Evidence for the interaction of the human metapneumovirus G and F proteins during virus-like particle formation.
BACKGROUND
Human metapneumovirus (HMPV) is now a major cause of lower respiratory infection in children. Although primary isolation of HMPV has been achieved in several different cell lines, the low level of virus replication and the subsequent recovery of low levels of infectious HMPV have hampered biochemical studies on the virus. These experimental methodologies usually require higher levels of biological material that can be achieved following HMPV infection. In this study we demonstrate that expression of the HMPV F, G and M proteins in mammalian cells leads to HMPV virus-like particles (VLP) formation. This experimental strategy will serve as a model system to allow the process of HMPV virus assembly to be examined.


METHODS
The HMPV F, G and M proteins were expressed in mammalian cell lines. Protein cross-linking studies, sucrose gradient centrifugation and in situ imaging was used to examine interactions between the virus proteins. VLP formation was examined using sucrose density gradient centrifugation and electron microscopy analysis.


RESULTS
Analysis of cells co-expressing the F, G and M proteins demonstrated that these proteins interacted. Furthermore, in cells co-expression the three HMPV proteins the formation VLPs was observed. Image analysis revealed the VLPs had a similar morphology to the filamentous virus morphology that we observed on HMPV-infected cells. The capacity of each protein to initiate VLP formation was examined using a VLP formation assay. Individual expression of each virus protein showed that the G protein was able to form VLPs in the absence of the other virus proteins. Furthermore, co-expression of the G protein with either the M or F proteins facilitated their incorporation into the VLP fraction.


CONCLUSIONS
Co-expression of the F, G and M proteins leads to the formation of VLPs, and that incorporation of the F and M proteins into VLPs is facilitated by their interaction with the G protein. Our data suggests that the G protein plays a central role in VLP formation, and further suggests that the G protein may also play a role in the recruitment of the F and M proteins to sites of virus particle formation during HMPV infection.
Division of Molecular Genetics and Cell Biology, School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore. rjsugrue@ntu.edu.sg.
Liat Hui
LH
Loo
Muhammad Raihan
MR
Jumat
Yi
Y
Fu
Teck Choon
TC
Ayi
Pui San
PS
Wong
Nancy W S
NW
Tee
Boon Huan
BH
Tan
Richard J
RJ
Sugrue
D002460:Cell Line; D002499:Centrifugation, Density Gradient; D002648:Child; D002675:Child, Preschool; D015870:Gene Expression; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D029121:Metapneumovirus / Q000235:genetics*; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D011485:Protein Binding; D012367:RNA, Viral / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014760:Viral Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D022701:Virosomes / Q000378:metabolism*
C484233:G glycoprotein, human metapneumovirus; D006023:Glycoproteins; D012367:RNA, Viral; D011994:Recombinant Proteins; D014760:Viral Fusion Proteins; D014764:Viral Proteins; D022701:Virosomes