Dept of Molecular Virology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, Peoples Republic of China1
Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, Peoples Republic of China2
Dept of Virus Research, University of Military Supplies, Changchun 130062, Peoples Republic of China3
Laboratory of Immunobiology, Dana-Farber Cancer Institute and Dept of Medicine, Harvard Medical School, Boston, MA 02115, USA4
Laboratory of Structural Biology and MOE Laboratory of Protein Sciences, Tsinghua University, Beijing 100084, Peoples Republic of China5
Laboratory of Molecular Medicine, Childrens Hospital, Dept of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 320 Longwood Avenue, Boston, MA 02115, and Dept of Molecular and Cellular Biology, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138, USA6
Authors for correspondence: (i) George Gao. Present address: Room 7508, Nuffield Dept of Clinical Medicine, John Radcliffe Hospital, University of Oxford, Headington, Oxford OX3 9DU, UK. Fax +44 1865 220993. e-mail ggao66{at}yahoo.com (ii) Po Tien. e-mail tienpo@sun.im.ac.cn
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Abstract |
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The F protein is synthesized initially as a precursor, F0, which is cleaved into a disulfide-linked heterodimer of F1 and F2 by a furin-like enzyme of the host cell (Homma & Ohuchi, 1973 ; Scheid & Choppin, 1974
). During the attachment and fusion process, the F protein undergoes conformational changes that expose the fusion peptide in F1 and results in the fusion peptide embedding in the host membrane, with or without the help of the HN protein. The F protein contains at least three heptad repeat regions (HR1, HR2 and HR3) (Young et al., 1997
, 1999
; Ghosh et al., 1998
; Dutch et al., 1999
; Matthews et al., 2000
; Sergel et al., 2000
). HR1 is located at the carboxyl terminus of the fusion peptide, while HR2 is located adjacent to the amino terminus of the transmembrane domain. Both HR1 and HR2 are important for fusion function, but the importance of HR3 is yet to be unravelled. Mutations of amino acids in each region of HR1 and HR2 decrease the fusion activity (Buckland et al., 1992
; Sergel-Germano et al., 1994
; Reitter et el., 1995
; Sergel et al., 2001
). Studies on enveloped viruses show that there are similar domains to HR1 (also called HR-A, N-peptide) and HR2 (also called HR-B, C-peptide) in many other enveloped viruses (see review by Bentz, 2000
; Eckert & Kim, 2001
).
Fusion of NDV does require both F and HN proteins. Previous studies on HR1 and HR2 of the NDV F protein indicated that HR2 could inhibit the F protein-mediated fusion (Young et al., 1997 , 1999
), whereas the inhibitive effect of HR1 could only be seen if it was added to cells prior to the cleavage of the F0 protein in an in vitro transfection system, which represents an artificial state. Nonetheless it is not clear whether the HR1 and HR2 of NDV could form a heterotrimer (trimer of the HR1/HR2 heterodimer) and whether it adopts a similar fusion mechanism to other enveloped viruses because of its absolute requirement for HN protein in the fusion process. However, a recent crystal structure of F0 in the metastable state indicates the possible existence of different conformation states of the NDV F protein (Chen et al., 2001
).
In this article, by using an E. coli expression system, we have expressed, purified and characterized HR1 and HR2 of the F proteins derived from a virulent and an avirulent NDV strain. The F gene clones of a Chinese virulent isolate, F48E9 (GenBank no. AF079172), and a Chinese avirulent isolate, Changchun (GenBank no. AF400614), were used in this study. The helical wheels of the HR1 (amino acids 137198) and HR2 (amino acids 449503) are depicted in Fig. 1(A). The HR regions for virulent and avirulent strains were named v-HR1, v-HR2 and a-HR1, a-HR2 respectively. Using the known HR1/HR2 structure from SV5 (sequence identities compared to NDV in the regions are 32·3% and 34·6% respectively, but the a and d sites of the helical wheel are highly conserved) as a template (Baker et al., 1999
), the modelling program (Modeller, http://guitar.rockefeller.edu/modeller/modeller.html) yields a model of the NDV HR1/HR2 heterotrimer structure (Fig. 1B
). This structure shows clearly an anti-parallel coiled coil and the same model has been obtained for a-HR1/HR2 and v-HR1/HR2, though there are three amino acid differences between v-HR1 and a-HR1 (S139A, N145K, N192K) and six amino acid differences between v-HR2 and a-HR2 (L451Q, V456I, W461D, D479N, S486R, G497S) in our sequences used for the study. These different amino acids locate either in the outside part of the coiled coil, which would not have any effect on the overall structure (at b, c, g sites of the helical wheel for HR1; or c, e, f sites for HR2; but not at the important sites a and d), or would not be included in the model.
