Imaging analysis of human metapneumovirus-infected cells provides evidence for the involvement of F-actin and the raft-lipid microdomains in virus morphogenesis

Virology Journal

Richard J Sugrue³  Geoffrey L Toms²  Fiona Fenwick²  Boon Huan Tan¹  Liat Hui Loo³  Puisan Wong¹  Tra Nguyen Huong³  Muhammad Raihan Jumat³ 

[1] Detection and Diagnostics Laboratory, DSO National Laboratories, 27 Medical Drive, Singapore 117510, Republic of Singapore;School of Clinical Medical Sciences, The University of Newcastle, Newcastle upon Tyne NE24HH, UK;Division of Molecular Genetics and Cell Biology, School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Nanyang 637551, Republic of Singapore

关键词: F-actin;    Lipid-raft;    Virus assembly;    Virus filaments;    Paramyxovirus;    Human metapneumovirus;   

Others  :  1148129
DOI  :  10.1186/s12985-014-0198-8

 received in 2014-09-01, accepted in 2014-11-05,  发布年份 2014

【 摘 要 】

Backgound

Due to difficulties of culturing Human metapneumovirus (HMPV) much of the current understanding of HMPV replication can be inferred from other closely related viruses. The slow rates of virus replication prevent many biochemical analyses of HMPV particles. In this study imaging was used to examine the process of HMPV morphogenesis in individually infected LLC-MK2 cells, and to better characterise the sites of HMPV assembly. This strategy has circumvented the problems associated with slow replication rates and allowed us to characterise both the HMPV particles and the sites of HMPV morphogenesis.

Methods

HMPV-infected LLC-MK2 cells were stained with antibodies that recognised the HMPV fusion protein (F protein), attachment protein (G protein) and matrix protein (M protein), and fluorescent probes that detect GM1 within lipid-raft membranes (CTX-B-AF488) and F-actin (Phalloidin-FITC). The stained cells were examined by confocal microscopy, which allowed imaging of F-actin, GM1 and virus particles in HMPV-infected cells. Cells co-expressing recombinant HMPV G and F proteins formed virus-like particles and were co-stained with antibodies that recognise the recombinant G and F proteins and phalloidin-FITC and CTX-B-AF594, and the distribution of the G and F proteins, GM1 and F-actin determined.

Results

HMPV-infected cells stained with anti-F, anti-G or anti-M revealed a filamentous staining pattern, indicating that the HMPV particles have a filamentous morphology. Staining of HMPV-infected cells with anti-G and either phalloidin-FITC or CTX-B-AF488 exhibited extensive co-localisation of these cellular probes within the HMPV filaments. This suggested that lipid-raft membrane domains and F-actin structures are present at the site of the virus morphogenesis, and are subsequently incorporated into the HMPV filaments. Furthermore, the filamentous virus-like particles that form in cells expressing the G protein formed in cellular structures containing GM1 and F-actin, suggesting the G protein contains intrinsic targeting signals to the sites of virus assembly.

Conclusions

These data suggest that HMPV matures as filamentous particles and that virus morphogenesis occurs within lipid-raft microdomains containing localized concentrations of F-actin. The similarity between HMPV morphogenesis and the closely related human respiratory syncytial virus suggests that involvement of F-actin and lipid-raft microdomains in virus morphogenesis may be a common feature of the Pneumovirinae.

