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1 Institute of Virology, University of Leipzig, Johannisallee 30, 04103 Leipzig, Germany
2 Department of Molecular and Medical Virology, Ruhr University Bochum, Bochum, Germany
Correspondence
Claudia Claus
claudia.claus{at}medizin.uni-leipzig.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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).
The rubella virion consists of a (+)-sense, single-stranded RNA genome enclosed in a quasispherical capsid and the lipid envelope, in which the two type I membrane glycoproteins, E2 and E1, are embedded as a heterodimeric spike complex. Within this complex, E2 is assumed to be hidden underneath E1 (Ho-Terry & Cohen, 1980). Katow & Sugiura (1988) have shown that E1 can bind to liposomes in the absence of E2 and is important for membrane fusion in the endosomal compartment. Additionally, the internal E1 hydrophobic domain was shown to be involved in cell–cell fusion (Yang et al., 1998). However, the role of E2 during viral entry remains to be determined.
The structural proteins are translated from the 24S subgenomic RNA as a polyprotein in the order NH2–capsid (C)–E2–E1–COOH. During translocation of the polyprotein into the endoplasmic reticulum (ER), C–E2 and E2–E1 cellular peptidase-mediated cleavages occur, leaving the hydrophobic E2 (SPE2) and E1 (SPE1) signal peptides attached to the C termini of C and E2, respectively (Frey, 1994). Due to an ER-retention signal that requires the E1 transmembrane (TM) and cytoplasmic (CT) domains (Hobman et al., 1997), the E1 protein, if expressed alone, accumulates in a post-ER/pre-Golgi compartment composed of tubular smooth membrane elements, supposedly representing amplified ER-exit sites (Hobman et al., 1998). E2 and E1 form disulfide-linked heterodimers in the ER, facilitating further transport to the Golgi apparatus (Baron & Forsell, 1991) where the heterodimeric complex itself is retained (Hobman et al., 1995), converting Golgi membranes into platforms of viral particle maturation and budding (Risco et al., 2003).
Maturation and release of RV particles depend on coexpression of E2 and E1, constraining their functional analysis. In this report, pseudotyped lentiviral vector particles were used to analyse E2- and E1-mediated entry separately from post-entry events. Viral vector pseudotypes comprise an envelope protein of a non-related virus and a replication-deficient lentiviral genome that usually contains a reporter gene. As the heterologous envelope glycoprotein determines receptor binding and membrane fusion, pseudotypes represent a valuable tool for the functional characterization of viral envelope glycoproteins.
A panel of plasmids encoding native and C-terminally modified E2 and E1 variants was used for pseudotyping of lentiviral vectors based on Simian immunodeficiency virus (SIV). Additionally, membrane fusion as one of the early steps of infection was analysed by a cell–cell fusion assay. The results presented here imply that both proteins are able to mediate cell entry and cell–cell fusion in the absence of the other. Coexpression of E2 and E1 with the capsid protein in cis was shown to enhance cell–cell fusion. For the first time, it was possible to analyse cell-entry functions of both proteins separately from each other, assigning not only E1, but also E2, distinct properties in viral entry.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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RV titration.
RV (strain M33) was titrated by plaque assay as described elsewhere (Hemphill et al., 1988). Briefly, serial dilutions of the virus were added to Vero cell monolayers (60 mm2 Petri dishes). After adsorption for 60 min at 37 °C, 5 ml agarose overlay medium (0.6 % agar, DMEM, 0.15 % sodium bicarbonate and 0.1 ng DEAE ml–1) was added. Dishes were incubated for 7 days at 37 °C, the agar overlay was removed and plaques were counted following staining with crystal violet (0.1 % in 3.7 % formaldehyde).
Plasmid constructs.
Generally, PCRs were performed with a total volume of 100 µl containing up to 250 ng template, 20 pmol of the respective primer, 2 mM MgCl2 and 5 U PlatinumTaq DNA polymerase (Invitrogen). The RV full-length cDNA clone Robo302 was used as template for the generation of native and truncated RV cDNA constructs. Robo302 and the vesicular stomatitis virus glycoprotein G (VSV G) expression plasmid pHIT-G (Schnell et al., 2000) were used as templates for the synthesis of chimeric RV cDNA constructs by overlap-extension PCR using equimolar amounts of the respective gel-purified PCR fragments. These constructs (Fig. 1) were subsequently cloned into the pcDNA3.1 plasmid (Invitrogen).
