Department of Virology, Haartman Institute, PO Box 21, FIN-00014 University of Helsinki, Finland
Correspondence
Sami K. J. Kukkonen
sami.kukkonen{at}iki.fi
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ABSTRACT |
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INTRODUCTION |
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Of the four hantavirus structural proteins, the localization of the two glycoproteins and the N protein has been studied. G1 has been found to have a Golgi retention signal that directs both glycoproteins G1 and G2 to the virus assembly site on the membranes of the Golgi into which the virus buds (Shi & Elliott, 2002). The Seoul hantavirus (SEOV) G2 accumulates in the Golgi from 8 h post-infection (p.i.) onwards (Kariwa et al., 2003
). The budding of the virus requires the presence of the two glycoproteins and the N protein at the virus assembly site, since these are the viral proteins required for formation of virus-like particles (Betenbaugh et al., 1995
).
The N protein, which encapsidates RNA, is required for RNA synthesis together with the L protein in a Hantaan virus minireplicon system (Flick et al., 2003), thus being the only viral protein required for both virus budding and RNA synthesis. The N protein of Black Creek Canal (BCCV), Seoul (SEOV) and Puumala (PUUV) hantaviruses is expressed in the perinuclear region, and BCCV N protein was further reported to co-localize with Golgi markers (Ravkov & Compans, 2001
). In another study, the N protein of SEOV was reported not to co-localize with the Golgi markers but to surround the Golgi (Kariwa et al., 2003
). The BCCV N protein was found to be associated with membranes in an assay in which microsomal membranes were separated from cytosolic proteins (Ravkov & Compans, 2001
). Of other Bunyaviridae, the N protein of CrimeanCongo haemorrhagic fever virus (CCHFV), genus Nairovirus, was found to be perinuclear but it did not localize in the Golgi (Andersson et al., 2004
). The CCHFV N protein interacts with actin, and the disruption of actin filaments abolished the perinuclear localization of the protein (Andersson et al., 2004
). The BCCV N protein was also found to interact with actin (Ravkov et al., 1998
).
L protein is the putative RNA transcriptase and replicase of the virus. In contrast to the N protein and the glycoproteins, very little is known about it. Hantaan virus L protein has been detected only in virions purified from the growth medium of metabolically labelled Vero E6 cells and the size of the protein was reported to be 200 kDa (Elliott et al., 1984). However, the protein has not been detected in hantavirus-infected cells. Therefore, it is not known whether the whole L ORF is used to encode a protein and whether there is post-translational cleavage of the protein product.
It is known that RNA replication of all positive-strand viruses is associated with membranes (den Boon et al., 2001). Positive-strand RNA viruses cause the formation of vesicles and appressed membranes that sequester viral RNA replication intermediates; this parallels the genome replication of double-stranded RNA viruses and retroviruses (Schwartz et al., 2002
). The poliovirus RNA polymerase forms oligomeric arrays on membrane surfaces (Lyle et al., 2002
). Of negative-strand viruses, the L RNA polymerase protein of Sendai virus has also recently been found to form oligomers (Smallwood et al., 2002
; Cevik et al., 2003
) but whether these are associated with membranes remains to be determined. The localization of influenza A virus RNA polymerase subunits of negative-strand viruses has been studied (Akkina et al., 1987
) and for La Crosse virus, genus Bunyavirus, family Bunyaviridae, the site of RNA synthesis has been localized to the cytoplasm (Rossier et al., 1986
), but there have been no studies on the localization of any hantavirus L protein or the possible membrane association of the protein.
In this report, we describe the development of antibodies against TULV L protein and the detection of the protein in virions, infected cells and transfected cells expressing recombinant L protein. These antibodies enabled the monitoring of the protein expression levels, determination of its size, and they were used to study the membrane association of the L protein. The localization of the L protein in cells was further characterized using expression of autofluorescent fusion proteins.
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METHODS |
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PCR and cloning.