【 授权许可】

   
2014 Jumat et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

【 参考文献 】

• [1]van den Hoogen BG, de Jong JC, Groen J, Kuiken T, de Groot R, Fouchier RA, Osterhaus AD: A newly discovered human pneumovirus isolated from young children with respiratory tract disease. Nat Med 2001, 7:719-724.
• [2]Kahn JS: Epidemiology of human metapneumovirus. Clin Microbiol Rev 2006, 19:546-557.
• [3]Sugrue RJ, Tan BH, Loo LH: The emergence of human metapneumovirus. Future Virol 2008, 3:363-371.
• [4]Schowalter RM, Smith SE, Dutch RE: Characterization of human metapneumovirus F protein-promoted membrane fusion: critical roles for proteolytic processing and low pH. J Virol 2006, 80:10931-10941.
• [5]Liu L, Bastien N, Li Y: Intracellular processing, glycosylation, and cell surface expression of human metapneumovirus attachment glycoprotein. J Virol 2007, 81:13435-13443.
• [6]Collins PL, Crowe JE Jr: Respiratory Syncytial Virus and Metapneumovirus. In Fields Virology. Edited by Fields BN, Knipe DM, Howley PM. Wolters Kluwer Health/Lippincott Williams & Wilkins, Philadelphia; 2007.
• [7]van den Hoogen BG, Bestebroer TM, Osterhaus AD, Fouchier RA: Analysis of the genomic sequence of a human metapneumovirus. Virology 2002, 295:119-132.
• [8]Biacchesi S, Skiadopoulos MH, Boivin G, Hanson CT, Murphy BR, Collins PL, Buchholz UJ: Genetic diversity between human metapneumovirus subgroups. Virology 2003, 315:1-9.
• [9]van den Hoogen BG, Herfst S, Sprong L, Cane PA, Forleo-Neto E, de Swart RL, Osterhaus AD, Fouchier RA: Antigenic and genetic variability of human metapneumoviruses. Emerg Infect Dis 2004, 10:658-666.
• [10]Njenga MK, Lwamba HM, Seal BS: Metapneumoviruses in birds and humans. Virus Res 2003, 91:163-169.
• [11]Chan PK, Tam JS, Lam CW, Chan E, Wu A, Li CK, Buckley TA, Ng KC, Joynt GM, Cheng FW, To KF, Lee N, Hui DS, Cheung JL, Chu I, Liu E, Chung SS, Sung JJ: Human metapneumovirus detection in patients with severe acute respiratory syndrome. Emerg Infect Dis 2003, 9:1058-1063.
• [12]Deffrasnes C, Cote S, Boivin G: Analysis of replication kinetics of the human metapneumovirus in different cell lines by real-time PCR. J Clin Microbiol 2005, 43:488-490.
• [13]Ingram RE, Fenwick F, McGuckin R, Tefari A, Taylor C, Toms GL: Detection of human metapneumovirus in respiratory secretions by reverse-transcriptase polymerase chain reaction, indirect immunofluorescence, and virus isolation in human bronchial epithelial cells. J Med Virol 2006, 78:1223-1231.
• [14]Boivin G, Abed Y, Pelletier G, Ruel L, Moisan D, Cote S, Peret TC, Erdman DD, Anderson LJ: Virological features and clinical manifestations associated with human metapneumovirus: a new paramyxovirus responsible for acute respiratory-tract infections in all age groups. J Infect Dis 2002, 186:1330-1334.
• [15]Ishiguro N, Ebihara T, Endo R, Ma X, Shirotsuki R, Ochiai S, Ishiko H, Kikuta H: Immunofluorescence assay for detection of human metapneumovirus-specific antibodies by use of baculovirus-expressed fusion protein. Clin Diagn Lab Immunol 2005, 12:202-205.
• [16]Fenwick F, Young B, McGuckin R, Robinson MJ, Taha Y, Taylor CE, Toms GL: Diagnosis of human metapneumovirus by immunofluorescence staining with monoclonal antibodies in the North-East of England. J Clin Virol 2007, 40:193-196.
• [17]Ravi LI, Liang L, Wong PS, Brown G, Tan BH, Sugrue RJ: Increased hydroxymethylglutaryl coenzyme A reductase activity during respiratory syncytial virus infection mediates actin dependent inter-cellular virus transmission. Antiviral Res 2013, 100:259-268.
