1 Department of Respiratory and Sleep Medicine, Monash Medical Centre, 246 Clayton Road, Clayton 3168, Australia
2 Departments of Microbiology and Medicine, Monash University, Clayton, Australia
3 Department of Environmental Biology, RMIT University, Melbourne, Australia
4 Macfarlane Burnet Institute for Medical Research and Public Health, Melbourne, Australia
5 Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, MA, USA
Correspondence
Reena Ghildyal
reena.ghildyal{at}med.monash.edu.au
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ABSTRACT |
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Present address: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, PA, USA.
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MAIN TEXT |
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RSV G glycoprotein (G) is a type II membrane protein and the major attachment protein of RSV (Levine et al., 1987). Recent studies have shown that RSV lacking the G gene can replicate efficiently in cell culture, implying that RSV has an auxiliary attachment function, probably involving the F protein (Karron et al., 1997
; Techaarpornkul et al., 2001
; Teng et al., 2001
). Genetically engineered RSV lacking the entire G gene replicates efficiently in some cell lines but not in others, and is attenuated in BALB/c mice (Teng et al., 2001
). Thus, although G is not essential for replication in cell lines, it is required for full infectivity and in vivo pathogenicity, and a largely intact G gene has been found to be present in all RSV field isolates analysed to date. The RSV matrix protein (M) is postulated to play a major role in virus assembly through its interactions with various components of the virus, as well as with membranes of infected cells (Ghildyal et al., 2002
; Henderson et al., 2002
).
As observed in other paramyxoviruses, it is proposed that the coalescence of RSV components at budding sites is facilitated by interactions between the cytoplasmic domains of the envelope glycoproteins and M. This is supported by the observation that M associates with cellular membranes by itself, but that the nature of that association is modified in the presence of F (Henderson et al., 2002).
We investigated the interaction of M with RSV envelope glycoproteins, specifically G. We have provided evidence of an interaction between M and G that involves the G cytoplasmic domain and, in particular, its first 6 aa.
HEp2 cells (Victorian Infectious Disease Reference Laboratory, Melbourne, Australia) were infected with RSV subgroup A, strain A2 (a gift from Paul Young, University of Queensland, Brisbane, Australia) or recombinant RSV lacking the cytoplasmic domain and one-third of the transmembrane domain of G (sG-RSV; Teng et al., 2001) (Fig. 1
). In wild-type RSV, G is expressed as a full-length, membrane-associated form that initiates at the first ATG in the 298-codon ORF and as a secreted form that initiates at the second ATG (codon 48), one-third of the way into the transmembrane domain (Roberts et al., 1994
). sG-RSV expressed only the secreted form of G due to the engineered deletion of codons 147 (Teng et al., 2001
).
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As shown in Fig. 1(a, b), G, but not M, was detected on the surface of infected cells as expected [Fig. 1a
(i) and (ii)]. In infected, permeabilized cells, G was observed throughout the cytoplasm with specific localization in patches, suggesting its association with intracellular membranes [Fig. 1b
(i)]. M was observed within the cytoplasm, in patches similar to G, and in characteristic cytoplasmic inclusions as reported previously [Fig. 1b
(ii)] (Ghildyal et al., 2002
). The computer-generated merge showed yellow patches, indicative of co-localization of M and G [Fig. 1b
(iii)], which we suggest is occurring at intracellular membranes such as the Golgi. However, not all areas with cytoplasmic G had demonstrable M.
Fig. 1(c, d) shows the surface and intracellular localization of M and sG in sG-RSV-infected HEp2 cells. There was apparent surface localization of sG in cells infected with sG-RSV [Fig. 1c
(i)]. Additionally, we routinely detected sG in the culture supernatant, indicating that it was transported to and across the plasma membrane, as has been reported previously (Teng et al., 2001
). Detection of sG at the cell surface was unexpected, but may indicate that it accumulates at least transiently at that site prior to secretion or that secreted sG remains associated with the cell via interaction with cell-surface components (Shields et al., 2003
). Interestingly, its cellular distribution was subtly different from that of wild-type G, as it was concentrated in what appeared to be surface projections. The significance of this is unknown, although it may be that the greater spread of wild-type G along the cell surface is due to its longer residence time. As expected, M was not detected on the surface of infected cells [Fig. 1c
(ii)]. sG was observed throughout the cytoplasm, with specific localization in patches [Fig. 1d
(i)]. There was also some low-level staining in cytoplasmic inclusions, which may or may not be specific, as we did not observe this localization with full-length G. M was observed in the cytoplasm in patches (similar to sG), as well as in cytoplasmic inclusions [Fig. 1d
(ii)]. However, the computer-generated merge showed no distinct regions of yellow colour [Fig. 1d
(iii)]. Thus, M and sG did not co-localize in cells infected with sG-RSV. The observation that wild-type G, but not sG, co-localized with M in the context of an authentic RSV infection suggests that the two proteins interact and that this interaction requires the cytoplasmic and/or transmembrane domains of G. Both antibodies (C781 and R
G) were specific for their respective proteins and did not bind to any proteins in mock-infected cells (Fig. 1e
).