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Under the same conditions, neither the GST fusion nor the free form of HR1 (a-HR1 or v-HR1) or the heterotrimer showed any inhibition activity in viruscell fusion or virus plaque formation (Fig. 4).
At the molecular level, the details of viruscell fusion mechanisms of paramyxovirus are largely unknown. Conformational changes of fusion proteins are probably involved in the fusion process (Lamb, 1993 ). A common post-fusion thermo-stable homologous HR1/HR2 heterotrimer structure has been observed in the fusion proteins of a number of enveloped viruses, e.g. retrovirus [including human and simian immunodeficiency viruses (HIV and SIV)], Ebola virus, influenza virus (see review by Bentz, 2000
; Eckert & Kim, 2001
). Moreover, the heterotrimer structures of the HR1/HR2 of the F proteins of paramyxoviruses SV5 and RSV have been unravelled lately (Baker et al., 1999
; Zhao et al., 2000
). A similar post-fusion structure has been proposed for the NDV F protein due to common characteristics of the HR1/HR2 region. Nevertheless, NDV and SV5 or RSV are fundamentally different in terms of fusion as HN protein is absolutely required for NDV, but not for SV5 or RSV. A recent crystal structure of NDV F0 protein (Chen et al., 2001
) in the metastable (pre-fusion) state has shown that its specific characters, in addition to the common features, could be compared to well-studied influenza virus (HA for influenza A, HEF for influenza C), e.g. the central triple-stranded coiled coil in the NDV F protein and influenza HA is oriented in the opposite directions in these two molecules. Therefore, a generalized extrapolation of structural conformational changes might not be appropriate and more experimental data need to be accumulated. In this study, our data showed the assembly of such a heterotrimer complex for NDV. Our data also indicate that creating GST fusion proteins is an effective way of preparing virus envelope HR peptide inhibitors. Other HR-homologue inhibitors should be pursued in the same way for quick and cost-effective preparation. This is especially relevant to HIV fusion inhibition peptides, e.g. T-20 in clinical trials (Kilby et al., 1998
).
Consistent with the previous report (Young et al., 1997 ), our study showed that HR2 gave strong inhibition activity against viruscell fusion, while HR1 did not show any. More importantly, in our system the GST fusion HR2 showed a similar inhibition effect to free HR2. Young et al. (1999)
did not see any fusion inhibition by HR1 but found that an amino acid change (F115G) at the F0 cleavage site (F0 into F1 and F2), in a system in which the F0 cleavage was controlled by externally added trypsin, could lead to the inhibition by HR1 of fusion mediated by F and HN proteins only if the HR1 was added before the F0 was cleaved by trypsin. As F0 is cleaved into F1 and F2 by a furin-like enzyme in the Golgi membrane and the virus surface F protein is processed into F1 and F2, these results may represent an artefact. It is noteworthy that all the other viruses whose HR1 or HR1 homologues show fusion inhibition do not have HN or HN homologues, e.g. HIV/SIV, Ebola virus. The HR1 derived from paramyxovirus SV5 does show some fusion inhibition (Joshi et al., 1998
), whereas HR1 derived from other paramyxoviruses, e.g. Sendai virus and measles virus, does not show any (Rapaport et al., 1995
; Wild & Buckland, 1997
). HR1 proteins derived from all those paramyxoviruses whose fusion absolutely requires HN protein do not appear to have any fusion inhibition, whereas the HR1 from SV5, whose fusion does not absolutely require HN protein, shows the fusion inhibition effect. Therefore it is possible that HN protein (or its homologues) plays an important role in the HR1/HR2 domain exposure in the pre-hairpin state of the F protein. This could be due to either the direct steric constraint of HN protein for the HR1/HR2 accessibility or HN-induced conformational change of the F protein during the fusion process if HN is absolutely required. This should be addressed in the future by testing HR1 inhibition of more paramyxoviruses, e.g. by including more HN (or homologue)-independent and -dependent viruses, and of NDV F protein mutants (Sergel et al., 2000
) whose fusion does not require HN protein.
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References |
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Received 26 July 2001;
accepted 25 October 2001.