Functional comparison of the two gene products of Thogoto virus segment 6

Kathrin Hagmaier1,{dagger}, Hans R. Gelderblom2 and Georg Kochs1

1 Abteilung Virologie, Institut für Medizinische Mikrobiologie und Hygiene, Universität Freiburg, D-79008 Freiburg, Germany
2 Robert Koch-Institut, Berlin, Germany

Correspondence
Georg Kochs
georg.kochs{at}uniklinik-freiburg.de


   ABSTRACT
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The sixth genomic segment of Thogoto virus (THOV) encodes two proteins, the viral matrix protein (M) and an accessory protein with an interferon (IFN)-antagonistic function named ML. M and ML are shown in this study to be structural components of the virion. Using an in vivo system based on the reconstitution of functional THOV ribonucleoprotein complexes from cloned cDNAs, it was demonstrated that M has an inhibitory effect on the viral RNA-dependent RNA polymerase (RdRP) and is essential for the formation of virus-like particles (VLPs). The functional domain responsible for the regulation of RdRP activity resides within the C-terminal half of M, while full-length M protein is required for VLP formation. The ML protein cannot complement M with respect to either RdRP downregulation or particle formation, although it is identical to M apart from a 38 aa extension at the C terminus. In contrast, ML, but not M, is able to prevent the induction of IFN-{beta} by double-stranded RNA. This function is contained within the C-terminal half of ML. These data suggest major structural differences between M and ML that could explain the different activities of the two proteins.

{dagger}Present address: Centre for Biomolecular Sciences, University of St Andrews, St Andrews, UK.


   INTRODUCTION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The family Orthomyxoviridae consists of three genera of influenza viruses, the genus Isavirus, whose only representative so far is Infectious salmon anemia virus (ISAV; Clouthier et al., 2002), and the genus Thogotovirus, with the species Thogoto virus (THOV) and Dhori virus (DHOV) (van Regenmortel et al., 2000). In contrast to the other orthomyxoviruses, thogotoviruses are arboviruses that replicate in both mammals and ticks (Jones & Nuttall, 1989). Their genome consists of six single-stranded RNA segments of negative polarity that have a coding capacity for seven proteins. The three subunits (PB1, PB2 and PA) of the viral RNA-dependent RNA polymerase (RdRP) and the viral nucleoprotein (NP) associate with the genomic RNA segments to form viral ribonucleoprotein complexes (vRNPs). The transmembrane glycoprotein (GP) mediates entry into the host cell. Furthermore, a matrix protein (M) and an additional protein called ML have been described (Hagmaier et al., 2003; Haller & Kochs, 2002). While the five larger RNA segments each encode only one gene product, the sixth segment encodes both M and ML. The 266 aa M protein is translated from a spliced transcript of segment 6. The stop codon that terminates the M reading frame is created by the splicing process (Kochs et al., 2000). The 304 aa ML protein is translated from the unspliced transcript and is an elongated version of M that contains an additional 38 aa at the C terminus. We recently showed that ML functions as an interferon (IFN) antagonist (Hagmaier et al., 2003). Influenza A virus (FLUAV) also encodes a protein with IFN-antagonistic function, NS1 (Garcia-Sastre et al., 1998). However, despite the similarity in function, FLUAV NS1 and THOV ML have no similarity with respect to sequence and coding strategy. While ML is encoded on the same RNA segment as the THOV M protein and shares most of its sequence, the NS1 protein is encoded on the same genomic segment as the FLUAV NS2/NEP gene.

Replication and transcription of the THOV genome occur within the nucleus of infected cells (Siebler et al., 1996). vRNPs represent the minimal replication-competent entity of the virus. Accumulation of M at a late stage of THOV infection is thought to be responsible for the switch from transcription and replication to nuclear export and packaging of the newly synthesized vRNPs into progeny virions. Matrix proteins of negative-stranded RNA viruses are multifunctional proteins crucial for the regulation of viral RdRP activity, virus assembly and budding (Finke et al., 2003; Lenard, 1996). The M1 protein of FLUAV is involved in the regulation of various steps of the virus replication cycle. Firstly, it has an inhibitory effect on the activity of the viral polymerase (Perez & Donis, 1998; Watanabe et al., 1996). Secondly, M1 is involved in export of newly synthesized vRNPs from the nucleus to the plasma membrane, the site of assembly of new virus particles (Bui et al., 2000), and prevents their re-import back into the nucleus (Whittaker et al., 1996). These two activities – polymerase inhibition and nuclear exclusion – are responsible for the fact that cells expressing recombinant M1 do not support productive replication of FLUAV (Martin & Helenius, 1991). Thirdly, M1 plays an important role in the assembly of progeny virus particles and their budding from the cell membrane (Gomez-Puertas et al., 2000). It interacts with both the vRNPs and the inside of the plasma membrane, mediating contact between these two components (Ruigrok et al., 2000; Ye et al., 1999; Zhang & Lamb, 1996). Finally, M1 has been shown to influence the morphology of progeny virions (Bourmakina & Garcia-Sastre, 2003; Elleman & Barclay, 2004; Roberts et al., 1998; Smirnov et al., 1991).

The biological role of the M and ML proteins of THOV in the virus replication cycle has not been investigated so far. Given that M and ML are identical except for the C-terminal 38 aa of ML, we expected that they would be at least partially redundant in function. However, our recent work has indicated that M is not able to substitute for ML with respect to its IFN-antagonistic activity (Hagmaier et al., 2003). Here, we compared the functional activity of the two gene products of THOV segment 6 with respect to their role in virus budding, regulation of the viral polymerase and inhibition of IFN induction, and defined the functional domains responsible for these activities.


   METHODS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cells and viruses.
VeroCH, mouse 3T3, human 293 and baby hamster kidney (BHK-21) cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10 % fetal calf serum. A cell culture-adapted mutant of THOV SiAr126, termed Sicily/FR (Hagmaier et al., 2003; Kochs et al., 2000), the Portuguese THOV isolate PoTi503 (kindly provided by Armindo Filipe; Filipe & Calisher, 1984), DHOV strain India/1313/61 (Anderson & Casals, 1973) and FLUAV strain FPV-B (Israel, 1979) were propagated on BHK cells and particles were isolated from cell-culture supernatants by ultracentrifugation through a 30 % (v/v) glycerol cushion. Recombinant vaccinia virus Ankara (MVA-T7; kindly provided by Gerd Sutter; Sutter et al., 1995) was used to provide the T7 RNA polymerase and Newcastle disease virus (NDV) strain H53 (Bazzigher et al., 1992) to activate the IFN-{beta} promoter.

