Institut für Virologie, Stiftung Tierärztliche Hochschule Hannover, Bünteweg 17, D-30559 Hannover, Germany
Correspondence
Gert Zimmer
gert.zimmer{at}tiho-hannover.de
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ABSTRACT |
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Details of primers used in construction of expression plasmids are available as supplementary material in JGV Online.
The GenBank/EMBL/DDBJ accession number for the HEF gene sequence of influenza C virus C/Johannesburg/1/66 is AJ872181.
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INTRODUCTION |
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In this study, the capacity of HEF to mediate infection of a heterologous virus in the absence of any other influenza C virus proteins was investigated. Incorporation of native and chimeric HEF into recombinant replication-incompetent vesicular stomatitis virus (VSV) that lacked the VSV-G glycoprotein gene was studied. With native HEF, infectious VSV pseudotypes were produced that required trypsin for activation and had cell tropism similar to that of influenza C virus. Unlike VSV-G glycoprotein, HEF allowed apical infection of polarized epithelial cells. Pseudotyping of viral vectors with HEF might be advantageous for the selective targeting of polarized or non-polarized cells expressing 9-O-acetylated sialic acids. In addition, pseudotyping with HEF will be a powerful tool to investigate HEF functional domains.
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METHODS |
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Antibodies.
A multiple antigenic peptide was synthesized by anchoring residues 121 (MSSLKKILGLKGKGKKSKKLG) of the VSV matrix protein onto an immunogenically inert core molecule of radially branching lysine dendrites. The antigen was repeatedly applied to rats by subcutaneous injections at 3-weekly intervals. The animals were bled and serum was prepared by centrifugation of the coagulated blood. Polyclonal antisera were produced by immunization of rabbits with sucrose-gradient-purified influenza C virus or VSV. Hybridomas producing the monoclonal antibodies I-14 and I-1 (Lefrancois & Lyles, 1982a, b
) were kindly provided by Volker ter Meulen (Würzburg, Germany).
Oligonucleotides.
Details of primers used in construction of expression plasmids are available in a supplementary table in JGV Online.
Construction of expression plasmids.
The G protein gene of VSV (strain Indiana) was excised from the pTM1-VSVG plasmid (Köhl et al., 2004) using EcoRI and XhoI endonucleases and ligated into plasmid pCDNA3.1 (Invitrogen) to give pCDNA3.1-VSVG. For cloning of the VSV-G gene into the pGeneC vector (Invitrogen), the gene was amplified from pTM1-VSVG by PCR with oligonucleotides VSVG-S and VSVG-AS. Taking advantage of the BamHI and EcoRI restriction sites included in the primers, the PCR product was inserted into pGeneC to give pGeneC-VSVG. The total open reading frame (ORF) of G was sequenced and found to be identical to the published sequence (GenBank/EMBL/DDBJ accession no. NC_001560). For cloning of the influenza C virus (JHB/1/66) HEF gene, total RNA was prepared from infected MDCK-I cells and reversed transcribed. The ORF of HEF was amplified from the cDNA by PCR using oligonucleotides HEF-S and HEF-AS. The PCR product was treated with KpnI and XhoI endonucleases and ligated into plasmid pCDNA3.1(+) (Invitrogen) to give pCDNA3.1-HEF. The DNA sequence of three clones was determined and compared with the published sequence (GenBank/EMBL/DDBJ accession no. M17868). A new entry in the EMBL nucleotide sequence database was created (GenBank/EMBL/DDBJ accession no. AJ872181) to account for the point mutations detected.
Amino acid exchanges T284I, T284L and T286I were introduced into pCDNA3.1-HEF by an overlapping PCR technique (Schlender et al., 2003; Köhl et al., 2004
) using oligonucleotides T284I-S, T284I-AS, T284L/T286I-S and T284L/T286I-AS. The PCR product was digested with BamHI and PflMI and this fragment was used to replace the corresponding region in pCDNA3.1-HEF. The DNA sequence of the cloned fragment was determined to verify the nucleotide exchanges. An overlapping PCR technique was also used to generate chimeric glycoprotein genes. HEF gene segments were amplified from pCDNA3.1-HEF or from pCDNA3.1-HEF(T284I) using the HEF-S primer in combination with either HEFG(T)-AS, HEFG(TM)-AS, HEFG(STM)-AS or HEFCD4-AS. To amplify regions of the VSV-G or CD4 cDNA, oligonucleotides HEFG(T)-S, HEFG(TM)-S, HEFG(STM)-S and HEFCD4-S were used in combination with VSVG-AS(XhoI) or CD4-AS, respectively. Hybridization of the PCR fragments was performed as described previously (Schlender et al., 2003
; Köhl et al., 2004
). The PflMIXhoI restriction fragments of the hybrid genes were used to replace the corresponding segment in pCDNA3.1-HEF.
