MRC Virology Unit, Institute of Virology, Church Street, Glasgow G11 5JR, UK
Correspondence
John McLauchlan
j.mclauchlan{at}vir.gla.ac.uk
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ABSTRACT |
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These authors contributed equally to this work.
Present address: Ingenza Ltd, King's Buildings, Edinburgh EH9 3JJ, UK.
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MAIN TEXT |
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To examine NS4B in live cells, the polypeptide was attached to the C terminus of EGFP. A PCR product encoding NS4B (aa 17121973 of HCV 1a strain H77) was amplified from pCV-H77C (kindly supplied by Dr J. Bukh; Yanagi et al., 1997) using forward and reverse primers (5'-GGGAGAATTCAGATCTCAGCACTTACCGTACATCGAG-3' and 5'-GGGAAGCTTCTAGAGGATCCGCTAGCATGGAGTGGTACACTCCGAGC-3', respectively) that incorporated restriction enzyme sites for cloning purposes and a stop codon (underlined). The PCR product was introduced into pGEM-1 following digestion with EcoRI and XbaI, to generate plasmid pGEM-1/NS4B. A BglII/XbaI DNA fragment from pGEM-1/NS4B was inserted into pEGFP-C1 (Clontech) to create pGFP-NS4B. pGFP-NS4B was transfected into tissue culture cells estimated to be between 30 and 60 % confluent using Lipofectamine 2000 (Invitrogen). To express untagged protein, an oligonucleotide (5'-AATTCTAGGATCCTCATTGATGA-3') that incorporated an initiator methionine codon was introduced between the EcoRI and BglII sites in pGEM-1/NS4B to produce pGEM-1/atg-NS4B. A BamHI DNA fragment from pGEM-1/atg-NS4B was inserted into the BamHI site in the Semliki Forest virus vector pSFV-1, generating pSFV/NS4B. RNA was transcribed from pSFV/NS4B and electroporated into tissue culture cells as described previously (Patel et al., 1999
; Hope & McLauchlan, 2000
). Untagged NS4B was detected using a polyclonal antiserum called R1061, which was raised against two synthetic peptides, LAEQFKQKALGLLQTA and QTNWQKLEVFWAKH, derived from aa 1328 and 4053, respectively, of the strain H77 polypeptide (NCBI accession number NP_751926). For live studies, cells were seeded onto glass-bottomed 35 mm Petri dishes (MatTek). Photobleaching was performed in Dulbecco's modified Eagle's medium lacking phenol red (Invitrogen), supplemented with 1 % fetal calf serum and 30 mM HEPES at 37 °C on a heated stage. Confocal microscopy was carried out with a Zeiss LSM510 META microscope.
Transfection of pGFP-NS4B into cells followed by examination under live conditions revealed that the fusion protein was distributed in a thread-like pattern, consistent with ER localization, and at small foci. This distribution was evident in BHK, HuH-7 [Fig. 1a, panels (i), (ii), (iv) and (v)] and Vero cells (data not shown) and therefore was not dependent on cell origin, although we did observe greater numbers of foci in BHK cells compared with HuH-7 cells. Using a specific NS4B antiserum, R1061, indirect immunofluorescence of cells that had been electroporated with RNA transcribed from pSFV/NS4B confirmed that both distributions were indicative of NS4B localization and did not arise from fusion to GFP [Fig. 1a
, panels (iii) and (vi)]. We also verified that the thread-like pattern denoted association of NS4B with the ER membrane using an anti-calnexin antibody (kindly supplied by B. Martoglio, ETH, ETH Hoenggerberg, 8093 Zurich, Switzerland; Fig. 1b
). These data agree with previously published studies (Lundin et al., 2003
). At higher magnification, the foci were coincident with the ER membrane [Fig. 1a
, panels (ii) and (v)], which demonstrated their close connection with the ER network. We propose that these NS4B-induced structures are referred to as membrane-associated foci (MAFs).