Construction of plasmids.
Plasmids used for the THOV minireplicon system have been described previously (Wagner et al., 2000; Weber et al., 2000). The M1 cDNA of FLUAV WSN/33 was amplified from plasmid {Pi}AM1 (kindly provided by P. Palese, Mount Sinai School of Medicine, New York) and cloned into the pBSK vector (Stratagene) or the mammalian expression vector pSC (Georgiev et al., 1996). pSC contains the sequence for a FLAG tag (Hopp, 1988), which was fused to the 5' end of the M1 open reading frame (ORF). The plasmids pCAGGS-M-HA encoding the M protein of THOV Sicily/FR with a C-terminal HA tag, pCAGGS-M5xT{Delta}SA encoding only ML and pCAGGS-M5xT encoding both M and ML have been described previously (Hagmaier et al., 2003). The various mutants of THOV M and ML are shown in Fig. 1. Full-length M and ML ORFs were obtained by RT-PCR of the spliced and unspliced transcripts of THOV Sicily/FR, respectively, using primers complementary to nt 1–24 and nt 936–909, resulting in plasmids encoding M (pBSK-THOV-M), FLAG–M (pSC-THOV-M) or both M and ML (pBSK-M5xT). Three deletion mutants were produced by PCR and corresponded to the N-terminal half of the M ORF (aa 1–141), the C-terminal half of the M ORF (aa 142–266) or the C-terminal half of the ML ORF (aa 142–304) using the respective primers. All deletion constructs were N-terminally fused to the FLAG-tag and cloned into either the pSC vector or the pcDNA3 vector (Invitrogen), resulting in plasmids pSC-M({Delta}C), pSC-M({Delta}N), pcDNA-M({Delta}C), pcDNA-M({Delta}N) and pcDNA-ML({Delta}N). Smaller C-terminal fragments of ML were produced by PCR and cloned in-frame into the pEGFP-C1 expression vector (Clontech), resulting in pEGFP(142–304), pEGFP(227–304) and pEGFP(265–304).



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 1. M and ML expression constructs. The M protein (shaded bar) has a length of 266 aa. The 38 aa C-terminal extension of the ML protein is indicated as a hatched box. M({Delta}C) corresponds to the N-terminal 141 aa of M and ML, M({Delta}N) to the C-terminal 124 aa of M and ML({Delta}N) to the C-terminal 162 aa of ML. All deletion constructs were N-terminally fused to a FLAG tag, indicated as a small flag, or to EGFP, indicated as a filled box.

 
Immunofluorescence analysis.
Mouse 3T3 cells were transfected with 2 µg expression plasmid and infected 24 h later at an m.o.i. of 10 with THOV or DHOV for 10 h, or with FLUAV for 6 h. Cells were fixed with 3 % paraformaldehyde and permeabilized with 0·5 % Triton X-100 and then stained for viral M proteins using the anti-FLAG M2 mouse monoclonal antibody (Sigma) and for the viral nucleoproteins with rabbit polyclonal antisera raised against bacterially expressed NP of THOV, DHOV or FLUAV. Primary antibodies were detected with Cy2-conjugated goat anti-mouse and Cy3-conjugated goat anti-rabbit antibodies (Amersham/Pharmacia).

Western blot.
Cells were infected with virus at an m.o.i. of 5 for 36 h. Purified virus particles from the cell supernatants and lysates of infected cells were analysed by SDS-PAGE and Western blotting. Viral M and NP were detected using polyclonal rabbit antisera directed against recombinant M and NP expressed in Escherichia coli (Kochs et al., 2000). FLAG-tagged M fragments were detected using a monoclonal antibody (M2) directed against the FLAG peptide (Sigma) and green fluorescent protein (GFP) fusion proteins were detected using a polyclonal rabbit antiserum against GFP (Molecular Probes).

THOV minireplicon system.
VeroCH cells were infected in 35 mm wells with MVA-T7 at an m.o.i. of 10. At 1 h post-infection, cells were transfected with T7-driven expression plasmids encoding the THOV polymerase subunits PA, PB1 and PB2 (75 ng each of pBS-PB1, pBS-PB2 and pG7-PA), NP (500 ng pG7-NP) and a chloramphenicol acetyltransferase (CAT) minigenome driven by the RNA polymerase I promoter (250 ng pPolI-THOV/CAT) as described previously (Wagner et al., 2000). As an internal expression control, a T7–luciferase construct (100 ng pBS-T7/Luc) was added to the plasmid mixture. To determine the activity of the different M proteins, the respective expression constructs were co-transfected. Transfections were performed using 3·5 µl Lipofectamine (µg DNA)–1 (Gibco-BRL). At 30 h post-transfection, cells were harvested, lysed in 100 µl CAT ELISA lysis buffer (Roche) and analysed for CAT enzyme activity by a standard procedure as described previously (Gorman et al., 1982). The production of radiolabelled products was quantified using a phosphorimager (Fuji BAS1000). For each reaction, CAT activity was normalized to the luciferase activity. Expression of the various M and ML constructs in transfected cells was confirmed by Western blotting.

To allow the formation of virus-like particles (VLPs), an expression construct for the THOV glycoprotein (pBS-T7-GP_THOV) was included in the transfection mixture as described previously (Wagner et al., 2000). Transfected cells were harvested 50 h post-transfection and CAT and luciferase activities were determined in the cell lysates. The supernatants of transfected cells were passaged on to indicator VeroCH cells that had been infected with THOV Sicily/FR (m.o.i. of 5) 1 h earlier. After 90 min, the supernatant was removed and cells were incubated in culture medium for 24 h. Cells were then lysed and CAT activity was determined.