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Production of pseudotyped lentiviral particles.
A plasmid system based on SIV was used, comprising (i) the self-inactivating gene-transfer vector ViCG
BH, encoding enhanced green fluorescent protein (EGFP), and (ii) the gag–pol packaging construct Sgp2 (Schnell et al., 2000). Briefly, 293T/17 cells were cotransfected with equal amounts of each of the components of the lentiviral vector-packaging system. At 24 h post-transfection, fresh packaging medium (DMEM, 1 % FBS) was added. At 48 and 72 h post-transfection, medium containing vector particles was collected and clarified by both centrifugation for 10 min at 1500 g and filtration through 0.45 µm pore-size membranes. Supernatants containing SIV pseudotypes were concentrated 50-fold by filtration through Amicon Ultra-15 centrifuge filter devices (Millipore Corporation).
Vector titration.
Target cells were seeded into 96-well culture plates (4x104 cells per well) 24 h before infection. Tenfold serial dilutions of viral stocks were made in 200 µl (final volume) DMEM with 1 % FBS. Cells were incubated at 37 °C for 48 h. Vector titre was determined by end-point dilution, calculating the number of EGFP-expressing cells. Titres were expressed as transducing units (TU) (ml vector supernatant)–1.
Neutralization of pseudovirions.
RV pseudotyped vector particles were preincubated with serial dilutions of a human RV-seropositive serum or -seronegative control serum for 1 h at 37 °C. The human RV-seropositive serum was obtained from a convalescent patient. The preincubated pseudotype–serum mixture was subsequently used to transduce target cells in the presence of the same concentration of serum. Vector titres were determined 72 h post-infection. In order to assess the neutralization of wild-type RV infection, 50 p.f.u. RV strain M33 was incubated with the same dilutions of the human RV-seropositive serum as applied for the neutralization of pseudotype particles. The infectivity of wild-type RV was determined by plaque assay in the presence of the same concentration of serum.
Immunofluorescence assays.
For cell-surface localization, transfected 293T/17 cells grown on glass coverslips were fixed at 48 h post-transfection with 2 % (w/v) paraformaldehyde/PBS for 10 min at room temperature. Cells were incubated for 90 min at 37 °C simultaneously with a 1 : 200 dilution of the mouse monoclonal antibody to E2 (mAb-26-24; Viral Antigens Inc.) or E1 (M-1B9-IgG; Roche) and 50 µg ml–1 of fluorescein isothiocyanate (FITC)-labelled cholera toxin B subunit (ChX; Sigma). After washing with PBS, tetramethylrhodamine B isothiocyanate (TRITC)-conjugated rabbit anti-mouse immunoglobulins were applied for 45 min at 37 °C at a final dilution of 1 : 50. Coverslips were mounted on glass slides with Entellan (Merck). Cell preparations were analysed by using the 488 and 568 nm bands of argon and argon–krypton lasers, respectively, of a Leica TCS SP2 confocal laser-scanning microscope. Optical sections in the slices were taken in a two-channel mode (double labelling). To avoid cross-talk, dual images were recorded in sequential mode (Leica TCS Confocal Systems user manual). The lasers were programmed to scan over successive focal planes (0.2 µm intervals). Post-acquisition processing of digital images was performed with TCS and Adobe Photoshop 5.5 software with minimal alterations to contrast and background.
Low pH-dependent cell–cell fusion assay.
