Abteilung Virologie, Institut für Medizinische Mikrobiologie und Hygiene, Universität Freiburg, D-79008 Freiburg, Germany1
Author for correspondence: Georg Kochs. Fax +49 761 2036562. e-mail KOCHS{at}UKL.UNI-FREIBURG.DE
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Several systems have been established to generate virus-like particles (VLPs) of Influenza A virus (FLUAV; Mena et al., 1996 ; Neumann et al., 2000
). In these systems, a minireplicon consisting of a reporter gene flanked by viral promoter sequences was cotransfected with plasmids encoding the structural proteins of FLUAV. The minireplicon was amplified by the reconstituted viral polymerase and packaged into infectious VLPs. Recently, successful generation of recombinant FLUAV entirely from cloned cDNAs was achieved by replacing the minireplicon with eight plasmids containing full-length sequences of the genomic segments (Neumann et al., 1999
; Fodor et al., 1999
). This system was a breakthrough in the field of orthomyxovirus research and allows manipulation of all genomic segments of FLUAV.
It is conceivable that such a rescue system could also be established for tick-borne orthomyxoviruses. This system may be a useful tool for several reasons. Firstly, THOV can serve as a simple model for study of the orthomyxovirus life-cycle since it encodes only the basic set of proteins required for multiplication and propagation of an orthomyxovirus genome, but lacks additional proteins such as the M2 ion channel, the NS2/NEP nuclear export factor and the nonstructural protein NS1 of FLUAV. Secondly, unlike FLUAV, THOV does not cause shutoff of host-cell protein synthesis (Siebler et al., 1996 ). Finally, THOV has been established as an ideal tool to investigate the function of the antivirally active, interferon-induced Mx proteins (Haller et al., 1995
; Frese et al., 1995
; Kochs & Haller, 1999
; Weber et al., 2000
).
We recently demonstrated the reconstitution of the functional RNA polymerase of THOV by expression of the three subunits of the viral polymerase (PB1, PB2 and PA) from cDNA plasmids in eukaryotic cells. Together with recombinant NP, the polymerase complex was able to transcribe a minigenome RNA containing THOV promoter sequences (Weber et al., 1998 , 2000
). In the present study, we employed an RNA polymerase I-based expression system for intracellular generation of minireplicons and extended the system by additionally expressing GP and M to generate infectious THO-VLPs.
To detect newly formed VLPs at a single cell level, we generated a negative-sense minireplicon construct encoding the green fluorescent protein (GFP), pPolI-THOV/GFP(-). A cDNA encoding the GFP reporter gene in negative-sense flanked by the noncoding end sequences of THOV segment 5 was inserted into the BsmBI site of pHH21 between the human RNA polymerase I promoter and terminator regions (kindly provided by Gerd Hobom, Justus Liebig-University, Giessen, Germany; Neumann et al., 1999 ). As a first step, this reporter plasmid was cotransfected with four T7-driven protein expression plasmids encoding the four components of the viral nucleocapsids (pG7-PA, pBS-PB1, pBS-PB2, pG7-NP) in Vero cells using LipofectAMINE (GibcoBRL). Six hours post-transfection, the cells were infected with MVA-T7, a recombinant vaccinia virus expressing the T7 RNA polymerase (kindly provided by Gerd Sutter, GSF-Neuherberg, Germany; Sutter et al., 1995
), at an m.o.i. of 10 and incubated for 40 h. Subsequent examination of the transfected cells by fluorescence microscopy revealed high level expression of GFP (data not shown), indicating that the GFP minigenome was accepted by the recombinant polymerase complex and that transcriptionally active nucleocapsids were reconstituted.
We then added expression plasmids encoding GP and M of THOV to the transfection mixture. The ORFs encoding GP and M were amplified from THOV vRNA by RTPCR and cloned into pBluescript under the control of the T7 RNA polymerase promoter to generate pBS-GP and pBS-M, respectively. In this extended system, cells were transfected with seven different plasmids. The highly attenuated recombinant vaccinia virus allowed long incubation times (over 50 h) to be used without any cytopathic effect. The expression of the two additional recombinant proteins had no obvious effects on the accumulation of GFP in the transfected cells (Fig. 1A). In order to demonstrate formation of THO-VLPs, supernatant from the transfected cells was transferred to a fresh Vero cell monolayer (indicator cells). Passaging the supernatants did not lead to detectable GFP signals in the indicator cells (data not shown). Easily detectable amounts of GFP, however, were produced when cells were infected with THOV [Sicilian isolate (SiAr126); Albanese et al., 1972
] prior to incubation with the VLP-containing supernatants. Pre-infection with THOV at an m.o.i. of 5 appeared to lead to amplification and transcription of the transferred minireplicon by the polymerase complex of the helper virus (Fig. 1B
). Quantification of the GFP-expressing indicator cells showed that the transfected cells produced approximately 7x103 infectious VLPs per ml of supernatant. Pre-infection of the indicator cells was also required for the production of detectable amounts of chloramphenicol acetyltransferase (CAT) activity in an analogous experiment using pPolI-THOV/CAT(-) (see Fig. 3B
, lanes 1 and 2), indicating that the activity of the recombinant polymerase complex packaged into the VLPs is too low to express detectable amounts of the reporter protein. No minigenome packaging could be detected when the GP expression plasmid was omitted from the transfection mixture (Fig. 1C
, D
).
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In summary, this plasmid-based reverse genetics system for THOV allows the reconstitution of functional vRNPs that can be packaged into infectious VLPs. All six structural proteins of THOV were required for this rescue. In previous experiments with FLUAV, the nine viral structural proteins were required for the generation of infectious FLUA-VLPs (Mena et al., 1996 ; Gomez-Puertas et al., 1999
; Neumann et al., 2000
). THOV has only six structural proteins, all of which are needed for VLP formation, indicating that functions required for FLUA-VLP formation (e.g. the M2 ion channel or the NS2/NEP nuclear export protein) might be dispensable for THOV passaging or might be encoded by one of the six THOV genes. This THOV system can be used to further analyse the function of the six viral proteins and of cis-acting sequence elements regulating virus replication, transcription, as well as particle morphogenesis. Furthermore, the system described here might serve as a basis for the rescue of a recombinant THOV entirely from cloned cDNAs.
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References |
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Received 13 June 2000;
accepted 24 August 2000.