Induction of the IFN-{beta} promoter.
293 cells in 35 mm wells were transfected with 1 µg p125Luc reporter plasmid (Yoneyama et al., 1998) encoding the firefly luciferase (FF-Luc) gene under the control of the IFN-{beta} promoter (kindly provided by Takashi Fujita, Tokyo Metropolitan Institute of Medical Science, Japan) and 0·1 µg of the control plasmid pRL-SV40 (Promega) containing the Renilla luciferase (REN-Luc) gene under the control of the constitutive SV40 promoter (Weber et al., 2002). Expression plasmids encoding the different M and ML variants were co-transfected. After 24 h, 1 µg poly(I : C) (Sigma) was transfected. Transfections were performed using 10 µl DAC30 (Eurogentec) per well. Alternatively, cells were infected with NDV at an m.o.i. of 1. At 24 h after treatment, cells were lysed in 200 µl luciferase lysis buffer (Promega). A sample (10 µl) of the lysate was used to measure FF-Luc and REN-Luc activities using the Dual-luciferase Assay kit (Promega). FF-Luc was normalized to REN-Luc activities.

Electron microscopy.
For electron microscopy, BHK-21 cells were grown on 60 mm Petri dishes and infected with THOV PoTi503 (ML+) or THOV Sicily/FR (ML) for 60 h at 37 °C. At this time point, the culture showed a clear cytopathic effect. Cell layers were fixed with 2·5 % glutaraldehyde in PBS and then removed from the plates using a cell scraper. After agarose enclosure, blocks of cells were post-fixed with 1 % osmium tetroxide and embedded following routine protocols (Gelderblom et al., 1987). Ultrathin sections were post-stained with lead citrate and evaluated using a ZEISS EM 10 A transmission electron microscope.


   RESULTS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
THOV M specifically inhibits THOV replication
First, we tested whether THOV M plays a regulatory role in the viral infection cycle, as has been described for the M1 protein of FLUAV. 3T3 fibroblasts were transfected with expression constructs encoding either FLAG-tagged THOV M or FLUAV M1. At 18 h post-transfection, cells were infected with THOV Sicily/FR for 10 h and subsequently analysed for viral protein expression by double immunofluorescence. Fig. 2 shows that no viral NP was produced in cells expressing the recombinant THOV M (Fig. 2a, b) indicating that no productive infection had occurred in these cells. In contrast, non-transfected cells and cells transfected with the FLUAV M1 expression construct were fully permissive for THOV, as demonstrated by the nuclear accumulation of viral NP (Fig. 2c, d). A quantitative analysis of the experiments is shown in Fig. 2(i). Overall, approximately 60 % of FLUAV M1-expressing cells, but only approximately 10 % of THOV M-expressing cells, were productively infected with THOV.



View larger version (44K):
[in this window]
[in a new window]
 
Fig. 2. M protein of THOV specifically prevents THOV replication. (a)–(h) Mouse 3T3 cells were transiently transfected with 1 µg of an expression construct encoding FLAG-tagged THOV M (pSC-THOV-M) or FLUAV M1 (pSC-FLUAV-M1). At 24 h post-transfection, cells were infected at an m.o.i. of 10 with either THOV Sicily/FR or DHOV India/1313/61 for 10 h, or with FLUAV FPV-B for 6 h. Cells were fixed and analysed by double immunofluorescence for the accumulation of recombinant M proteins using the anti-FLAG antibody ({alpha}-FLAG) and with specific antisera directed against the different viral NPs ({alpha}-NP). (i) Quantitative analysis of transfection/infection experiments. The percentage of infected cells expressing recombinant M proteins is shown. The overall infection rates for non-transfected cells in the same experiment are indicated below.

 
To demonstrate that the inhibitory effect was THOV specific, we infected THOV M-expressing cells with two other orthomyxoviruses. As shown in Fig. 2(e–h), replication of neither DHOV nor FLUAV was affected by the recombinant THOV M. Approximately 90 % of M-expressing cells showed accumulation of DHOV NP or FLUAV NP (Fig. 2i). These results indicated that THOV M specifically prevented the replication of THOV.

M, but not ML, blocks reporter gene expression in a THOV minireplicon system
To investigate the inhibitory effect of M in more detail, we used a THOV minireplicon system that allows reconstitution of the active THOV polymerase complex from cloned cDNAs (Wagner et al., 2000; Weber et al., 2000). The three subunits of the viral polymerase complex (PA, PB1 and PB2) and NP were co-expressed with an RNA minireplicon. The minireplicon contained the CAT gene in negative orientation, flanked by THOV-specific regulatory sequences of segment 5. This is transcribed by the cellular RNA polymerase I in the cell nucleus, where it assembles with the recombinant viral proteins to form functional vRNPs that direct the synthesis of CAT mRNA. CAT activity measured in the cell lysate thus reflects the transcriptional activity of the reconstituted vRNPs (Fig. 3a; +Ctrl). When one of the structural components of the vRNPs, e.g. NP, was omitted, no CAT expression was observed, indicating that all four components of the polymerase complex were required (Fig. 3a; –Ctrl). An FF-Luc reporter construct was co-transfected as an internal expression control to account for possible differences in transfection efficiency. All CAT values were normalized with respect to luciferase expression and are indicated as ratios of CAT/Luc.



View larger version (52K):
[in this window]
[in a new window]
 
Fig. 3. Activity of THOV M in a THOV minireplicon system. VeroCH cells were transfected with T7-driven expression plasmids encoding the components of the THOV minireplicon system. (a) THOV M (pBSK-THOV-M) or FLUAV M1 (pBSK-FLUAV-M1) was expressed under the control of the T7 promoter using increasing amounts of expression plasmids as described previously (Wagner et al., 2000; Weber et al., 2000). CAT activity determined in the cell lysates was normalized with respect to luciferase activity expressed from a T7 promoter and is indicated as CAT/Luc ratios. The CAT/Luc ratio for experiments without M (+Ctrl) were assigned a value of 1. As a negative control, the NP expression plasmid was omitted (–Ctrl). The result shows one of three independent experiments. (b) The experiment was carried out as in (a), but with an expression plasmid encoding the viral GP included in the minireplicon system. Increasing amounts of THOV M (pBSK-THOV-M) or THOV ML (pBSK-M5xT) plasmid were expressed as indicated and the CAT and luciferase activities were determined as in (a).