Generation of a stable cell line for conditional expression of VSV-G.
BHK-21 cells were co-transfected with the plasmids pSwitch (Invitrogen) and pGeneC-VSVG and grown for 14 days in selection medium containing hygromycin B (500 µg ml1) and zeocin (1 mg ml1). Cell clones were isolated by limiting dilution and analysed for mifepristone-induced VSV-G expression by immunofluorescence and Western blotting. One cell clone, BHK-G43, efficiently supported replication of VSV*-G-G (the asterisk denotes the eGFP gene; see below) and was used throughout the study.
Immunofluorescence analysis and flow cytometry.
BHK-21 cells grown on 12-mm-diameter coverslips (2·5x105 cells) were transfected with 1 µg plasmid DNA and 2 µl Lipofectamine 2000 transfection reagent. At 20 h post-transfection, cells were fixed with 3 % paraformaldehyde and permeabilized with 0·2 % Triton X-100 if intracellular antigen was to be detected. Cells were incubated first with rabbit polyclonal anti-influenza C virus serum (1 : 2000) and then with FITC-conjugated goat anti-rabbit IgG serum (1 : 500; Sigma). For flow cytometric analysis, BHK-21 cells grown in 24-well dishes (3x105 cells per well) were transfected as described above. At 20 h post-transfection, adherent cells were stained at 4 °C with the antibodies described above and finally suspended in PBS. Cells were analysed using a Beckman Coulter Epics XL cytometer equipped with Expo 32 ADC software.
Generation of VSV pseudotypes.
For generation of replication-incompetent recombinant VSV, a previously published strategy (Takada et al., 1997) was followed. The gene for the enhanced green fluorescent protein (eGFP) was cloned into pVSV-XN2 (Schnell et al., 1996b
) taking advantage of the single restriction sites MluI and NheI. In this way, the VSV-G gene of the genome was replaced and the resulting vector was therefore designated pVSV*-
G (the asterisk denotes the eGFP gene). BSR-T7/5 cells were transfected with pVSV*-
G, pCDNA3.1-VSVG and three plasmids driving the expression of the viral polymerase complex (Schnell et al., 1996b
). Two days after transfection, VSV*-
G-G was recovered from the cell culture supernatant and propagated on mifepristone-treated BHK-G43 cells.
For generation of HEF-pseudotyped VSV (VSV*-G-HEF), BHK-21 cells grown in 35-mm-diameter dishes were transfected with 5 µg HEF or VSV-G expression plasmid and 10 µl Lipofectamine 2000 transfection reagent. At 20 h post-transfection, cells were inoculated with VSV*-
G-G (10 p.f.u. per cell) for 1 h at 37 °C and then with medium containing a polyclonal rabbit anti-VSV serum to neutralize the helper virus. Following incubation for 20 h at 37 °C in the absence of FCS, the cell culture supernatant was harvested, clarified by low-speed centrifugation and treated with 5 µg acetylated trypsin (Sigma) ml1 for 1 h at 37 °C to activate the HEF glycoprotein. The reaction was stopped by adding FCS (1 % final concentration). VSV*-
G-HEF was titrated on MDCK-I cells grown on 12-mm coverslips by inoculating cells with serial dilutions of the cell culture supernatant for 90 min at 37 °C. At 16 h post-infection, cells were fixed with paraformaldehyde and the number of eGFP-positive cells was determined.
Pseudotype virus infection.