The fusogenic activity of RV envelope glycoproteins was assayed by monitoring polykaryon formation of transfected 293T/17 cells grown on glass coverslips as described elsewhere (Yang et al., 1998). Briefly, cells were incubated with low-pH buffer [Eagle's medium containing 10 mM MES (Sigma) and 10 mM HEPES (GIBCO) (pH 5.1)] for 15 min at 37 °C. Thereafter, cells were incubated for 4 h with DMEM and 1 % FBS at 37 °C. RV-mediated cell–cell fusion was determined for RV (strain M33)-infected Vero cells (m.o.i.=0.1). Fusion was induced 4 days post-infection. The degree of cell–cell fusion was analysed by using immunofluorescence analysis. Cells were fixed with 2 % (w/v) paraformaldehyde/PBS and stained with ChX as described above. Cells were permeabilized with ice-cold methanol for intracellular staining. The mouse monoclonal antibodies to E2 and E1 proteins were diluted 1 : 200 and that to the capsid protein (mAb-2-36; Viral Antigens Inc.) was diluted 1 : 100. Antibodies were washed off and cells were then incubated with TRITC-conjugated rabbit anti-mouse immunoglobulins. Nuclei were counterstained with Hoechst bisbenzamide 33258 fluorochrome stain (Sigma) at a concentration of 0.2 µg ml–1. Slides were examined with a Zeiss confocal laser-scanning microscope (LSM 510). The argon laser (488 nm) was used for FITC fluorescence. TRITC fluorescence was analysed with a helium–neon laser (543 nm) and a UV laser with an excitation wavelength of 364 nm was used for excitation of Hoechst 33285 fluorescence. Post-acquisition processing of digital images was performed with Zeiss and Adobe Photoshop 5.5 software with minimal alterations to contrast and background. Fusion was quantified by determination of the number of stained cells (ncell, positive immunofluorescence signal for the respective RV protein) and the number of nuclei present in these cells (nnuclei, stained with Hoechst dye). The fusion index fi was calculated by using the formula fi=[1–(ncell/nnuclei)] as described elsewhere (White et al., 1981). Fusion events in seven random x40 fields on coverslips from two independent experiments were quantified.
Cell–cell fusion activity of cell-free RV-like particles (fusion from without).
RV-like particles (RLPs) released from transfected 293T/17 cells were tested for their ability to induce cell–cell fusion from without. Glass coverslips of 293T/17 cells were cooled to 4 °C and washed with cold binding medium (DMEM, pH 6.5, 0.2 % BSA). The RLPs were allowed to attach to the cells for 90 min at 4 °C. The supernatant was removed and cell monolayers were washed with cold binding medium. After that, cell monolayers were treated with pH 5.0 fusion medium for 15 min in a 37 °C incubator and returned to growth medium for a 4 h period of incubation at 37 °C. Cells were fixed with 2 % (w/v) paraformaldehyde/PBS and labelled with ChX. Nuclei were counterstained with Hoechst 33258. Fusion was quantified as described above.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Pseudotyping of SIV vector particles was not established in 293T/17 cells expressing E2 or E1 and SIV vector components (Table 1).
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CT), and second, the CT domain of E1 was replaced by the corresponding region of the VSV G protein, singly or in conjunction with the TM domain (E1GCT and E1GTMCT, respectively; Fig. 1
). All C-terminally modified E1 proteins were able to assemble into infectious pseudotyped vector particles with similar functional vector titres (Table 1). Pseudotype titre was not increased by coexpression of C-terminally modified E1 variants with E2 or the TM and CT domains of E2. Among the E1 C-terminally modified constructs, the E1GTMCT protein was used in further experiments for pseudotyping of SIV vector particles. It was conceivable to exchange both the TM and CT domain as dominant ER-retention signals. The most permissive cell lines for RV infection are Vero and BHK-21. The reason(s) for the non-permissiveness of several mammalian cell lines for a productive RV infection are not known. The transduction efficiency did not vary remarkably between cell lines that are permissive (Vero and BHK-21 cell lines) and cell lines that are non-permissive (293T/17, 293A and HeLa) for a productive RV infection (data not shown). The 293T/17 cell line was used for subsequent experiments as transduction efficiency and reporter-gene expression were slightly higher in this cell line.
Based on the results of pseudotyping of SIV vector particles with C-terminally modified E1 variants, the E2 CT domain was substituted by the CT domain of VSV G. No difference in functional vector titres of SIV vectors pseudotyped with either E2GCT or E1GTMCT was observed (Table 1).