 
When increasing amounts of an expression plasmid for THOV M were co-transfected, a proportional decrease in CAT activity was observed (THOV M; Fig. 3a), indicating that expression of the minireplicon was inhibited in the presence of M. Expression of FLUAV M1 did not affect CAT values (Fig. 3a; FLUAV M1). We concluded that THOV M had the potential specifically to inhibit the THOV polymerase complex.

Since the complete M sequence is contained within the sequence of ML (see Fig. 1), it was conceivable that ML could have a similar inhibitory effect. Therefore, we tested an expression plasmid encoding THOV ML in the minireplicon system as described above. Fig. 3(b) shows a comparison of the effects of M and ML. While increasing amounts of M led to a decrease in CAT synthesis, expression of ML did not show any consistent effect on CAT activity. The results therefore showed that ML, in contrast to M, did not inhibit the viral polymerase.

M, but not ML, is sufficient to support the formation of VLPs
In order to determine the requirements for the formation of virus particles, we expanded the minireplicon system to allow the formation of VLPs. Therefore, in addition to the vRNP components and M, an expression construct for the viral GP was included in the transfection mixture (Wagner et al., 2000). Expression of GP allows packaging of the CAT minireplicon into infectious particles that are released into the medium. Supernatants of transfected cells were passaged on to indicator cells that had been infected with helper THOV to provide sufficient polymerase for amplification and transcription of the minireplicon. CAT activity measured in these cells indicated that VLPs containing the CAT segment had been produced by the transfected cells. The CAT values shown in Fig. 4 were measured after passaging the supernatants of the transfected cells described in the previous paragraph (Fig. 3b). In the absence of NP (Fig. 4; –Ctrl), no CAT activity was passaged since no functional vRNPs could be formed in the transfected cells. Likewise, no CAT activity was passaged in the absence of GP (not shown). In the presence of NP and GP (Fig. 4; +Ctrl), a low background activity was measured in the indicator cells, probably due to random formation of vesicles containing the CAT minireplicon in the absence of M. Efficient packaging of the CAT segment could be observed only when GP as well as M constructs were co-transfected (Fig. 4; THOV M), with small amounts of M being sufficient to support the formation of VLPs. By contrast, expression of ML instead of M did not support packaging of the CAT segment (Fig. 4; THOV ML). These results showed that ML could not functionally substitute for M, with respect to either regulation of the viral polymerase complex or packaging of vRNPs into progeny virions.



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 4. M, but not ML, supports formation of infectious particles. Supernatants of the transfected cells shown in Fig. 3(b) were passaged onto indicator VeroCH cells infected with THOV. At 24 h after passage, the indicator cells were harvested and CAT activity was determined in 10 µl of the cell lysates. To allow a comparison of individual sets of experiments, CAT activities in the samples with 500 ng M plasmid were assigned a value of 1. Activities were determined in triplicate. Mean values±SD are shown.

 
The C-terminal moiety of M is sufficient for inhibition of THOV polymerase, but not for formation of VLPs
In order to investigate which part of THOV M was responsible for inhibition of the viral polymerase, we constructed expression vectors encoding either the N-terminal half of M [M({Delta}C), aa 1–141] or the C-terminal half of M [M({Delta}N), aa 142–266]. A schematic representation of the various deletion mutants is given in Fig. 1. These plasmids were co-transfected together with the minireplicon system as described above, and the activity of the viral polymerase was determined 24 h later. Fig. 5(a) shows that the N-terminal half of M had no effect on the replication and transcription of the minireplicon RNA. In contrast, the inhibitory activity of the C-terminal half of M was as strong as that of the full-length protein. This result indicated that the C-terminal part of M was sufficient to interact with the viral polymerase complex and to interfere with its activity. Western blot analysis of the cell lysates confirmed expression of the full-length and truncated versions of M (Fig. 5b). Although the M({Delta}N) signal was weaker than the band for full-length M or M({Delta}C), the expression level of this fragment was still sufficient to suppress the activity of the polymerase complex completely.



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 5. Activity of THOV M deletion mutants in the THOV minireplicon system. VeroCH cells were co-transfected with 1 µg of an expression plasmid for full-length M (pSC-M) or the C- and N-terminal deletion mutants of M [pSC-M({Delta}C) or pSC-M({Delta}N), respectively] and the components of the minireplicon system. (a) CAT and luciferase activities were measured in 0·2 µl cell lysates and are indicated as CAT/Luc ratios as in Fig. 3. The CAT/Luc ratio of experiments without M (+Ctrl) were taken as a positive control and assigned a value of 1. For the negative control, the NP expression plasmid was omitted (–Ctrl). (b) Western blot analysis of the recombinant full-length and M deletion mutants in lysates of transfected cells using an anti-FLAG antibody. (c) The supernatants of the transfected cells shown in (a) were passaged onto indicator cells as in Fig. 4. The CAT turnover reached after transfection of wild-type M was taken as a positive control and assigned a value of 1. The figure shows one of three independent experiments.

 
In order to assay the ability of the mutant M proteins to drive packaging of the CAT minireplicon into VLPs, supernatants from transfected cells were passaged on to indicator cells. As shown in Fig. 5(c), only full-length M was sufficient to support the formation of infectious VLPs.

The C-terminal moiety of ML is sufficient to inhibit IFN-{beta} induction
We have recently shown that THOV ML, but not M, is able to suppress the induction of IFN-{beta} in response to virus infection or double-stranded RNA (dsRNA) treatment (Hagmaier et al., 2003). In order to investigate which part of THOV ML was responsible for this inhibitory effect, we used expression vectors encoding either the N-terminal half of M [M({Delta}C), aa 1–141], the C-terminal half of M [M({Delta}N), aa 142–266] or the C-terminal half of ML [ML({Delta}N), aa 142–304] (see Fig. 1). These plasmids were co-transfected with a reporter construct encoding the FF-Luc gene under the control of the IFN-{beta} promoter and a control construct encoding the REN-Luc gene under the control of the constitutive SV40 promoter. At 24 h post-transfection, the IFN-{beta} promoter was stimulated by transfection of poly(I : C), which represents an artificial dsRNA. Luciferase activities were measured 24 h later and are shown in Fig. 6(a). The FF-Luc values were normalized with respect to the REN-Luc values to account for general differences in gene expression.