To analyse the cell tropism of VSV*-G-HEF, cell lines were seeded on 12-mm coverslips (3x105 cells) and infected with 105 infectious units of pseudotype virus in 250 µl medium for 90 min at 37 °C. In some experiments, BHK-21 and U373 cells were pretreated with 250 µl affinity-purified neuraminidase from Clostridium perfringens (1 U ml1; Sigma) for 60 min at 37 °C, prior to inoculation with 250 µl medium containing 105 infectious units of pseudotype virus for 60 min at 37 °C. Cells were then incubated for 30 min at 37 °C with anti-FluC or anti-VSV serum. At 16 h post-infection, cells were fixed with paraformaldehyde and analysed by fluorescence microscopy to monitor eGFP expression. For analysis of polarized virus entry, MDCK cells were seeded on Falcon cell culture inserts containing porous (1 µm), 6·5-mm-diameter membranes (Becton Dickinson) and maintained for 3 days. Trypsin-activated pseudotype viruses were concentrated by ultracentrifugation and 2x106 infectious units were inoculated for 5 h at 37 °C with either the apical or the basolateral site of the filter. eGFP reporter expression was detected 16 h post-infection. For virus neutralization, serial dilutions (125 µl) of anti-VSV or anti-FluC serum were incubated for 30 min at room temperature with 125 µl of either VSV*-
G-HEF (106 infectious units ml1) or trypsin-activated VSV*-
G-HEF (4x105 infectious units ml1), prior to infection of BHK-21 or MDCK-I cells seeded in 24-well dishes (2x105 cells per well). The ratio of eGFP-positive cells was determined by flow cytometry 16 h post-infection. Virus that was not pre-incubated with antiserum served as a control; its titre was set to 100 %.
Analysis of VSV particles.
VSV pseudotypes from the cell culture supernatant of two 35-mm-diameter dishes were pelleted through a 25 % (w/w) sucrose cushion by ultracentrifugation (105 000 g, 60 min, 4 °C). The pelleted particles were dissolved in 100 µl 2x SDS sample buffer. Solubilized proteins (10 µl samples) were separated by 12 % SDS-PAGE under non-reducing conditions and transferred to nitrocellulose by semi-dry blotting. The nitrocellulose membranes were incubated for 60 min at 4 °C with either a rat monospecific antiserum directed to VSV-M (1 : 2000), a rabbit polyclonal anti-influenza C virus serum (1 : 2500) or a mouse monoclonal antibody (I-14) directed towards VSV-G (1 : 100). The blots were washed and subsequently incubated with biotinylated secondary antibodies that were specific for the IgG fraction of the respective species (1 : 1000; Sigma). Finally, the blots were incubated for 60 min with a streptavidinperoxidase complex (1 : 1000; Amersham Biosciences) and the antigens were visualized by enhanced chemiluminescence (Roche Diagnostics).
Analysis of HEF esterase activity.
BHK-21 cells grown in 35-mm-diameter dishes (8x105 cells per well) were transfected with 5 µg HEF expression plasmid and 10 µl Lipofectamine 2000 transfection reagent. Twenty hours after transfection, cells were lysed and HEF was immunoprecipitated with an antiserum directed to influenza C virus according to published procedures (Zimmer et al., 2001). The immunoprecipitates were run on a 10 % SDS-PAGE gel and blotted to nitrocellulose membranes. Esterase activity was detected as described previously (Döll et al., 1993
). For detection of HEF antigen, cell lysates were analysed by Western blotting as described above.
Haemadsorption.
Freshly prepared chicken erythrocytes were labelled with octadecyl rhodamine B chloride (R18; Molecular Probes) as reported previously (Fischer et al., 1998). The R18-labelled chicken erythrocytes were suspended in ice-cold PBS to obtain a 0·2 % cell suspension and incubated for 60 min at 4 °C with BHK-21 cells grown in 24-well dishes (2x105 cells per well) that had been transfected with HEF expression plasmids (1 µg per well). The cells were rinsed several times with ice-cold PBS and bound erythrocytes were visualized by fluorescence microscopy.