For antigen production, the L ORF was PCR amplified from earlier constructs (Kukkonen et al., 1998) and cloned into the vector pET15b (Novagen) under a T7 promoter. This created the three constructs: pET15b/TLa (nt 372190, aa 1718), pET15b/TLb (nt 21914344, aa 7191436) and pET15b/TLc (nt 43456495, aa 14372153). For localization studies, the constructs pTM1/L, pTM1/EGFPL, pTM1/LEGFP, pTM1/EGFP and pcDNA3/S were made. The L ORF was cloned into the vector pTM1 (Moss et al., 1990
) to create pTM1/L. The EGFP sequence was PCR amplified from the vector pEGFPC1 (Clontech) and inserted into the NcoI site of pTM1 to generate pTM1/EGFP and to the 5' and 3' ends of the L ORF of pTM1/L to create pTM1/EGFPL and pTM1/LEGFP, respectively. The ORF of TULV S was PCR amplified and cloned into the vector pcDNA3 to create pcDNA3/S. The pET15b/L construct used for in vitro synthesis of the L protein and was generated by transferring the L ORF from pTM1/L to pET15b using restriction sites NcoI and XhoI. All cloned sequences were verified by automatic sequencing with ABI PRISM (PerkinElmer) carried out by the sequencing facility of the Haartman Institute, University of Helsinki, Finland. All primer sequences are available upon request.
Antigen expression and immunization.
Protein expression and inclusion body purification was done according to the manufacturer's instructions (Novagen). Briefly, E. coli [strain BL21(DE3)pLysS] transformed with pET15b/TLb was propagated in LB medium (containing 50 µg ampicillin ml1 and 34 µg chloramphenicol ml1) at 37 °C. IPTG was added at 1 mM to induce T7 RNA polymerase expression and subsequent target protein expression. The bacteria were pelleted (15 min, 4 °C, 6000 g), resuspended in washing buffer (20 mM Tris/HCl pH 7·5, 10 mM EDTA, 1 % Triton X-100) and lysed by sonication on ice. The lysate was washed twice with washing buffer, resuspended in PBS, pelleted and then resuspended in PBS containing 30 mM HEPES. The purified inclusion bodies were used for immunization of two rabbits, which was carried out by Davids Biotechnologie (Regensburg, Germany) according to their immunization protocol.
Affinity purification of antibodies.
The antigen used to immunize rabbits, TLb, was coupled to CNBr-activated Sepharose 4B according to the manufacturer's instructions (AmershamPharmacia). The immune serum was allowed to bind to a TLb/Sepharose 4B column in TTBS buffer [0·5 M NaCl, 20 mM Tris/HCl pH 7·8, 0·1 % (v/v) Tween 20, 0·01 % NaN3], the column was washed with the same buffer, and the bound antibodies were eluted in fractions with 0·2 M glycine pH 2·8, containing 0·02 % NaN3 and neutralized with 1 M Tris/HCl pH 8·5. The fractions with the highest protein content were pooled. The antibody solutions were dialysed against PBS with 0·02 % NaN3 and stored at 70 °C in 50 % glycerol.
Immunoblotting.
Protein samples were resuspended in Laemmli buffer (Laemmli, 1970), denatured by heating for 3 min at 70 °C, run in denaturing 6 % and 10 % SDS-PAGE gels, and transferred onto PROTRAN nitrocellulose filters (Schleicher and Schuell). Unspecific binding was blocked by preincubating the filters for 24 h at 4 °C in 3 % non-fat milk in TEN-Tween 20 buffer [20 mM Tris/HCl pH 8·0, 1 mM EDTA, 50 mM NaCl, 0·05 (v/v) Tween 20]. The filters were stained with primary antibodies, washed and stained with horseradish peroxidase conjugated secondary antibodies (Dako). Detection of the protein bands was done with enhanced chemical luminescence. To determine copy numbers of L and N proteins, protein bands of TULV infected cell lysates were compared to serial dilutions of known amounts of TLb and purified TULV N protein, respectively. Protein amounts were determined using an image analysis program IMAGEJ (U.S. National Institutes of Health, http://rsb.info.nih.gov/ij/). Anti-L antiserum was used for the detection of the L protein and the monoclonal antibody 6A6 (Lundkvist et al., 1996
) for the N protein detection. The other antibodies used in immunoblotting were anti-GFP (Covance), anti-
-tubulin (Sigma), anti-
-actin (Sigma) and anti-BAP31 (Määttä et al., 2000
).
Purification of microsomal membranes.
