfluenza virus 5 (PIV5) belong to the genus Rubulavirus; Mumps virus (MuV) is not a parainfluenza virus but is a member of the latter genus (1). Newcastle disease virus (NDV) is one of the 10 avian paramyxoviruses of the genus Avulavirus (1). These viruses have two kinds of glycoprotein spikes on the envelope: hemagglutinin-neuraminidase (HN) protein tetramers and fusion (F) protein ...

Section: Discussionmentioning

confidence: 99%

The Fusion Protein Specificity of the Parainfluenza Virus Hemagglutinin-Neuraminidase Protein Is Not Solely Defined by the Primary Structure of Its Stalk Domain

Tsurudome

Ito

Ohtsuka

et al. 2015

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“…Most recently, the HN specificity domains were further localized into two smaller regions, called M1 (amino acids from 234 to 246) and M2 (amino acids from 278 to 285) (48). Of note, these HN specificity domains were located in the surface of the F trimer based on the predicted structure (48). A similar region in the F protein of canine distemper virus (CDV) mediates the interaction with the homologous H protein (28).…”

Section: Discussionmentioning

confidence: 99%

“…Previous studies using chimeric F proteins derived from hPIV2 and simian virus 41 (SV41) have shown that a region composed of 144 amino acids (from positions 227 to 370) in HPIV2 F protein harbors the site(s) that determines the specificity of the interaction with homologous HN protein (47). Most recently, the HN specificity domains were further localized into two smaller regions, called M1 (amino acids from 234 to 246) and M2 (amino acids from 278 to 285) (48). Of note, these HN specificity domains were located in the surface of the F trimer based on the predicted structure (48).…”

Section: Discussionmentioning

confidence: 99%

“…12C and F), the majority of residues in region 2a (residues 170 to 338) are located at the surface of the F trimer. Interestingly, the corresponding region in the F protein of the Paramyxovirinae subfamily has been shown to specifically interact with the homologous attachment protein for fusion promotion (28,47,48). Previous studies using chimeric F proteins derived from hPIV2 and simian virus 41 (SV41) have shown that a region composed of 144 amino acids (from positions 227 to 370) in HPIV2 F protein harbors the site(s) that determines the specificity of the interaction with homologous HN protein (47).…”

Section: Discussionmentioning

confidence: 99%

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Localization of a Region in the Fusion Protein of Avian Metapneumovirus That Modulates Cell-Cell Fusion

Wei

Feng²,

Yao³

et al. 2012

eThe genus Metapneumovirus within the subfamily Pneumovirinae of the family Paramyxoviridae includes two members, human metapneumovirus (hMPV) and avian metapneumovirus (aMPV), causing respiratory tract infections in humans and birds, respectively. Paramyxoviruses enter host cells by fusing the viral envelope with a host cell membrane. Membrane fusion of hMPV appears to be unique, in that fusion of some hMPV strains requires low pH. Here, we show that the fusion (F) proteins of aMPV promote fusion in the absence of the attachment protein and low pH is not required. Furthermore, there are notable differences in cell-cell fusion among aMPV subtypes. Trypsin was required for cell-cell fusion induced by subtype B but not subtypes A and C. The F protein of aMPV subtype A was highly fusogenic, whereas those from subtypes B and C were not. By construction and evaluation of chimeric F proteins composed of domains from the F proteins of subtypes A and B, we localized a region composed of amino acid residues 170 to 338 in the F protein that is responsible for the hyperfusogenic phenotype of the F from subtype A. Further mutagenesis analysis revealed that residues R295, G297, and K323 in this region collectively contributed to the hyperfusogenicity. Taken together, we have identified a region in the aMPV F protein that modulates the extent of membrane fusion. A model for fusion consistent with these data is presented.T he subfamily Pneumovirinae of the family Paramyxoviridae includes a number of significant human and animal respiratory pathogens, such as human respiratory syncytial virus (hRSV), bovine RSV (bRSV), human metapneumovirus (hMPV), avian metapneumovirus (aMPV), and pneumonia virus of mice (PVM). The major cytopathic effect in paramyxovirus-infected culture cells is the formation of multinucleate syncytia. This is mediated by membrane fusion induced by surface glycoprotein spikes (reviewed in reference 24). For viruses in the Paramyxovirinae subfamily, it is firmly established that membrane fusion requires a specific interaction between two glycoproteins, the attachment protein (HN, H, or G) and the fusion (F) protein (1,9,14,15,23,39), and such fusion occurs at neutral pH (4,27). Specifically, it is thought that the binding of the attachment protein to cell surface receptors triggers major conformational changes in F, which in turn activates membrane fusion (1,27,29,30,39).However, membrane fusion of pneumoviruses appears to be unique among the paramyxoviruses, in that fusion is accomplished by the F protein alone without help from the attachment glycoprotein (6,7,17,21,42,43). This suggests that the F proteins of pneumoviruses possess dual functions, receptor binding and fusion promotion. To date, RSV F is arguably the best characterized F protein within the Pneumovirus subfamily. The F protein of RSV is capable of causing membrane fusion and initiating virus infection in the absence of its attachment G glycoprotein at neutral pH (6, 57). Furthermore, it has been demonstrated that RSV F specifically recognize...