View larger version (32K):
[in this window]
[in a new window]
 
Fig. 6. Suppression of dsRNA-mediated induction of the IFN-{beta} promoter by THOV ML deletion mutants. 293 cells were transfected with an IFN-{beta} promoter reporter plasmid together with a REN-Luc expression plasmid under the control of a constitutive promoter as described previously (Weber et al., 2002). An expression plasmid (1 µg) encoding HA-tagged M (pCAGGS-M-HA), full-length ML (pCAGGS-M5xT{Delta}SA), FLAG-tagged C- and N-terminal deletion mutants of M and ML [pcDNA-M({Delta}C), pcDNA-M({Delta}N) and pcDNA-ML({Delta}N)] or empty pcDNA vector (Ctrl), or GFP expression plasmids fused to C-terminal deletion mutants of ML [GFP(142–304), GFP(227–304) and GFP(265–304)] were co-transfected. At 24 h post-transfection, cells were treated with poly(I : C) (a) or infected with NDV at an m.o.i. of 1 (b) for a further 24 h. Cell lysates were prepared and analysed for reporter gene expression by luciferase activity assays. FF-Luc values were normalized with respect to the REN-Luc activity and are indicated as fold induction compared with the untreated control. One of three independent experiments is shown. (c) Western blot analysis of recombinant M fragments in the lysates of the transfected cells used in (b). Proteins were detected with antibodies directed against the FLAG peptide or against GFP.

 
Treatment of cells with poly(I : C) induced high expression of FF-Luc compared with untreated control cells (Fig. 6a; Ctrl). As expected, co-expression of M did not influence this induction, whereas induction was dramatically reduced when ML was co-expressed. Interestingly, co-expression of the C-terminal half of ML reduced the IFN-{beta} promoter activity in a manner comparable to wild-type ML [Fig. 6a; ML({Delta}N)]. The N-terminal half of M and ML did not show such an inhibitory activity, nor did the C-terminal half of M [Fig. 6a; ML({Delta}C) and M({Delta}N), respectively].

In a second experiment, we addressed the question of whether the C-terminal 38 aa (aa 267–304) unique to ML were sufficient to prevent IFN-{beta} promoter activation. Different C-terminal fragments of ML were fused to GFP and co-transfected with the luciferase reporter constructs as described above. To stimulate the IFN-{beta} promoter, cells were infected with NDV as an IFN inducer (Fig. 6b; Ctrl). The C-terminal half of ML efficiently prevented IFN-{beta} promoter activation, whether or not it was fused to GFP [Fig. 6b; ML({Delta}N) and GFP(142–304)]. Interestingly, co-transfection of two smaller fragments of the C-terminal region of ML did not reduce IFN-{beta} promoter activation [Fig. 6b; GFP(227–304) and GFP(265–304)]. Western blot analysis confirmed expression of the ML fragments in the transfected cells (Fig. 6c). Therefore, although the unique 38 aa at the C terminus were essential for the inhibitory activity of ML, they were functional only within the context of the entire C-terminal moiety of the ML protein.

ML is a structural protein
In order to investigate whether ML was packaged into viral particles, we analysed two strains of THOV that differed in their capacity to express ML. THOV PoTi503 (Filipe & Calisher, 1984) is a wild-type isolate from Portugal that encodes both M and ML. THOV Sicily/FR is a laboratory strain that has lost the capacity to express the ML protein due to an insertion mutation within the intron region of genomic segment 6 (Hagmaier et al., 2003). We compared infected cells and purified virus particles by Western blotting. M and ML were detected using a polyclonal antiserum against M. As expected, cell lysates infected with THOV Sicily/FR contained only M, whereas those infected with THOV PoTi503 contained both M and ML (Fig. 7a; Cell lysate). ML was also present in particle preparations of THOV PoTi503, indicating that ML is a structural component of the virion (Fig. 7a; Virus particles). While M and ML were present in similar amounts in the cell lysates, M was strongly enriched in the particles. As a control for cellular contamination, we analysed particles and cell lysates with an antibody against cellular tubulin. As shown in Fig. 7(b), tubulin was readily detected in infected-cell lysates, while no tubulin could be detected in the particle preparations, indicating that the ML band in the virions was not due to insufficient purification of the particles.



View larger version (95K):
[in this window]
[in a new window]
 
Fig. 7. ML is a structural component of the virion. Virus particles of THOV Sicily/FR and THOV PoTi503 were produced in BHK-21 cells and purified from the supernatant by ultracentrifugation through a glycerol cushion. Infected BHK-21 cells were lysed by repetitive freezing and thawing in 250 mM Tris/HCl buffer, pH 7·5. Virus particles and cell lysates were analysed by Western blotting using an antiserum directed against THOV M (a) or a monoclonal antibody against cellular {beta}-tubulin (b). (c) Electron micrographs of ultrathin sections showing the release of THOV strains PoTi503 or Sicily/FR from BHK-21 cells. At 60 h post-infection, cells were fixed, stained and prepared for electron microscopy. The virions formed showed surface spikes, an electron-dense protein shell and internal profiles of viral RNPs and thus closely resembled regular orthomyxoviruses. Bars, 100 nm (upper panels); 200 nm (lower panels).

 
To investigate a possible effect of ML on virion morphogenesis and fine structure, ultrathin sections of THOV PoTi503- and Sicily/FR-infected cells were studied by thin-section electron microscopy. Cell profiles showed multiple enveloped particles budding from the plasma membrane and a number of released particles with a morphology typical of orthomyxoviruses (Compans & Dimmock, 1969; Ruigrok et al., 2000) (Fig. 7c). The membranes of budding and released particles were lipid bilayers studded with surface projections 10–12 nm in length, probably representing glycoprotein spikes. Beneath the bilayer, an electron-dense shell of 5–10 nm thickness was visible, probably representing a layer of M. The interior of budding and released particles revealed several profiles of vRNPs with a diameter of 5 nm, sectioned at different angles. Released particles appeared spherical or slightly heterogeneous with a diameter of 100–120 nm. A comparison of THOV PoTi503 and Sicily/FR did not reveal profound differences in the assembly process and in virus fine structure (Fig. 7c). It appeared that incorporation of ML into THOV particles occurred as a random process during budding and was not required for the assembly process and formation of infectious virions.