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RESULTS |
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Incorporation of HEF glycoproteins into VSV particles
For generation of VSV pseudotypes, a recombinant replication-incompetent VSV was used with the G gene replaced by the gene encoding eGFP (Takada et al., 1997). VSV-G protein was provided in trans and the resulting virus, VSV*-
G-G, was propagated on a transgenic helper cell line expressing VSV-G after induction by mifepristone. VSV*-
G-G was used to infect BHK-21 cells transiently expressing unmodified or chimeric HEF glycoproteins. The virus particles released into the cell culture supernatant during the following 20 h were pelleted through a sucrose cushion by ultracentrifugation and analysed by Western blotting (Fig. 4a
). Infection of mock-transfected BHK-21 cells with VSV*-
G-G resulted in the release of virus particles devoid of any viral glycoprotein: the sample reacted with a monospecific antibody directed to the matrix protein of VSV, but not with an antibody directed to influenza C virus or an antibody to VSV-G (Fig. 4a
, lane 2). When cells were transfected with HEF cDNA without subsequent infection by VSV*-
G-G, release of some HEF antigen into the cell culture supernatant was observed (Fig. 4a
, lane 3). However, when HEF-transfected cells were infected with VSV*-
G-G, HEF release into the cell culture supernatant was much more efficient, indicating that VSV particles containing HEF had been formed (Fig. 4a
, lane 4). Likewise, the three HEF chimeras containing VSV-G sequences at the C terminus were incorporated into VSV particles (Fig. 4a
, lanes 57). HEF glycoprotein appeared as a 100 kDa band under non-reducing conditions, indicating that the pseudotypes contain predominantly the proteolytically unprocessed precursor HEF0 (Herrler et al., 1979
). However, when the cell culture supernatant was treated with trypsin (5 µg ml1) and then collected by ultracentrifugation, Western blot analysis revealed that most HEF0 had been proteolytically cleaved, as indicated by the characteristic 80 kDa band representing HEF1,2 (Fig. 4b
). Like authentic HEF, the mutant and chimeric HEF glycoproteins were cleaved by trypsin, giving an 80 kDa band. Trypsin-mediated degradation, which is sometimes observed with misfolded proteins (Zimmer et al., 2001
), was not detected. The low level of HEFCD4 found in VSV particles (Fig. 4b
) was also observed in untreated samples (data not shown), indicating that this chimeric glycoprotein is not incorporated with the same efficiency as the other constructs.
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DISCUSSION |
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VSV pseudotypes have proven to be of value for the characterization of envelope glycoproteins from several viruses including Ebola virus (Takada et al., 1997; Ito et al., 2001
), measles virus (Tatsuo et al., 2000
), hepatitis C virus (Meyer et al., 2000
; Matsuura et al., 2001
; Burioni et al., 2002
; Lagging et al., 1998
; Beyene et al., 2004
), bunyaviruses (Ogino et al., 2003
), human T cell leukaemia virus (Okuma et al., 2001
) and Borna disease virus (Perez et al., 2001
). In this study, recombinant replication-incompetent VSV were pseudotyped to characterize HEF, the major spike protein of influenza C virus. It was demonstrated that native HEF is efficiently incorporated into VSV particles resulting in infectious pseudotypes. This finding indicates that the shortness of the HEF cytoplasmic domain, which is predicted to consist of only three amino acids, is not critical for glycoprotein uptake. In contrast, the homotypic VSV-G protein has been shown to lose its capacity to complement a VSV mutant with a temperature-sensitive VSV-G protein when the cytoplasmic domain of 29 amino acids was shortened to three amino acids (Whitt et al., 1989
). Using VSV-G proteins with truncated cytoplasmic domains and cytoplasmic domains from heterologous proteins, Schnell et al. (1998)
reasoned that a non-specific glycoprotein cytoplasmic domain sequence of between one and nine amino acids is required to drive efficient budding of VSV. Our study shows that, at least in the case of HEF, a cytoplasmic domain of three amino acids is sufficient for efficient assembly of VSV. Uptake of HEF was not improved if the HEF cytoplasmic domain was replaced by the corresponding VSV-G domain. Likewise, chimeric HEF glycoprotein containing the VSV-G transmembrane and cytoplasmic domains was incorporated into VSV particles with the same efficiency as native HEF. These results confirm previous studies showing that VSV-G does not contain any specific sequences in its C-terminal domain that specify glycoprotein incorporation (Robison & Whitt, 2000