To define further the region of G that is required for interaction with M, we co-expressed M with full-length G or truncated derivatives thereof (sG, G30, G
13 and G
6; Fig. 2a
). G constructs were cloned into the Semliki Forest virus replicon system (Gibco-BRL) as described previously (JMPpSFV; Ghildyal et al., 1999
; Peroulis et al., 1999
). Cloning of the full-length G and a variant lacking the first 47 aa (G and sG, respectively) within this vector has been described previously (Peroulis et al., 1999
). The G construct was used as template for generating the various mutants by PCR, using the same C-terminal primer combined with specific N-terminal primers. The M gene was subcloned from the pET30(a) construct (described below) into the pSD4.2 expression vector (a gift from Douglas S. Lyles, Wake Forest University, Winston-Salem, NC, USA) (Desforges et al., 2002
). We and others have found that the paramyxovirus M is difficult to observe by immunological means, especially in transfected cells, as it is not very immunogenic and is usually expressed at low levels (Faaberg & Peeples, 1988
). To facilitate the detection of low levels of M, we added an influenza HA-epitope tag to the C terminus of M (M-HA). Recombinant JMPpSFV or M-HA plasmid DNA was linearized, followed by transcription using an mMessage mMachine SP6 transcription kit (Ambion). Freshly prepared mRNA [1 (24-well plates) or 5 (six-well plates) µg per well] was transfected into HEp2 cells using Lipofectamine 2000 (Invitrogen). Transfected cells were fixed at 15 h post-transfection as described above. Immunofluorescence assays were performed as described above, except that C781 was replaced with anti-HA (rabbit anti-influenza HA antibody; Sigma); co-localization of G variants and M was analysed by CLSM.
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To validate further the CLSM data, we assessed the binding of G variants to M in a cell-free ELISA. G variants were expressed in HEp2 cells by transfection and the level of expression of each variant was assessed by Western blotting (Fig. 2c) prior to use in the ELISA. Transfected cells were lysed 20 h later in lysis buffer as described previously (Marty et al., 2004
). Proteins were separated by 10 % SDS-PAGE (Laemmli, 1970
) and transferred on to Hybond-C membrane (Amersham Biosciences). Membranes were blocked with BSA, probed with an anti-G-specific mAb (mAb30; a gift from Geraldine Taylor, Institute for Animal Health, UK) and bound antibody was detected with horseradish peroxidase-conjugated goat anti-mouse antibody (Dako) followed by ECL (Amersham Biosciences). Full-length G and its derivatives were expressed at similar levels (Fig. 2c
). M was expressed in bacteria and purified prior to use. The M gene was PCR-amplified from total RNA extracted from RSV-infected cells and cloned into the pET30(a) vector (Novagen), which introduces a 6-His fusion tag. M was expressed in Escherichia coli and purified under denaturing conditions by using Ni+ affinity chromatography (Qiagen). We also used a recombinant hepatitis C virus NS3 protein, likewise produced in bacteria and purified with a 6-His tag, as an irrelevant control protein. The purity of both M and NS3 proteins was determined by Coomassie blue staining (Fig. 2c
).
Approximately 100 ng of either M or NS3 protein was immobilized onto microtitre plates, followed by overnight incubation with cell lysates. Bound G was detected with mAb30 followed by horseradish peroxidase-conjugated goat anti-mouse antibody (Dako) and tetramethylbenzidine hydrochloride (TMB; Sigma Chemicals). As shown in Fig. 2(d), full-length G bound to M, whereas mock-transfected cell lysate and cell lysates containing each of the G derivatives did not bind to M. None of the lysates bound to the unrelated NS3 protein (Fig. 2e
). These results further substantiated our CLSM data showing that the N-terminal 6 aa of G are important for its interaction with M. At present, we cannot explain the low absorbance values recorded in the ELISA, but they may be a consequence of the low avidity of the antibodies used, low levels of G in the whole-cell lysate or the presence of G as multimers, making epitopes inaccessible to mAb30.
We next assessed whether a peptide homologous to the N-terminal 36 aa of G (with a C-terminal biotin; 92 % pure peptide; Mimotopes) would bind to M and inhibit binding of G. Recombinant M was immobilized onto microtitre plates, followed by overnight incubation with peptide or with a mix of peptide and full-length purified G (a gift from Dan Speelman, Lederle Praxis Biochemicals, USA) (Hancock et al., 2000). Bound peptide was detected with streptavidinperoxidase (Dako) followed by TMB. The peptide bound to M in a dose-dependent fashion and 80 ng peptide was able to inhibit 5070 % of the binding of 20 ng purified G to 100 ng M (data not shown).
The effect of alanine substitution of individual amino acids at positions 27 of the G cytoplasmic domain was then examined (Fig. 3a). All of the alanine mutants were expressed in BHK-21 cells and cell lysates were used in a GM binding ELISA, as shown in Fig. 2(d)
. The mutants and full-length G were expressed at similar levels, as shown by Western blotting with mAb30 (Fig. 3b
). sG bound to M at levels equal to or less than the mock cell lysate, as did the two mutants GS2A and GD6A, whereas the other mutants bound to M at various levels above that of mock cell lysate (Fig. 3c
). Thus, loss of either serine at position 2 or aspartate at position 6 in G largely abrogated its interaction with M. These results were also confirmed by CLSM studies of M and G co-expression (data not shown).
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As mentioned above, G is not essential for the formation of infectious virus (Karron et al., 1997; Techaarpornkul et al., 2001
; Teng et al., 2001
) or for the formation of virus-like particles (Teng & Collins, 1998
). It is possible that M interacts independently with both G and F and that, whilst interaction with a glycoprotein is essential for assembly, either glycoprotein alone will suffice. In support of this hypothesis, we have observed co-localization of M and F in cells infected with sG-RSV or with a recombinant RSV lacking the entire G gene (data not shown).
In conclusion, we have demonstrated for the first time that RSV M interacts with G, and that this interaction is mediated through the cytoplasmic tail of G. How this interaction relates to other aspects of RSV assembly remains to be determined.
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ACKNOWLEDGEMENTS |
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Received 15 December 2004;
accepted 30 March 2005.
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