Microsomal membranes were purified as described by Ravkov & Compans (2001). Briefly, Vero E6 cells infected with TULV or HeLa cells in 75 cm2 flasks infected with vTF7-3 and then transfected with pTM1/L or pTM1/EGFPL were washed and scraped into PBS 7 days p.i. (for TULV) or 12 h post-transfection (for vTF7-3). Cells were pelleted and resuspended in hypotonic buffer containing 5 mM Tris/HCl pH 7·4, 0·5 mM MgCl2, Complete protease inhibitor cocktail (Roche) and incubated on ice for 10 min. The cells were homogenized with a Dounce homogenizer. Sucrose was added to 0·25 M and cell debris and nuclei were removed by centrifugation (2000 g, 10 min, 4 °C; twice). Following ultracentrifugation (100 000 g, 2 h, 4 °C), the supernatant (cytosolic fraction) was collected. The pellet (membrane fraction) was washed with the hypotonic buffer and dissolved overnight at 4 °C in SDS buffer containing 10 mM Tris/HCl pH 6·8, Complete, 1 % SDS.
Membrane flotation.
Five days p.i., Tula-infected Vero E6 cells were washed and scraped into PBS. The cells were pelleted and resuspended in hypotonic lysis buffer (10 mM Tris/HCl pH 7·5, 10 mM KCl, 5 mM MgCl2, 20 µg RNase A ml1, Complete). Following Dounce homogenization, the nuclei were removed by centrifugation. The post-nuclear supernatant was treated on ice for 30 min with 1 % Triton X-100, 1 % Nonidet P-40, 1 M NaCl, 15 mM Na2CO3 pH 11·5, or left untreated. The cell lysate (500 µl) was then mixed with 85 % sucrose (1·2 ml, final 60 %) or 57 % OptiPrep (final 40 %), and layered with 2·5 ml 55 % and 1 ml 10 % sucrose or 1·5 ml 30 %, 1·5 ml 25 % and 1 ml 5 % OptiPrep. Following ultracentrifugation (100 000 g, 18 h, 4 °C), eight fractions were collected from the top; the proteins were precipitated with methanolchloroform and analysed by immunoblotting.
Localization of L protein in cells.
Vero E6 cells were transfected with the autogene pCMV/T7-T7pol (Brisson et al., 1999) that produces T7 RNA polymerase. The cells were then transfected with either pTM1/EGFP or pTM1/LEGFP. HeLa cells in a 24-well plate were infected with 10 p.f.u. per cell of vTF7-3 and then transfected with either pTM1/EGFP or pTM1/EGFPL constructs using the transfection reagent Fugene 6 (Boehringer Mannheim). After 48 h (12 h for the vTF7-3-infected cells), the cells were fixed with 3·7 % paraformaldehyde at room temperature, permeabilized with 0·5 % Triton X-100, blocked with 1 % BSA/PBS and incubated with antibodies diluted in 1 % BSA/PBS for 1 h at 37 °C. The primary antibodies used were a 1 : 100 dilution of polyclonal anti-PUUV N antiserum for TULV nucleocapsid protein and a 1 : 500 dilution of monoclonal anti-GM130 (BD Biosciences) for Golgi staining. These antibodies were detected with Texas red-conjugated secondary antibodies. Hoechst stain was used to stain the nucleus. An Axioplan 2 optical microscope (Carl Zeiss) was used for fluorescence microscopy and a Leica SP2 (Leica Microsystems) for confocal microscopy.
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RESULTS |
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TLb could not be expressed in the soluble form in E. coli and, therefore, we decided to use purified inclusion bodies to immunize two rabbits. Immune and pre-immune sera were first tested for their ability to recognize the antigen itself. Both rabbits produced immune serum that recognized the antigen, TLb, in immunoblotting (data not shown). However, as the immune serum from rabbit number 2 had higher affinity for the antigen, it was used for all subsequent experiments.
