1 International Centre for Genetic Engineering and Biotechnology, Padriciano 99, 34012 Trieste, Italy
2 Lay Line Genomics SpA, via di Castel Romano 100, 00128 Roma, Italy
3 Scuola Internazionale Superiore di Studi Avanzati, Ed. B, Padriciano 99, 34012 Trieste, Italy
Correspondence
Oscar R. Burrone
burrone{at}icgeb.org
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ABSTRACT |
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Present address: Institute Curie, INSERM U520, 12 Rue Lhomond, 75005 Paris, France.
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MAIN TEXT |
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As a reverse-genetic system is not yet available in rotavirus, we decided to use intracellular antibodies or intrabodies (ICAbs) as a protein knockout strategy to investigate the role of NSP5 during the virus replicative cycle. A novel and powerful technology to obtain intracellular-competent antibodies, termed intracellular antibody-capture technology, has recently been developed (Visintin et al., 1999, 2002
). It makes use of the two-hybrid system in yeast to select antibodies that are capable of binding antigens intracellularly. Although some recombinant antibodies expressed in the cell cytoplasm are able to maintain selective-binding properties against their antigens, a large number are unable to do so in the reducing cytoplasmic environment. Problems of folding and stability, mainly due to the lack of intrachain disulphide bonds of the light- and heavy-chain variable (VL and VH) domains (Biocca et al., 1995
), occur very often, resulting in non-functional molecules with low expression levels and a short half-life (Cattaneo & Biocca, 1997
, 1999
). However, some VL and VH domains, despite a lack of disulphide bonds, retain their antigen-binding capacity in non-natural reducing environments, such as the cytoplasm and nucleus of the cell (Proba et al., 1997
, 1998
). In particular, the single-chain Fv format (scFv) of antibodies (Bird et al., 1988
) has been shown to be suitable for intracellular expression (Marasco et al., 1993
; Tavladoraki et al., 1993
).
To select for anti-NSP5 ICAbs, we constructed an scFv library in the VL-linker-VH orientation from mice that had been immunized with recombinant GST-NSP5. The immunization protocol is described previously (Eichwald et al., 2002). The scFv library was fused to the transactivator protein VP16 and used in the yeast two-hybrid selection method as described by Hollenberg et al. (1995)
and Visintin et al. (1999)
. The target antigen (fused to the DNA-binding domain of LexA) was
2, an NSP5 deletion mutant (lacking aa 3480), because the wild-type NSP5 showed transactivation of the two reporter genes histidine (HIS3) and
-galactosidase (data not shown) (F. Vascotto & O. R. Burrone, unpublished data). From two different selections, we obtained five clones that were specific for the target protein
2, named A19, A22, D24, 14 and 20, which were well-expressed in the cytoplasm of yeast cells. For expression in mammalian cells, the scFv cassettes were subcloned into pcDNA3 (Invitrogen). When cotransfected into MA104 cells as described previously (Eichwald et al., 2002
), all five selected ICAbs retained specificity for the NSP5 mutant used for library selection (data not shown). However, to characterize their activity against the wild-type NSP5 protein, they were cotransfected with a construct encoding NSP5 fused to EGFP (NSP5EGFP) (Eichwald et al., 2004
). To divert localization, ICAbs were targeted to the nucleus by adding a C-terminal nuclear localization signal (NLS) (Persic et al., 1997
). In addition, all scFvs contained the 12 aa SV5 tag (Hanke et al., 1992
) for detection by immunofluorescence, as described previously (Fabbretti et al., 1999
). As a negative control, we used an irrelevant ICAb [3b(NLS)], which was selected from the library and validated as an intracellularly competent scFv (M. Visintin & A. Cattaneo, unpublished data). As shown by confocal microscopy, on transfection in MA104 cells, all five ICAbs showed nuclear localization, as expected, whereas NSP5EGFP had a homogeneous cytoplasmic distribution (Fig. 1a
). However, when ICAbs and NSP5EGFP were coexpressed in the same cell, three ICAbs (A19, D24 and 20) showed a dramatic change in their original cellular distribution and complete colocalization: ICAbs were retained in the cytoplasm and NSP5EGFP was no longer distributed homogeneously (Fig. 1b
). For these three selected ICAbs, we clearly detected characteristic intracellular structures that were reminiscent of aggresomes (Kopito & Sitia, 2000
), formed by their specific interaction with the antigen (NSP5EGFP). Two other ICAbs, A22(NLS) and 14(NLS), as well as the negative control, 3b(NLS), did not show any interaction, with complete nuclear localization and NSP5EGFP retaining its cytoplasmic distribution (Fig. 1b
). Furthermore, as expected, all five ICAbs selected did not recognize another non-structural rotavirus protein, NSP2 (NSP2EGFP) (Eichwald et al., 2004
). Fig. 1(c)
shows A19(NLS) coexpressed with NSP2EGFP as an example.
