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
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ABSTRACT |
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Present address: Centre for Biomolecular Sciences, University of St Andrews, St Andrews, UK.
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INTRODUCTION |
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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.
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METHODS |
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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
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
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 124 and nt 936909, resulting in plasmids encoding M (pBSK-THOV-M), FLAGM (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 1141), the C-terminal half of the M ORF (aa 142266) or the C-terminal half of the ML ORF (aa 142304) 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(
C), pSC-M(
N), pcDNA-M(
C), pcDNA-M(
N) and pcDNA-ML(
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(142304), pEGFP(227304) and pEGFP(265304).
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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 T7luciferase 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- 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-
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.
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RESULTS |
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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.
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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.
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The C-terminal moiety of ML is sufficient to inhibit IFN- induction
We have recently shown that THOV ML, but not M, is able to suppress the induction of IFN- 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(
C), aa 1141], the C-terminal half of M [M(
N), aa 142266] or the C-terminal half of ML [ML(
N), aa 142304] (see Fig. 1
). These plasmids were co-transfected with a reporter construct encoding the FF-Luc gene under the control of the IFN-
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-
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.
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In a second experiment, we addressed the question of whether the C-terminal 38 aa (aa 267304) unique to ML were sufficient to prevent IFN- 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-
promoter, cells were infected with NDV as an IFN inducer (Fig. 6b
; Ctrl). The C-terminal half of ML efficiently prevented IFN-
promoter activation, whether or not it was fused to GFP [Fig. 6b
; ML(
N) and GFP(142304)]. Interestingly, co-transfection of two smaller fragments of the C-terminal region of ML did not reduce IFN-
promoter activation [Fig. 6b
; GFP(227304) and GFP(265304)]. 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.
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DISCUSSION |
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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-
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-
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.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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---|
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, 1228912294.
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, 102108.[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, 154160.[Medline]
Bourmakina, S. V. & Garcia-Sastre, A. (2003). Reverse genetics studies on the filamentous morphology of influenza A virus. J Gen Virol 84, 517527.
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, 1143911444.
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, 17811786.
Clouthier, S. C., Rector, T., Brown, N. E. & Anderson, E. D. (2002). Genomic organization of infectious salmon anaemia virus. J Gen Virol 83, 421428.
Compans, R. W. & Dimmock, N. J. (1969). An electron microscopic study of single-cycle infection of chick embryo fibroblasts by influenza virus. Virology 39, 499515.[Medline]
Elleman, C. J. & Barclay, W. S. (2004). The M1 matrix protein controls the filamentous phenotype of influenza A virus. Virology 321, 144153.[CrossRef][Medline]
Filipe, A. R. & Calisher, C. H. (1984). Isolation of Thogoto virus from ticks in Portugal. Acta Virol 28, 152155.[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, 16131621.
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, 324330.[CrossRef][Medline]
Gaudier, M., Gaudin, Y. & Knossow, M. (2002). Crystal structure of vesicular stomatitis virus matrix protein. EMBO J 21, 28862892.
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, 171176.[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, 165167.[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, 1153811547.
Gorman, M., Moffat, L. F. & Howard, B. H. (1982). Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol 2, 10441051.[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, 27472752.
Haller, O. & Kochs, G. (2002). Thogotovirus. In The Springer Index of Viruses, pp. 615619. 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, 25962601.[Abstract]
Hopp, T. P. (1988). A short polypeptide marker sequence useful for recombinant protein identification and purification. Biotechnology 6, 12041210.
Israel, A. (1979). Preliminary characterization of the particles from productive and abortive infections of L cells by fowl plague virus. Ann Microbiol 130B, 85100.
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, 129140.[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, 10571065.[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, 1078510789.
Lamb, R. A. & Krug, R. M. (2001). Orthomyxoviridae: the viruses and their replication. In Fields Virology, 4th edn, pp. 14871532. 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, 289298.[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, 117130.[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, 5261.[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, 127137.[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, 289298.[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, 67326741.
Sha, B. & Luo, M. (1997). Structure of a bifunctional membraneRNA binding protein, influenza virus matrix protein M1. Nat Struct Biol 4, 239244.[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, 15871594.[Medline]
Smirnov, Y. A., Kuznetsova, M. A. & Kaverin, N. V. (1991). The genetic aspects of influenza virus filamentous particle formation. Arch Virol 118, 279284.[Medline]
Sutter, G., Ohlmann, M. & Erfle, V. (1995). Non-replicating vaccinia vector efficiently expresses bacteriophage T7 RNA polymerase. FEBS Lett 371, 912.[CrossRef][Medline]
Timmins, J., Scianimanico, S., Schoen, G. & Weissenhorn, W. (2001). Vesicular release of Ebola virus matrix protein VP40. Virology 283, 16.[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, 28492853.
Watanabe, K., Handa, H., Mizumoto, K. & Nagata, K. (1996). Mechanism for inhibition of influenza virus RNA polymerase activity by matrix protein. J Virol 70, 241247.[Abstract]
Weber, F., Haller, O. & Kochs, G. (2000). MxA GTPase blocks reporter gene expression of reconstituted Thogoto virus ribonucleoprotein complexes. J Virol 74, 560563.
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, 79497955.
Whittaker, G., Bui, M. & Helenius, A. (1996). Nuclear trafficking of influenza virus ribonucleoproteins in heterokaryons. J Virol 70, 27432756.[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, 74677473.
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, 239246.[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, 10871095.
Zhang, J. & Lamb, R. A. (1996). Characterization of the membrane association of the influenza virus matrix protein in living cells. Virology 225, 255266.[CrossRef][Medline]
Received 21 May 2004;
accepted 2 September 2004.
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