Detection of L protein in virions, infected cells and in vitro translation products
We tested the anti-L antiserum we had raised for its ability to recognize native L protein in TULV virions and TULV-infected Vero E6 cells. TULV was concentrated from the growth medium of infected Vero E6 cells. Growth medium of mock-infected control cells was handled in a similar way to get a control for immunoblotting. A single protein of approximately 250 kDa could be detected in concentrated TULV (Fig. 1a). Total cell lysates of TULV- and mock-infected Vero E6 cells were also immunoblotted and the 250 kDa protein band was the one band detected only in the TULV-infected cells (Fig. 1b
). This corresponds to the deduced size of the protein encoded by the single large ORFs in the L RNA segment of TULV (Kukkonen et al., 1998
) and is larger than the size reported for Hantaan virus L protein (Elliott et al., 1984
). The nature of the band of approximately 55 kDa (Fig. 1b
) remains unknown. We cannot exclude that it is a degradation product of TULV L.
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Amount of viral proteins in TULV-infected cells
In order to determine the expression levels of TULV proteins in infected Vero E6 cells, cells were infected with TULV and collected 18 days p.i. Actin was used as a control and the amount of actin in the cells remained stable (Fig. 2). The glycoproteins G1 and G2 are translated from the same M mRNA; G2 levels, therefore, reflect expression levels of both proteins. The expression patterns of all three viral proteins are similar with expression increasing until peaking at 46 days p.i. (Fig. 2
).
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L protein pellets with microsomal membranes
The RNA synthesis of all positive-strand RNA viruses is associated with membranes (see Schwartz et al., 2002, and references therein). However, little is known of the possible membrane association of RNA synthesis in negative-strand viruses. The new antiserum against TULV L protein allowed us to determine whether the protein is membrane-associated.
In the first method, microsomal membranes and associated proteins were separated from cytosolic proteins by pelleting the membranes by ultracentrifugation. Briefly, Vero E6 cells were infected with TULV, the cells were lysed by Dounce homogenization, nuclei were removed, microsomal membranes were separated from the cytosolic fraction by ultracentrifugation and proteins were detected by immunoblotting. As controls for the purity of the membrane and cytosolic fractions, we used BAP31 as a marker for membrane proteins and -tubulin as a marker for cytosolic proteins. BAP31 is an integral membrane protein of the endoplasmic reticulum (Ng et al., 1997
) and
-tubulin is a cytosolic protein (Taverna et al., 2002
) that forms tubulin filaments with
-tubulin. The controls behaved as expected, with BAP31 found exclusively in the membrane and
-tubulin in the cytosolic fraction (Fig. 3
). We then determined how TULV L and N proteins fractionate. All of the detected L and N proteins were found in the membrane fraction (Fig. 3
). For the N protein this was expected, as the N protein of the hantavirus Black Creek Canal virus has previously been found to be membrane-associated (Ravkov & Compans, 2001
).
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We infected HeLa cells with the recombinant vaccinia virus, vTF7-3, transfected the cells with a construct expressing L protein (pTM1/L), and then detected the proteins from the microsomal and cytosolic fractions by immunoblotting. Recombinant L protein was detected exclusively in the microsomal fraction (Fig. 3). Therefore, the L protein is, in itself, a membrane-associated protein and interaction with the N protein is not required for the association. We also tested the membrane association of the protein constructs used to localize the L protein in cells, EGFP-L and EGFP (see below), and found the EGFP-L in the membrane fraction and the EGFP in the cytosolic fraction (Fig. 3
).
The membrane association of the L and N proteins was further confirmed by membrane flotation experiments, in which membrane-associated proteins in cell lysates float to less dense fractions of discontinuous sucrose or OptiPrep gradients, both of which were used. The L protein of TULV-infected cell lysates was dissociated from membranes following 1 M NaCl or 15 mM Na2CO3 pH 11·5 treatments. Both L and N proteins were dissociated from membranes by 1 % Triton X-100 and 1 % Nonidet P-40 treatments, whereas the glycoproteins remained membrane-associated (data not shown). In our membrane flotation experiments, a portion of L and N co-fractionated with cytosolic proteins. In membrane flotation assays, fractions of membrane proteins are commonly seen to fractionate with cytosolic proteins for reasons discussed by Simons & Toomre (2000). However, cytosolic proteins do not float.
Perinuclear localization of L protein
We next tried to use the antiserum to localize the L protein in TULV-infected cells. However, the antibodies could not detect the L protein in indirect immunofluorescence assays (IFA) and could not, therefore, be used to localize the L protein in cells. One possibility is that the antiserum only recognizes denatured linear epitopes. However, denaturing proteins with guanidine hydrochloride prior to IFA according to the protocol of Peränen et al. (1993), did not enable L protein detection.