   DISCUSSION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this report, we compared the functional characteristics of M and ML, the two gene products of THOV segment 6. M was originally identified as the matrix protein of THOV (Kochs et al., 2000). In addition to their role as a structural component of virions, orthomyxovirus matrix proteins also play a crucial role in regulating the virus replication cycle. Using an in vivo system for reconstitution of the THOV polymerase complex, we demonstrated that M had an inhibitory effect on the THOV polymerase. This inhibitory activity is relevant in that it terminates ongoing viral RNA synthesis before condensation and export of the nucleocapsids. Matrix proteins are synthesized in large amounts at a late stage during the orthomyxovirus infection cycle (Lamb & Krug, 2001). With increasing concentration, they block transcriptional activity of vRNPs and are involved in nuclear export of vRNPs, which can then be packaged into new virus particles at the plasma membrane (Bui et al., 2000; Gomez-Puertas et al., 2000; Perez & Donis, 1998; Watanabe et al., 1996). Thus, when THOV M was overexpressed experimentally before infection of the cells, the activity of the viral polymerase complex was inhibited at an early stage of the infection cycle and no productive replication of THOV could take place. The M1 protein of FLUAV has a similar inhibitory effect on transcription and replication of FLUAV vRNPs (Perez & Donis, 1998; Watanabe et al., 1996). However, expression of FLUAV M1 did not have any effect on THOV replication or expression of the THOV minigenome. Conversely, THOV M did not inhibit the replication of either FLUAV or DHOV, indicating that the interaction of M with the respective vRNP components was virus type-specific. This may be explained by the low sequence similarity between the matrix proteins of THOV, DHOV and FLUAV (20–25 %).

Viral matrix proteins, for example FLUAV M1 or Ebola virus VP40, are known to be required for the formation of virus particles (Gomez-Puertas et al., 2000; Timmins et al., 2001). Using the THOV in vivo reconstitution system, we previously demonstrated the formation of VLPs in the presence of small amounts of M (Wagner et al., 2000). Here, we have shown that the M protein supports packaging of vRNPs into infectious THOV particles, even in amounts that are sufficient to inhibit viral polymerase activity.

In order to separate the functional domains of THOV M, we generated deletion mutants and compared their activity with that of the full-length protein. Surprisingly, the C-terminal half of M was as efficient at inhibiting the THOV polymerase complex as wild-type M. In contrast, the N-terminal half of the protein did not show any effect on polymerase activity, but was required for particle formation, as only full-length M supported the formation of VLPs. Most probably, the N-terminal part is required for the interaction of M with the plasma membrane or with the cytoplasmic part of the viral glycoprotein, as is known to occur with FLUAV M1 (Baudin et al., 2001; Sha & Luo, 1997; Ye et al., 1987). Although Baudin et al. (2001) reported that the C-terminal moiety of M1 had no transcription-inhibition activity, a longer fragment used by Ye et al. (1987), comprising the C-terminal two-thirds of the M1 sequence, was sufficient for inhibition of the FLUAV polymerase in vitro. Thus, despite the low similarity of THOV M and FLUAV M1 in primary sequence, the two proteins seem to be comparable with respect to their domain organization. This arrangement as two separate functional domains may be a general feature of matrix proteins of negative-stranded RNA viruses, as a similar organization has also been reported for the Ebola virus VP40 (Scianimanico et al., 2000) and the matrix protein of vesicular stomatitis virus (Gaudier et al., 2002; Kaptur et al., 1991).

ML contains full-length M with an additional 38 aa at the C terminus. Analysis of the subcellular localization of M and ML in transfected cells showed no obvious differences between the two proteins. Both M and ML were equally distributed in the cytoplasm as well as in the nucleus of the transfected cells (S. Jennings and G. Kochs, unpublished results). It was therefore surprising that ML could not substitute for M with respect to either regulation of the polymerase complex or the formation of VLPs. Also, ML was not required for either of the two activities, which is in agreement with the fact that the ML-deficient THOV strain Sicily/FR is able to replicate well in both cell culture (Kochs et al., 2000) and mice (Haller et al., 1995). Instead, ML has been characterized as an accessory protein that exhibits IFN-antagonistic activity (Hagmaier et al., 2003). As we have shown here, the C-terminal half of ML was as efficient as full-length ML in preventing activation of the IFN-{beta} promoter, while the N-terminal half did not show this effect. Thus, in both ML and M, the C-terminal moieties contained the functionally active domains. Moreover, M was not able to substitute for ML as an antagonist of IFN-{beta} induction. It was therefore obvious that the C-terminal extension of 38 aa that distinguishes ML from M is responsible for fundamental structural differences between the two proteins. Alternatively, the C-terminal extension of ML may make inaccessible a region at the C terminus of M that is essential for M-specific functions. Surprisingly, the unique C terminus of ML was not sufficient to suppress IFN promoter activation when expressed as a fusion protein with GFP. Thus, although being required for the inhibitory activity of ML, these 38 aa are not functional on their own. Unfortunately, no known motifs have so far been identified within this C-terminal region that could explain why two proteins so similar in sequence are so strikingly different in function.