• [18]Low KW, Tan T, Ng K, Tan BH, Sugrue RJ: The RSV F and G glycoproteins interact to form a complex on the surface of infected cells. Biochem Biophys Res Commun 2008, 366:308-313.
• [19]Jeffree CE, Rixon HW, Brown G, Aitken J, Sugrue RJ: Distribution of the attachment (G) glycoprotein and GM1 within the envelope of mature respiratory syncytial virus filaments revealed using field emission scanning electron microscopy. Virology 2003, 306:254-267.
• [20]Roberts SR, Compans RW, Wertz GW: Respiratory syncytial virus matures at the apical surfaces of polarized epithelial cells. J Virol 1995, 69:2667-2673.
• [21]Parry JE, Shirodaria PV, Pringle CR: Pneumoviruses: the cell surface of lytically and persistently infected cells. J Gen Virol 1979, 44:479-491.
• [22]Bagnaud-Baule A, Reynard O, Perret M, Berland JL, Maache M, Peyrefitte C, Vernet G, Volchkov V, Paranhos-Baccala G: The human metapneumovirus matrix protein stimulates the inflammatory immune response in vitro. PLoS One 2011, 6:e17818.
• [23]Loo LH, Jumat MR, Fu Y, Ayi TC, Wong PS, Tee NW, Tan BH, Sugrue RJ: Evidence for the interaction of the human metapneumovirus G and F proteins during virus-like particle formation. Virol J 2013, 10:294. BioMed Central Full Text
• [24]Brown G, Rixon HW, Sugrue RJ: Respiratory syncytial virus assembly occurs in GM1-rich regions of the host-cell membrane and alters the cellular distribution of tyrosine phosphorylated caveolin-1. J Gen Virol 2002, 83:1841-1850.
• [25]Burke E, Dupuy L, Wall C, Barik S: Role of cellular actin in the gene expression and morphogenesis of human respiratory syncytial virus. Virology 1998, 252:137-148.
• [26]Bitko V, Oldenburg A, Garmon NE, Barik S: Profilin is required for viral morphogenesis, syncytium formation, and cell-specific stress fiber induction by respiratory syncytial virus. BMC Microbiol 2003, 3:9. BioMed Central Full Text
• [27]Kallewaard NL, Bowen AL, Crowe JE Jr: Cooperativity of actin and microtubule elements during replication of respiratory syncytial virus. Virology 2005, 331:73-81.
• [28]Jeffree CE, Brown G, Aitken J, Su-Yin DY, Tan BH, Sugrue RJ: Ultrastructural analysis of the interaction between F-actin and respiratory syncytial virus during virus assembly. Virology 2007, 369:309-323.
• [29]Radhakrishnan A, Yeo D, Brown G, Myaing MZ, Iyer LR, Fleck R, Tan BH, Aitken J, Sanmun D, Tang K, Yarwood A, Brink J, Sugrue RJ: Protein analysis of purified respiratory syncytial virus particles reveals an important role for heat shock protein 90 in virus particle assembly. Mol Cell Proteomics 2010, 9:1829-1848.
• [30]Ulloa L, Serra R, Asenjo A, Villanueva N: Interactions between cellular actin and human respiratory syncytial virus (HRSV). Virus Res 1998, 53:13-25.
• [31]Brown DA, London E: Functions of lipid rafts in biological membranes. Annu Rev Cell Dev Biol 1998, 14:111-136.
• [32]Scheiffele P, Rietveld A, Wilk T, Simons K: Influenza viruses select ordered lipid domains during budding from the plasma membrane. J Biol Chem 1999, 274:2038-2044.
• [33]Patch JR, Crameri G, Wang LF, Eaton BT, Broder CC: Quantitative analysis of Nipah virus proteins released as virus-like particles reveals central role for the matrix protein. Virol J 2007, 4:1. BioMed Central Full Text
• [34]Sugrue RJ, Brown C, Brown G, Aitken J, Mc LRHW: Furin cleavage of the respiratory syncytial virus fusion protein is not a requirement for its transport to the surface of virus-infected cells. J Gen Virol 2001, 82:1375-1386.