Neutralization of RV pseudotype transduction by human RV-seropositive serum
In order to confirm the specificity of E2GCT- and E1GTMCT-mediated transduction of 293T/17 cells, RV-specific serum from a convalescent patient was tested for the ability to neutralize pseudotype transduction. RV Env and VSV G pseudotypes were incubated with either a human RV-seropositive or -seronegative serum. As a positive control, the same dilutions of the human RV-seropositive serum were used to neutralize wild-type RV (strain M33) infection. An almost 100 % reduction of infectivity was achieved with a 1 : 50 dilution of the human RV-seropositive serum (Fig. 2).
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). With a 1 : 50 serum dilution, transduction by SIV (E1GTMCT) pseudotypes was reduced by >95 % in comparison to the untreated control (Fig. 2a), whereas SIV (E2GCT) pseudotype transduction was reduced by 85 % (Fig. 2b). The ability of different dilutions of the human RV-seropositive serum to neutralize the transduction by SIV (E1GTMCT) pseudotypes was similar to the neutralization of wild-type RV infection. The human RV-seropositive serum did not neutralize VSV G-mediated transduction of target cells. Thus, transduction of cells with SIV (RV Env) particles was specific and dependent on the presence of the RV glycoprotein.
Analysis of cell-surface expression of RV envelope glycoproteins
The degree of cell-surface expression of RV envelope glycoproteins was studied by immunofluorescence analysis in order to determine its influence on pseudotype formation. As illustrated in Fig. 3, E1-expressing cells that were double-labelled with the monoclonal antibody to E1 (red) and FITC-labelled ChX bound to the lipid-raft marker ganglioside GM1 (green) showed no E1 surface expression, as was expected (Fig. 3a). Upon coexpression with the E2 TM and CT domains, E1 (Fig. 3b) and E1GTMCT (Fig. 3c) were translocated efficiently to the cell surface. Native and chimeric E1 proteins were stained as isolated patches outside plasma-membrane regions with GM1 staining [Fig. 3b (right panel) and 3c (right panel)]. The surface expression of E1 upon coexpression with E2 TM and CT domains was also confirmed by flow-cytometric analysis (data not shown). Coexpression of E1 with E2 results in E1 surface expression. The degree of E1 surface expression upon coexpression with E2 (Fig. 3d) was comparable to the coexpression with only the TM and CT domains of E2, but E1 distribution within the plasma membrane was more homogeneous. E2 is expressed at the cell surface in the absence of E1. E2 was distributed evenly within the plasma membrane and localized in regions that were positive for GM1 staining. Colocalization of E2 (Fig. 3e) and E2GCT (Fig. 3f) with GM1 was observed by the appearance of a yellow fluorescent signal in the overlay images, irrespective of a low [Fig. 3e (right panel)] or high [Fig. 3f (right panel)] level of E2 surface staining.
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) or E1 (Fig. 4b). In mock-transfected cells and cells expressing the RV capsid protein, fusion of neighbouring cells was never observed. Syncytium formation was also detectable in cells expressing the E2 protein (Fig. 4c). The largest number of nuclei within syncytia was detectable upon coexpression of E2 and E1 with the capsid protein (Fig. 4d). The E1 (Fig. 4a, b), E2 (Fig. 4c) and capsid (Fig. 4d) proteins localized mainly to the centre of the syncytium in a perinuclear region.
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. The fi obtained for E1 was similar to that for E2. Upon coexpression of E2 and E1, an additive increase in membrane-fusion activity was observed. The coexpression of E2 with the capsid protein in the absence of the E1 ectodomain (CE2E1TMCT) resulted in doubling of fi compared with transfecting a plasmid encoding E2 alone. The coexpression of the N-terminal part of the E1 ectodomain (including the hydrophobic fusion domain) with capsid and E2 resulted in an fi similar to that obtained for CE2E1TMCT.