To study the localization of the L protein, we decided to make a fusion protein of L with enhanced green fluorescent protein (EGFP). Using EGFP as a tag has the advantage of enabling the detection of the fusion protein without antibodies and, in addition, EGFP-tagged proteins can be detected without fixing the cells, i.e. in live cells. We made constructs in which EGFP was in the C terminus, pTM1/LEGFP, but also a construct in which EGFP was in the N terminus of the fusion protein, pTM1/EGFPL (Fig. 4a). Both constructs were cloned in the T7 expression vector, pTM1, which has a T7 promoter followed by the 5' untranslated region of encephalomyocarditis virus, which enhances the translation of uncapped mRNAs. As a control we had a construct with EGFP alone in the vector pTM1, pTM1/EGFP (Fig. 4a
).
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We first tested by immunoblotting whether we could express the full-length EGFPL construct in cells. The vTF7-3-infected HeLa cells expressing EGFP or EGFPL were lysed 12 h after transfection and the cell lysates were immunoblotted using anti-L antiserum and anti-GFP antibodies. The 275 kDa LEGFP-fusion protein and 27 kDa EGFP were detected (Fig. 4b); the fusion protein had the expected size when compared to unmodified L protein (Fig. 1a, b
). The autogene pCMV/T7-T7pol-driven T7 expression system in Vero E6 cells produced less of the fusion L protein products and the background reactivity of the anti-GFP antibodies was high (data not shown).
We then used indirect immunofluorescence to study the localization of LEGFP and EGFP in Vero E6 cells using the plasmid-driven T7 expression system and EGFPL and EGFP in HeLa cells using the vaccinia-driven T7 expression system. We studied co-localization with TULV N protein and various markers for cellular organelles. The N protein was expressed by co-transfection with pcDNA3/S. Cells were fixed, permeabilized and the proteins were visualized by indirect immunofluorescence. Transfection efficiencies were low (<10 % of cells transfected).
EGFP had a diffuse expression pattern in the cytoplasm and the nucleus as is characteristic for this protein (Fig. 5a). In contrast, LEGFP and EGFPL had a more punctated expression pattern characteristic of membrane-associated proteins. The EGFP-tagged L proteins were localized in the perinuclear region. The only cellular marker with which we found any co-localization was the Golgi protein GM130, but the distribution of the proteins differed, with GM130 having wider distribution in the Vero E6 cells (Fig. 5b
). LEGFP had the same localization when expressed in TULV-infected Vero E6 cells (data not shown), indicating that the presence of the other viral proteins does not alter the localization. Confocal microscopy of the EGFPL construct expressed in HeLa cells also showed partial co-localization with the GM130 marker and also in HeLa cells GM130 had wider distribution than the EGFPL fusion construct (Fig. 5c
). In HeLa cells expressing both EGFP-tagged L protein and TULV N protein, confocal microscopy showed that the proteins had distinct expression patterns, but there was also partial co-localization of the two proteins in the perinuclear region (Fig. 5d
).
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DISCUSSION |
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A single 250 kDa protein was detectable in TULV-infected cells and concentrated virus particles (Fig. 1). The antiserum also detected PUUV L protein, which has 85 % aa sequence identity with TULV L protein (Kukkonen et al., 1998
), and it was found to be of identical size. The hantavirus L RNA large ORF of more than 6400 bases is, therefore, used in its entirety to encode a single L protein as has been previously shown for other Bunyaviridae (Jin & Elliott, 1992
; Adkins et al., 1995
; Lukashevich et al., 1997
). The antiserum also recognized recombinant L protein expressed by using in vitro translation and by vTF7-3-driven transient expression in transfected cells (Fig. 4b
).
Hantaviruses grow to low titres in cell culture. We typically observe a peak level of only 0·1 f.f.u. per cell. Immunoblots of nucleocapsid protein and glycoproteins (Pensiero et al., 1988) and Northern hybridization blots of vRNAs (Vapalahti et al., 1996
) indicate that all of these virion components are abundant in infected cells. We show that the L protein, with a copy number of 104, is also abundant enough not to explain the low virus titres. The 3' termini of genomic RNAs contain deletions (Kukkonen et al., 1998
; Meyer & Schmaljohn, 2000
; Padula et al., 2002
) but vRNAs with deletions can be packaged into virions (Kukkonen et al., 1998
). Taken together, this points to inefficient assembly of the viral particles as the most probable cause of low virus titres.