In the case of FLUAV, the matrix protein M1 and the IFN antagonist NS1 are encoded on two separate genomic segments and do not share a common sequence. The fact that THOV uses a completely different coding strategy for these two functions creates a certain dilemma for the virus: M and ML are encoded on the same RNA segment and are expressed by differential splicing (Hagmaier et al., 2003; Kochs et al., 2000). Thus, expression of the two proteins is regulated by the same promoter. The inhibitory activity of M demands expression late in infection in order to avoid untimely inhibition of the viral polymerase. In contrast, the IFN-suppressive activity of ML is required early in infection, preferably immediately after entry into the host cell, so as to prevent the initial induction of the antiviral IFN response. We therefore suspected that ML might be packaged into virions like other known viral IFN antagonists, such as pp65 of human cytomegalovirus (Browne & Shenk, 2003) or VP35 of Ebola virus (Basler et al., 2000). By analysing purified THOV particles, we demonstrated here that ML is indeed a component of the virion. Thus, in contrast to FLUAV NS1, a non-structural protein expressed early in the infection cycle, the strategy of THOV ML is more reminiscent of Ebola virus VP35 in that both represent virion proteins that are available immediately after entry and uncoating.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the Deutsche Forschungsgemeinschaft (Ko 1579/3-5) to G. K. We would like to thank Otto Haller for constant support and suggestions; Simone Gruber, Freya Kaulbars and Carsten Schwann for excellent technical assistance; and Peter Staeheli and Friedemann Weber for discussions and critical comments on the manuscript. We are grateful to Patricia A. Nuttall and Armindo R. Filipe for providing isolates of THOV and to Takashi Fujita for reporter plasmids.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Anderson, C. R. & Casals, J. (1973). Dhori virus, a new agent isolated from Hyalomma dromedarii in India. Indian J Med Res 61, 1416–1420.[Medline]

Basler, C. F., Wang, X., Mühlberger, E., Volchkov, V., Paragas, J., Klenk, H.-D., Garcia-Sastre, A. & Palese, P. (2000). The Ebola virus VP35 protein functions as a type I IFN antagonist. Proc Natl Acad Sci U S A 97, 12289–12294.[Abstract/Free Full Text]

Baudin, F., Petit, I., Weissenhorn, W. & Ruigrock, R. W. H. (2001). In vitro dissection of the membrane and RNP binding activities of influenza virus M1 protein. Virology 281, 102–108.[CrossRef][Medline]

Bazzigher, L., Pavlovic, J., Haller, O. & Staeheli, P. (1992). Mx genes show weaker primary response to virus than other interferon-regulated genes. Virology 186, 154–160.[Medline]

Bourmakina, S. V. & Garcia-Sastre, A. (2003). Reverse genetics studies on the filamentous morphology of influenza A virus. J Gen Virol 84, 517–527.[Abstract/Free Full Text]

Browne, E. P. & Shenk, T. (2003). Human cytomegalovirus UL83-coded pp65 virion protein inhibits antiviral gene expression in infected cells. Proc Natl Acad Sci U S A 100, 11439–11444.[Abstract/Free Full Text]

Bui, M., Wills, E. G., Helenius, A. & Whittaker, G. R. (2000). Role of the influenza virus M1 protein in nuclear export of viral ribonucleoproteins. J Virol 74, 1781–1786.[Abstract/Free Full Text]

Clouthier, S. C., Rector, T., Brown, N. E. & Anderson, E. D. (2002). Genomic organization of infectious salmon anaemia virus. J Gen Virol 83, 421–428.[Abstract/Free Full Text]

Compans, R. W. & Dimmock, N. J. (1969). An electron microscopic study of single-cycle infection of chick embryo fibroblasts by influenza virus. Virology 39, 499–515.[Medline]

Elleman, C. J. & Barclay, W. S. (2004). The M1 matrix protein controls the filamentous phenotype of influenza A virus. Virology 321, 144–153.[CrossRef][Medline]

Filipe, A. R. & Calisher, C. H. (1984). Isolation of Thogoto virus from ticks in Portugal. Acta Virol 28, 152–155.[Medline]

Finke, S., Mueller-Waldeck, R. & Conzelmann, K. (2003). Rabies virus matrix protein regulates the balance of virus transcription and replication. J Gen Virol 84, 1613–1621.[Abstract/Free Full Text]

Garcia-Sastre, A., Egorov, A., Matassov, D., Brandt, S., Levy, D. E., Durbin, J. E., Palese, P. & Muster, T. (1998). Influenza A virus lacking the NS1 gene replicates in interferon-deficient systems. Virology 252, 324–330.[CrossRef][Medline]

Gaudier, M., Gaudin, Y. & Knossow, M. (2002). Crystal structure of vesicular stomatitis virus matrix protein. EMBO J 21, 2886–2892.[Abstract/Free Full Text]

Gelderblom, H. R., Hausmann, E. H. S., Özel, M., Pauli, G. & Koch, M. A. (1987). Fine structure of human immunodeficiency virus (HIV) and immunolocalization of structural proteins. Virology 156, 171–176.[Medline]

Georgiev, O., Bourquin, J. P., Gstaiger, M., Knoepfel, L., Schaffner, W. & Hovens, C. (1996). Two versatile eukaryotic vectors permitting epitope tagging, radiolabelling and nuclear localisation of expressed proteins. Gene 168, 165–167.[CrossRef][Medline]

Gomez-Puertas, P., Albo, C., Perez-Pastrana, E., Vivo, A. & Portela, A. (2000). Influenza virus matrix protein is the major driving force in virus budding. J Virol 74, 11538–11547.[Abstract/Free Full Text]

Gorman, M., Moffat, L. F. & Howard, B. H. (1982). Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol 2, 1044–1051.[Medline]

Hagmaier, K., Jennings, S., Buse, J., Weber, F. & Kochs, G. (2003). Novel gene product of Thogoto virus segment 6 codes for an interferon antagonist. J Virol 77, 2747–2752.[Abstract/Free Full Text]

Haller, O. & Kochs, G. (2002). Thogotovirus. In The Springer Index of Viruses, pp. 615–619. Edited by C. A. Tidona & G. Darai. Berlin/Heidelberg/New York: Springer.

Haller, O., Frese, M., Rost, D., Nuttall, P. & Kochs, G. (1995). Tick-borne Thogoto virus infection in mice is inhibited by the orthomyxovirus resistance gene product Mx1. J Virol 69, 2596–2601.[Abstract]

Hopp, T. P. (1988). A short polypeptide marker sequence useful for recombinant protein identification and purification. Biotechnology 6, 1204–1210.

Israel, A. (1979). Preliminary characterization of the particles from productive and abortive infections of L cells by fowl plague virus. Ann Microbiol 130B, 85–100.