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With an RLP cell–cell fusion assay from without (corresponding to fusion induction by cell-free virus or virus-like particles), the question was addressed whether RLP secretion is the cause of the observed increase in the fi of E2E1 upon expression with C in cis. However, fusion from without was inefficient, with an fi of 0.03±0.01.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Efficient incorporation of foreign glycoproteins such as VSV G into retroviral pseudotypes was demonstrated for several viruses. However, coexpression of a heterologous envelope glycoprotein with retroviral vector components does not necessarily result in high-titre vector production. Pseudotyping of vector particles by the envelope glycoproteins of Hantaan and La Crosse viruses yielded titres of 103–104 TU ml–1 after 80-fold concentration (Ma et al., 1999). The maturation and assembly process of RV structural proteins is similar to that of these envelope glycoproteins, as reflected by low vector yield. Additionally, several viral envelope glycoproteins are only incorporated following modification of their CT domain (Höhne et al., 1999; Christodoulopoulos & Cannon, 2001; Kobayashi et al., 2003). Several models have been developed to explain the mechanisms underlying the process of pseudotyping and will be discussed in the context of the production of low-titre RV pseudotypes.
The passive model of envelope glycoprotein incorporation suggests that high surface expression at the site of viral budding is required. Although E1 surface expression was not detectable by immunofluorescence analysis (Fig. 3), E1-transfected cells formed syncytia, suggesting that E1 is, at least in trace amounts, present at the cell surface. The E1 protein is functionally incorporated in SIV pseudotypes as a result of E1 processing in the absence of E2. The E1 protein, which was fused to the EGFP protein at its C terminus, localized partly to the cis-Golgi and even to the trans-Golgi network (Ojala et al., 2004). The E2 TM and CT domains were reported to be important for heterodimerization preceding E2E1 transport from the ER to the Golgi (Garbutt et al., 1999). Localization to the Golgi shows correct protein processing and enables surface transport. Based on this, the E2TMCTE1 construct was used successfully to increase E1 surface expression. For the first time, it was possible to demonstrate that the E2 TM and CT domains alone are sufficient to overcome E1 ER retention. However, vector titre was not increased upon coexpression of E1GTMCT with E2 TM and CT domains. Although E2 is transported to the Golgi and the plasma membrane when expressed in the absence of E1 (Baron et al., 1992), SIV (E2GCT) titres were similar to that of SIV (E1GTMCT). Taken together, the degree of cell-surface expression of RV envelope glycoprotein variants or the localization within certain plasma-membrane microdomains does not seem to influence the outcome of SIV pseudotyping. Consistently, the mutation of the ER-retention signal of the human foamy virus envelope protein does not result in higher infectivity of pseudotyped murine leukemia virus (MuLV) particles, albeit increasing cell-surface expression (Lindemann et al., 1997). Human immunodeficiency virus was reported to form pseudotypes with hepatitis C virus envelope glycoproteins, despite ER retention of both proteins (Hsu et al., 2003).
A recent publication proposed a model in which the interactions between the envelope glycoproteins and the core components (Gag protein) do not occur at the plasma membrane, but in intracellular compartments (Sandrin et al., 2004). It is conceivable that the E2 and E1 CT domains contain an intracellular-transport signal inconsistent with the incorporation in SIV particles and that the VSV G CT domain redirects E2 and E1 transport, enabling pseudotype formation. However, there are probably additional sorting signals within the E1 ectodomain, as upon removal of the TM and CT domains, E1 is only secreted upon coexpression with E2 (Hobman et al., 1994).
The ability of the human RV-seropositive serum to neutralize transduction by SIV (E1) pseudotypes was comparable to neutralization of wild-type RV infection, as virion properties are mainly mediated by E1. The poor immune response to E2 (Nedeljkovic et al., 1999) might explain the reduced neutralization of SIV (E2) pseudotype transduction. The degree of E2 antibody production with neutralizing ability is unknown. However, as the immune response against E2 is lower than that against E1 as the main target for induction of an immune response (Katow & Sugiura, 1985), the level of neutralizing antibodies against E2 is generally lower than that against E1. This probably accounts for the difference in pseudotype neutralization.