With the new antiserum we were able to show hantavirus L protein to be membrane-associated in TULV-infected cells (Fig. 3) in addition to the N protein and the glycoproteins. The L protein was shown to be membrane-associated even in the absence of other viral proteins and viral RNA. Therefore, all the hantavirus structural proteins can independently interact with cellular components localizing the proteins on membranes. The L protein domain responsible for the membrane interaction and the cellular component with which it interacts remains to be determined. Our results obtained using membrane flotation indicate that the membrane association of the L protein can be inhibited by treatment with both 1 M NaCl and 15 mM Na2CO3 pH 11·5, indicating that the L protein is a peripheral membrane protein.
Having a viral RNA polymerase function on membranes could bring about the advantages of surface catalysis (Lyle et al., 2002). The freedom of motion of the RNA polymerase, RNA template and nucleotides is limited to two dimensions, substrate affinity is increased by binding of several RNA polymerases to the same RNA template, and, perhaps most importantly, reaction products, e.g. cRNA, are retained to be used in subsequent reactions, e.g. synthesis of vRNA (Lyle et al., 2002
). The membrane association of Tula virus L protein could be a result of viral RNA synthesis being sequestered within membrane structures. This would not only bring about the advantages of surface catalysis but also protect the virus from antiviral responses triggered by double-stranded RNA, as has been suggested for other RNA viruses (Schwartz et al., 2002
). Having RNA polymerases concentrated on membranes can also facilitate template switching and, thus, increase events of homologous recombination which have been observed also for hantaviruses (Plyusnin et al., 2002
).
To study the localization of the L protein in cells, we used autofluorescent fusion proteins having EGFP in either the N or C terminus (Fig. 4a). With EGFP in the N terminus it is possible to have prematurely terminated translation products having full EGFP and only partial L protein; therefore, we repeated the experiments with the LEGFP construct, even though the only EGFPL fusion protein band we could detect by immunoblotting was the full-length EGFPL (Fig. 4b
). Furthermore, by having both types of constructs we tried to minimize the probability of observing a misfolded protein caused by fusion with EGFP. However, it has even been possible to incorporate EGFP within the open reading frame of a viral RNA polymerase protein without abolishing its activity (Duprex et al., 2002
). Both of the fusion protein constructs localized in the perinuclear region (Fig. 5
), which has previously been found to be the localization site for the N protein of BCCV, Seoul and Puumala viruses (Ravkov & Compans, 2001
). We found that the LEGFP and EGFPL fusion proteins did have at least partial co-localization with the Golgi marker GM130 (Fig. 5
). However, the distribution of GM130 differed from those of the LEGFP and EGFPL fusion proteins. The L fusion protein also had an expression pattern distinct from that of the N protein (Fig. 5
), although the site of the L protein localization also had N protein. Based on these data, it is not yet possible to determine on what cellular structures the RNA polymerase complex is located.
It has been difficult to find any co-localization of the N protein with the Golgi-localized glycoproteins, even though these proteins must co-localize during virus assembly. For example, the Seoul virus G2 co-localizes with the Golgi markers, whereas the N protein does not but rather surrounds the Golgi (Kariwa et al., 2003). The glycoproteins are not required for RNA synthesis (Dunn et al., 1995
; Lopez et al., 1995
; Flick et al., 2003
) and our results, obtained by membrane flotation following detergent treatment, indicate that the glycoproteins are localized in lipid rafts whereas the L and N proteins are not. These data point to the site of RNA synthesis being distinct from the site of virus assembly.
Unlike positive-strand viruses, little is known about the localization of negative-strand RNA polymerases. Here, we provide the first evidence for the membrane association of an RNA polymerase of a negative-strand virus replicating in the cytoplasm. Thus, the membrane association of RNA synthesis can be a universal feature of RNA viruses. This awaits further investigation.
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ACKNOWLEDGEMENTS |
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Received 22 October 2003;
accepted 23 December 2003.
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