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Analysis of GFP-tagged proteins in live cells enables monitoring not only of the movement of subcellular structures but also of protein diffusion within an organelle by applying a method termed fluorescence recovery after photobleaching (FRAP) (Reits & Neefjes, 2001). In FRAP experiments, fluorescent molecules in pre-defined areas are irreversibly photobleached by a high-power focused laser beam. Subsequent diffusion of non-bleached molecules into the bleached area leads to a recovery of fluorescence. To test whether NS4B was mobile in ER membranes and MAFs, fluorescence intensity in selected regions in live cells expressing the GFP-tagged protein was measured before and after photobleaching [Fig. 2a
, panels (i) and (ii)]. For comparison, cells expressing GFP-tagged DNase X were examined under identical conditions [Fig. 2a
, panels (iii) and (iv)]. GFPDNase X targets the ER membrane (http://gfp-cdna.embl.de/loc-html/P49184.html) and has been used previously in live-cell studies (Targett-Adams et al., 2003
).
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Among the strategies that enable biophysical analysis of intracellular events, FRAP has emerged as a powerful method for monitoring the kinetics of protein movement (Reits & Neefjes, 2001). NS4B is mobile in the ER membrane, although its diffusion is slower compared with DNase X. Both NS4B (Lundin et al., 2003
) and DNase X (data not shown) are predicted to contain transmembrane domains and therefore are topologically related. More importantly, we have demonstrated that NS4B has reduced mobility in MAFs compared with the ER membrane. These characteristics for NS4B were identical, irrespective of whether GFP was located at the N or C terminus of the protein (data not shown). We concluded that there are two populations of NS4B with distinct relative mobilities in the ER membrane and MAFs.
Several factors may account for the reduced diffusion of NS4B in MAFs. NS4B is an integral membrane protein (Hugle et al., 2001), predicted to contain at least four transmembrane segments (Lundin et al., 2003
). Transmembrane regions behave as cylindrical molecules in a lipid environment and contribute considerably to diffusional movement. NS4B could form an oligomeric complex in MAFs that would effectively increase the bulk of transmembrane regions embedded in the membrane. Movement of an oligomeric complex in membranes would be retarded, although it should be noted that diffusion rate is proportional to the logarithm of the inverse of the radius of transmembrane segments (Saffman & Delbruck, 1975
; Vaz et al., 1982
). Thus, increasing the radius of transmembrane regions through oligomerization has less of an impact on diffusion than would be predicted intuitively. Alternatively, interactions between NS4B and cellular proteins in MAFs could be different from those in the ER membrane. Studies with GFP-tagged H2Ld, an MHC class I molecule involved in peptide presentation, have shown that two populations of the protein exist (Marguet et al., 1999
). One form diffused rapidly in membranes and apparently was not associated with the transporter associated with antigen processing (TAP) complex. The second form had a much lower capacity to diffuse and was associated with TAP. These slower GFPH2Ld molecules had a diffusion coefficient similar to TAP, leading to the conclusion that interactions between the two proteins constrained the mobility of H2Ld. Little is known about the interactions of NS4B with cellular components. If such interactions are established, it will be important to determine whether they could also influence the mobility of NS4B. Finally, interactions between NS4B and lipids present in MAFs could reduce its diffusion. For the cellular protein H-Ras, lateral diffusion is restricted through interaction with lipid rafts on the plasma membrane (Niv et al., 2002
). NS4B also associates with lipid rafts (Gao et al., 2004
), although we do not know whether NS4B in MAFs is indicative of its association with such membrane domains. None the less, MAFs could represent specialized regions in the ER membrane with a particular lipid composition that influences NS4B mobility.
The MAFs generated by NS4B alone could be equivalent to the foci present in cells harbouring the HCV replicon. Since MAFs and the ER membrane are apparently contiguous, we consider it likely that NS4B in MAFs originates from the pool of protein attached to the ER membrane that has greater mobility. However, after incorporation into MAFs, the movement of NS4B molecules may be blocked by the interactions that occur at these sites. Therefore, our results may have some bearing on the interpretation of the sites for viral RNA synthesis (Gosert et al., 2003). HCV RNA is detected in foci, suggesting that these structures represent locations of active replication complexes. Alternatively, replication complexes that are functional for RNA synthesis could diffuse rapidly along the ER membrane. Upon incorporation into foci, viral proteins and any associated viral RNA in these complexes would be restricted in their movement, resulting in accumulation at foci. Thus, foci may be sites at which synthesized RNA accumulates but not necessarily the locations for replication. Such a distinction is important for our understanding of HCV replication, and further experimental evidence is needed to test these possibilities.
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ACKNOWLEDGEMENTS |
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Received 19 November 2004;
accepted 24 January 2005.