Although the titres of vector particles pseudotyped with RV envelope glycoproteins were modest, they were sufficient to analyse the capacity of both proteins to promote infectious entry. The transduction by SIV (RV Env) particles showed that both envelope glycoproteins can mediate viral entry. There are other examples of heterodimeric envelope glycoproteins that can independently mediate infectivity upon pseudotyped vector particles. It was demonstrated that the hepatitis C virus envelope glycoproteins can assemble independently into infectious MuLV vectors (Bartosch et al., 2003). VSV pseudotypes bearing individual hepatitis C envelope proteins (E1–G and E2–G) were shown to be infectious, although infectivity was highest upon coexpression of both proteins (Matsuura et al., 2001). Pseudotyping of VSV particles with hepatitis B surface glycoproteins (HBs) revealed that all three HBs proteins assemble into infectious VSV pseudotypes (Saha et al., 2005).
The results presented here point to the capsid protein as an important factor influencing E2- and E1-mediated membrane fusion. The E2 signal peptide at the C terminus of the capsid protein mediates membrane anchorage and is required for virion assembly and secretion (Law et al., 2001). Only coexpression in cis results in localization of C to the Golgi complex together with E2 and E1 (Baron et al., 1992). Hence, C is only able to interact with E2 and E1 upon coexpression in cis, as emphasized by the observed increase in the fusion index. It is conceivable that the capsid protein induces conformational changes within E2 and E1 as a prerequisite to reach the complete fusogenic state. Additionally, the capsid protein could either stabilize the envelope glycoproteins, which was also reported for the Sindbis virus capsid protein (Lee et al., 1994), or their interactions. The capsid protein could exert this stabilizing effect on cell surface-expressed E2 and E1, as well as in rubella virions during virus entry. It cannot be ruled out that the capsid protein has a supportive function in cell-surface expression of E2 and E1, as was shown for Semliki Forest virus, by a block of proteolysis of surface-expressed envelope glycoproteins (Zhao & Garoff, 1992). This stabilization would result in a higher fi, as cell–cell fusion is influenced by the density of the fusion protein on the cell surface. However, in contrast to RV, alphaviruses bud from the plasma membrane and in trans coexpression of the Sindbis virus E1 and E2 envelope glycoproteins with the capsid protein leads to particle formation (Sanz et al., 2003). Thus, it seems more likely that the RV capsid protein supports E2 and E1 maturation or stabilizes either E2 and E1 or their interactions during intracellular transport to the cell surface. In agreement with our results, Qiu et al. (2000) reported an influence of C on E2E1 fusogenicity, supposedly by stabilizing E2E1 interactions.
The data presented here point not only to the expression of the three structural proteins in cis, but also to the presence of the C terminus of E1, as determinants for the degree of membrane fusion. This is in accordance with published data that the E1 TM and CT domains are important for transport of RLPs between the Golgi apparatus and the plasma membrane and, finally, for virus particle release (Garbutt et al., 1999). The requirement of the homologous capsid protein for E2E1 fusion may give an explanation for the low pseudotype vector yield.
This is the first report on lentiviral pseudotypes bearing RV envelope glycoproteins. With the results presented here, distinct biological functions in virus entry were assigned to E2. So far, E2 was shown to possess strain-specific epitopes (Dorsett et al., 1985) and at least one domain with neutralizing ability that is accessible on the virion surface (Green & Dorsett, 1986), pointing towards a potential role in the entry process. A potential fusion peptide of E2 has been identified by computer analysis (Blobel et al., 1992). Further studies are required to gain more information on the functional dependence of the structural proteins on each other. RV assembly and entry seem to be tightly regulated processes. Analysis of the functional interaction of the structural proteins will add to an understanding of the overall mechanism of viral pathogenesis.
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ACKNOWLEDGEMENTS |
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Received 16 March 2006;
accepted 16 June 2006.
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, Anti-RV serum, RV strain M33; 
, control serum, SIV [E1GTMCT (a) or E2GCT (b)]; 
, anti-RV serum, SIV (VSV G); 
, anti-RV serum, SIV [E1GTMCT (a) or E2GCT (b)].