; Schnell et al., 1996a
). Nevertheless, VSV-G was shown to be incorporated into VSV particles with significantly higher efficiency than any foreign viral glycoprotein. An explanation for this phenomenon was provided by a study showing that the membrane proximal stem region of VSV-G confers efficient virus assembly (Robison & Whitt, 2000
). It has been postulated that the VSV-G stem region induces membrane curvature at sites where budding occurs (Robison & Whitt, 2000
). However, incorporation of chimeric HEF was not significantly improved when this VSV-G stem domain was introduced. It is speculated that the HEF stem region per se meets the conformational requirements for induction of membrane curvature, so that insertion of the VSV-G stem region did not further improve glycoprotein uptake. Previous work has shown that the cellular protein CD4 or chimeric VSV-G containing the CD4 cytoplasmic domain are efficiently incorporated into recombinant VSV (Schnell et al., 1996a
, 1998
). A different result was obtained when the CD4 cytoplasmic domain was linked to HEF (HEFCD4); this glycoprotein was incorporated into VSV particles with lower efficiency than native HEF or either of the HEF/VSV-G chimeras. The reason for this phenomenon is not exactly clear. It may be that HEFCD4 adopts a different conformation that is suboptimal for uptake into virus particles.
A striking result of our studies is that chimeric HEF glycoproteins containing C-terminal domains from the VSV-G glycoprotein were efficiently transported to the cell surface and incorporated into VSV particles but, unlike authentic HEF, did not mediate efficient infection of the pseudotypes. Loss of infectivity was also observed when the cytoplasmic domain of the CD4 molecule was fused to HEF indicating that the effect does not depend on the sequence of the cytoplasmic tail. Our finding that the chimeric HEF glycoproteins tested positive for esterase and receptor-binding activities argues for the possibility that the fusion activity of these glycoproteins might not be functional. Indeed, observations pointing in this direction have been made with other viral fusion proteins, in which truncations or elongations of the respective cytoplasmic domains were shown to have profound effects on fusion activity (Bagai & Lamb, 1996; Ohuchi et al., 1998
; Tong et al., 2002
).
Our finding that native HEF is able to mediate infection of VSV pseudotypes implies that, to perform this function, HEF does not rely on the assistance of CM2, the putative ion channel protein of influenza C virus (Hongo et al., 2004). Since HEF is proteolytically activated by trypsin-like proteases, cleavage does not occur before the glycoprotein reaches the cell surface. Therefore, HEF passes the acidic Golgi compartment without the risk of early low-pH-triggered conformational change. CM2 might be involved in entry of influenza C virus by facilitating dissociation of the matrix protein and the ribonucleoprotein complex. It appears that this activity is not relevant in the context of a VSV pseudotype infection. However, pseudotyping of VSV might be a useful system for further characterization of HEF. Questions which can now be addressed are the role of the esterase activity in virus entry, the function of post-translational modifications and the effects of mutations on receptor affinity and cell tropism.
The present study suggests that the HEF glycoprotein of influenza C virus might be an interesting tool for pseudotyping viral vectors. In particular, the ability of HEF to mediate apical infection of polarized epithelial cells is an important aspect, as many viral vector systems commonly used in gene therapy, including retroviruses (Wang et al., 1998, 2002
), adenoviruses (Zabner et al., 1997
; Pickles et al., 1998
; Kitson et al., 1999
; Walters et al., 1999
), adeno-associated virus (Duan et al., 1998
; Bals et al., 1999
) and vaccinia virus (Rodriguez et al., 1991
), have been recognized to be rather ineffective in transduction of polarized epithelial cells from the apical side. Future studies will show whether it is feasible to use HEF for pseudotyping of retroviral and lentiviral vectors. The selective binding of HEF to glycoconjugates containing 9-O-acetylated sialic acids is also an interesting aspect, as 9-O-acetylation has been shown to be a tissue-specific and developmentally regulated modification of sialic acids (Varki, 1992
; Herrler et al., 1987
). The use of HEF for pseudotyping of viral vectors might therefore allow specific cell subsets to be targeted.
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ACKNOWLEDGEMENTS |
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Received 26 November 2004;
accepted 19 January 2005.
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