Jones, J. D. & Nuttall, P. A. (1989). The effect of virus-immune hosts on Thogoto virus infection of the tick, Rhipicephalus appendiculatus. Virus Res 14, 129–140.[CrossRef][Medline]

Kaptur, P. E., Rhodes, R. B. & Lyles, D. S. (1991). Sequences of the vesicular stomatitis virus matrix protein involved in binding to nucleocapsids. J Virol 65, 1057–1065.[Medline]

Kochs, G., Weber, F., Gruber, S., Delvendahl, A., Leitz, C. & Haller, O. (2000). Thogoto virus matrix protein is encoded by a spliced mRNA. J Virol 74, 10785–10789.[Abstract/Free Full Text]

Lamb, R. A. & Krug, R. M. (2001). Orthomyxoviridae: the viruses and their replication. In Fields Virology, 4th edn, pp. 1487–1532. Edited by D. M. Knipe & P. M. Howley. Philadelphia: Lippincott Williams and Wilkins.

Lenard, J. (1996). Negative-strand virus M and retrovirus MA proteins: all in a family. Virology 216, 289–298.[CrossRef][Medline]

Martin, K. & Helenius, A. (1991). Nuclear transport of influenza virus ribonucleoproteins: the viral matrix protein (M1) promotes export and inhibits import. Cell 67, 117–130.[Medline]

Perez, D. R. & Donis, R. O. (1998). The matrix 1 protein of influenza A virus inhibits the transcriptase activity of a model influenza reporter genome in vivo. Virology 249, 52–61.[CrossRef][Medline]

Roberts, P. C., Lamb, R. A. & Compans, R. W. (1998). The M1 and M2 proteins of influenza A virus are important determinants in filamentous particle formation. Virology 240, 127–137.[CrossRef][Medline]

Ruigrok, R. W. H., Barge, A., Durrer, P., Brunner, J., Ma, K. & Whittaker, G. R. (2000). Membrane interaction of influenza virus M1 protein. Virology 267, 289–298.[CrossRef][Medline]

Scianimanico, S., Schoehn, G., Timmins, J., Ruigrock, R. W. H., Klenk, H.-D. & Weissenhorn, W. (2000). Membrane association induces a conformational change in the Ebola virus matrix protein. EMBO J 19, 6732–6741.[Abstract/Free Full Text]

Sha, B. & Luo, M. (1997). Structure of a bifunctional membrane–RNA binding protein, influenza virus matrix protein M1. Nat Struct Biol 4, 239–244.[Medline]

Siebler, J., Haller, O. & Kochs, G. (1996). Thogoto and Dhori virus replication is blocked by inhibitors of cellular polymerase II activity but does not cause shutoff of host cell protein synthesis. Arch Virol 141, 1587–1594.[Medline]

Smirnov, Y. A., Kuznetsova, M. A. & Kaverin, N. V. (1991). The genetic aspects of influenza virus filamentous particle formation. Arch Virol 118, 279–284.[Medline]

Sutter, G., Ohlmann, M. & Erfle, V. (1995). Non-replicating vaccinia vector efficiently expresses bacteriophage T7 RNA polymerase. FEBS Lett 371, 9–12.[CrossRef][Medline]

Timmins, J., Scianimanico, S., Schoen, G. & Weissenhorn, W. (2001). Vesicular release of Ebola virus matrix protein VP40. Virology 283, 1–6.[CrossRef][Medline]

van Regenmortel, M. H. V., Fauquet, C. M., Bishop, D. H. L. & 8 other editors (2000). Virus Taxonomy: the Classification and Nomenclature of Viruses. Seventh Report of the International Committee on Taxonomy of Viruses. San Diego: Academic Press.

Wagner, E., Engelhardt, O. G., Weber, F., Haller, O. & Kochs, G. (2000). Formation of virus-like particles from cloned cDNA of Thogoto virus. J Gen Virol 81, 2849–2853.[Abstract/Free Full Text]

Watanabe, K., Handa, H., Mizumoto, K. & Nagata, K. (1996). Mechanism for inhibition of influenza virus RNA polymerase activity by matrix protein. J Virol 70, 241–247.[Abstract]

Weber, F., Haller, O. & Kochs, G. (2000). MxA GTPase blocks reporter gene expression of reconstituted Thogoto virus ribonucleoprotein complexes. J Virol 74, 560–563.[Abstract/Free Full Text]

Weber, F., Bridgen, A., Fazakerley, J. K., Streitenfeld, H., Kessler, N., Randall, R. E. & Elliott, R. M. (2002). Bunyamwera bunyavirus nonstructural protein NSs counteracts the induction of alpha/beta interferon. J Virol 76, 7949–7955.[Abstract/Free Full Text]

Whittaker, G., Bui, M. & Helenius, A. (1996). Nuclear trafficking of influenza virus ribonucleoproteins in heterokaryons. J Virol 70, 2743–2756.[Abstract]

Ye, Z., Liu, T., Offringa, D. P., McInnis, J. & Levandowski, R. A. (1999). Association of influenza virus matrix protein with ribonucleoproteins. J Virol 73, 7467–7473.[Abstract/Free Full Text]

Ye, Z., Pal, R., Fox, J. W. & Wagner, R. R. (1987). Functional and antigenic domains of the matrix (M1) protein of influenza virus. J Virol 61, 239–246.[Medline]

Yoneyama, M., Suhara, W., Fukuhara, Y., Fukuda, M., Nishida, E. & Fujita, T. (1998). Direct triggering of the type I interferon system by virus infection: activation of a transcription factor complex containing IRF-3 and CBP/p300. EMBO J 17, 1087–1095.[Abstract/Free Full Text]

Zhang, J. & Lamb, R. A. (1996). Characterization of the membrane association of the influenza virus matrix protein in living cells. Virology 225, 255–266.[CrossRef][Medline]

Received 21 May 2004; accepted 2 September 2004.



This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Hagmaier, K.
Articles by Kochs, G.
Articles citing this Article
PubMed
PubMed Citation
Articles by Hagmaier, K.
Articles by Kochs, G.
Agricola
Articles by Hagmaier, K.
Articles by Kochs, G.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
INT J SYST EVOL MICROBIOL MICROBIOLOGY J GEN VIROL
J MED MICROBIOL ALL SGM JOURNALS