“…On the other hand, our previous chimeric analyses of the F proteins of two closely related rubulaviruses, human parainfluenza virus 2 (HPIV2) and simian virus 41 (SV41), suggested that a 144-amino-acid region (designated the middle region) in the ectodomain of the HPIV2 F protein contains the site(s) that determines its specificity for the HPIV2 HN protein in the induction of cell-cell fusion (22). Recently, we found that replacement of two domains in the head region of the PIV5 F protein with the SV41 F counterparts bestowed on the PIV5 F protein the ability to induce cell-cell fusion on coexpression with the SV41 HN protein without significantly affecting its ability to induce fusion with the PIV5 HN protein (23). Similarly, mutations of four amino acids in the head region of the F protein of canine distemper virus (CDV) strain Onderstepoort impair the ability to induce cell-cell fusion with the co-expressed measles virus (MV) hemagglutinin (H) protein without significantly affecting the ability to induce fusion with the CDV H protein (24).…”

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confidence: 99%

Full Conversion of the Hemagglutinin-Neuraminidase Specificity of the Parainfluenza Virus 5 Fusion Protein by Replacement of 21 Amino Acids in Its Head Region with Those of the Simian Virus 41 Fusion Protein

Tsurudome

Nakahashi

Matsushima

et al. 2013

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dFor most parainfluenza viruses, a virus type-specific interaction between the hemagglutinin-neuraminidase (HN) and fusion (F) proteins is a prerequisite for mediating virus-cell fusion and cell-cell fusion. The molecular basis of this functional interaction is still obscure partly because it is unknown which region of the F protein is responsible for the physical interaction with the HN protein. Our previous cell-cell fusion assay using the chimeric F proteins of parainfluenza virus 5 (PIV5) and simian virus 41 (SV41) indicated that replacement of two domains in the head region of the PIV5 F protein with the SV41 F counterparts bestowed on the PIV5 F protein the ability to induce cell-cell fusion on coexpression with the SV41 HN protein while retaining its ability to induce fusion with the PIV5 HN protein. In the study presented here, we furthered the chimeric analysis of the F proteins of PIV5 and SV41, finding that the PIV5 F protein could be converted to an SV41 HN-specific chimeric F protein by replacing five domains in the head region with the SV41 F counterparts. The five SV41 F-protein-derived domains of this chimera were then divided into 16 segments; 9 out of 16 proved to be not involved in determining its specificity for the SV41 HN protein. Finally, mutational analyses of a chimeric F protein, which harbored seven SV41 F-protein-derived segments, revealed that replacement of at most 21 amino acids of the PIV5 F protein with the SV41 F-protein counterparts was enough to convert its HN protein specificity.