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To further investigate the effect of the selected ICAbs in virus replication, we used the retrovirus expression system (Zacchi et al., 2002) to increase the percentage of cells expressing ICAbs, as we were unable to select long-lived stable cell clones expressing ICAbs, probably due to toxicity effects. Retroviruses encoding two anti-NSP5 ICAbs, 20(NLS) and D24(NLS), and the negative control 3b(NLS), were used to infect the human lung carcinoma H1299 cell line. At 2 days post-infection, these cells were infected with rotavirus (m.o.i. approx. 1) and, 16 h later, cytopathic effect was analysed (Fig. 3a
) and viral dsRNAs were collected from infected cells and detected as described by Chen et al. (1990)
. As shown in Fig. 3(a)
, the strong cytopathic effect observed in control cells and in cells expressing the irrelevant ICAb 3b was suppressed in the presence of the two anti-NSP5-specific ICAbs. Moreover, they showed a partial, although significant, reduction (4045 %) in production of dsRNA compared with control cells (considered as 100 %) (Fig. 3b
). Despite this, we have not detected relevant differences in virus yields from cells expressing anti-NSP5 ICAbs in comparison with the negative control (data not shown). The effects observed may depend on the expression level of the different ICAbs, which was lower for the two anti-NSP5 ICAbs (Fig. 3c
).
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Temperature-sensitive mutants, genome reassortment and in vitro reconstitution studies have been the main approaches used to understand the function of rotavirus proteins, because of the limitations imposed by the lack of reverse genetics. However, no conclusive data on the role of NSP5 protein have been obtained so far.
Here, we describe the application of intracellular antibody-capture technology to select active cytoplasmic ICAbs as a protein knockout system, in order to investigate the function of rotavirus NSP5 (Visintin et al., 1999, 2002
). The phenotype observed upon NSP5 knockout by ICAbs is similar to those reported for the temperature-sensitive (ts) mutants affecting the structural rotavirus proteins related to replication, VP1 (tsC) and VP3 (tsB) (Chen et al., 1990
), as well as the for the non-structural protein NSP2 (tsE), which showed a lack of viroplasm formation and impaired viral ssRNA and dsRNA production at the non-permissive temperature (Chen et al., 1990
; Ramig & Petrie, 1984
; Taraporewala et al., 2002
). A similar conclusion was reported recently for NSP2 depletion by siRNA (Silvestri et al., 2004
). Our results indicate that NSP5 is also an essential element for the assembly of functional viroplasms and is therefore relevant for virus replication.
We have reached similar conclusions regarding the role of NSP5 in viroplasm formation and virus replication by means of an alternative knockout strategy using RNA interference specific for genomic segment 11 mRNA, which produced strong impairment of virus yield in MA104 cells (M. Campagna, C. Eichwald, F. Vascotto & O. R. Burrone, unpublished data). In contrast, ICAbs were not as efficient in blocking infective virus release, probably due to low levels of expression. A recent study using ICAbs specific for the NS1 protein of the bluetongue virus also demonstrated lack of virus inhibition, despite a clear reduction of the cytopathic effect (Owens et al., 2004).
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ACKNOWLEDGEMENTS |
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Received 1 March 2004;
accepted 21 July 2004.