Molecular Virology Laboratory, Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, LUMC E4-P, PO Box 9600, 2300 RC Leiden, The Netherlands
Correspondence
Eric Snijder
E.J.Snijder{at}LUMC.NL
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ABSTRACT |
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MAIN TEXT |
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Equine arteritis virus (EAV) is a +RNA virus belonging to the family Arteriviridae (order Nidovirales) and employs an elegant combination of translational and transcriptional regulation to coordinate the expression of its polycistronic genome (Fig. 1A; Snijder & Meulenberg, 2001
). EAV produces two replicase polyproteins that are cleaved into mature non-structural proteins (nsps) by proteinases contained in nsp1, nsp2 and nsp4 (Ziebuhr et al., 2000
). The structural proteins are encoded in the 3'-terminal quarter of the genome and are expressed from a nested set of subgenomic (sg) mRNAs. These sg RNAs (Fig. 1A
) consist of a common leader sequence, derived from the 5' end of the genome (de Vries et al., 1990
), and a body segment that represents a specific part of the 3'-proximal region of the genome, with one of the structural protein-coding regions at its 5' end. Our recent studies have indicated that the fusion of leader and body of EAV sg RNAs probably occurs by discontinuous minus-strand synthesis (Pasternak et al., 2000
; Sawicki & Sawicki, 1995
; van Marle et al., 1999
).
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Interestingly, the N-terminal cleavage product of the EAV replicase, nsp1, is completely dispensable for genome replication, but essential for sg RNA synthesis (Tijms et al., 2001; and unpublished data). The subgenomic RNA synthesis of an nsp1 deletion mutant could be restored by nsp1 expression from a locus in the 3'-proximal part of the genome. Both a predicted N-terminal zinc-finger domain (ZF; Fig. 1C
) and the structural integrity of nsp1, but not the proteolytic activity of its C-terminal papainlike cysteine proteinase domain (PCP
), were essential for this function (Fig. 1B
). Thus, nidovirus sg RNA synthesis and genome replication are separate processes and specific viral proteins can dedicate the RdRp complex to sg RNA synthesis (Tijms et al., 2001
).
The molecular details of EAV nsp1 function in sg RNA synthesis remain to be elucidated. Nsp1 may interact directly with the RdRp complex, template or nascent strand. Alternatively, nsp1 may act indirectly, e.g. by recruiting host factors to the RdRp complex. Interestingly, nsp1 is the only EAV replicase component that localizes both to the nucleus (Tijms et al., 2002) and to the viral replication complex, which is associated with modified cytoplasmic membranes (Pedersen et al., 1999
). Targeting of nsp1 to the nucleus is especially pronounced during the earlier stages of infection (Tijms et al., 2002
), but its implications remain unclear. The identification of viral or host proteins that interact with nsp1 might shed more light on the function(s) of nsp1 and/or the reason for its presence in the nucleus.
A yeast two-hybrid assay was used to screen for virus and host proteins that interact with EAV nsp1 (Chien et al., 1991; for reviews see Allen et al., 1995
; Fashena et al., 2000
). Nsp1 was expressed from the pAS2 vector (Clontech) as an N-terminal fusion to the GAL4 DNA-binding (DB) domain (nsp1DB). Experiments were carried out in Saccharomyces cerevisiae strains Y190 and AH109, yielding essentially identical results, and data obtained with Y190 will be presented here. Activation of lacZ and prototrophic growth reporter gene was tested by colony filter lift assays and growth on appropriate selective media respectively (Clontech Matchmaker manual; see also Serebriiskii & Golemis, 2001
). Expression of the nsp1DB bait did not activate histidine prototrophic growth (HIS3) and
-galactosidase (lacZ) reporter genes. The same was true on co-expression of the GAL4 trans-activation domain (TA) from the (empty) prey vector pACT2 (Clontech). Nsp1 was also expressed from the pACT2 vector (nsp1-pACT2) as an N-terminal fusion to the GAL4 TA (nsp1TA), which did not activate any of the reporter genes.
Nsp1DB and nsp1TA co-expression activated growth marker and -galactosidase expression in Y190, indicating that nsp1DB and nsp1TA interacted in the yeast two-hybrid system. The interaction depended on the N-terminal half of nsp1 (Table 1
), containing the putative ZF domain, but not on the integrity of ZF itself. Mutations that are assumed to disrupt ZF did not block the interaction, which was specific since nsp1 did not interact with the EAV nucleocapsid protein. This structural protein of arteriviruses also forms homomers in the yeast two-hybrid system (Table 1
; Wootton & Yoo, 2003
).
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Next, nsp1 was used as bait to screen a HeLa cell cDNA library (in pACT2; purchased from Clontech). Upon transfection with the EAV genome (van Dinten et al., 1997), HeLa cells support full EAV replication (L. C. van Dinten & E. J. Snijder, unpublished data), although they cannot be infected with EAV particles, probably due to the lack of appropriate receptors on their surface. S. cerevisiae was co-transformed with the pACT2-cDNA library and the nsp1-pAS2 bait, resulting in 6 million transformants, which approximated twice the complexity of this library. Thirty clones were recovered under growth conditions selective for the presence of prey and bait plasmids, 15 of which were also positive for activation of both ADE2 and HIS3 reporter genes. Upon retransformation of yeast cells with the isolated library plasmids and nsp1-pAS2, all clones remained positive for reporter gene activation. Sequence analysis and an NCBI database search subsequently identified 13 different potential interactors of nsp1 (Table 2
; see also below).
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The library clone encoded the C-terminal 229 amino acids of p100, constituting the tudor domain. Point mutations in ZF that knock-out the function of nsp1 in EAV sg RNA synthesis did not affect its interaction with p100, indicating it is probably independent of ZF (Tijms et al., 2001). Deletion of the C- or N-terminal half of nsp1, containing the PCP
and ZF domains, respectively, blocked the interaction with p100, suggesting that the structural integrity of nsp1 is important for interaction with p100, by analogy with its function in EAV sg RNA synthesis (Tijms et al., 2001
). Interactions of the p100 domain with other EAV nsps and the nucleocapsid protein were not detected (Table 1
), confirming the specificity of the nsp1p100 interaction. The only exception was the weak activation of the HIS3 reporter by co-expression of p100 and EAV nsp1112.
The observed nsp1p100 interaction might depend on yeast factors and/or the unusual nuclear localization of fusion proteins in this assay. To determine whether this interaction also occurred in the context of mammalian cells that can support EAV replication (RK-13 cells), a co-immunoprecipitation analysis was performed. Cells were transfected with vectors expressing the following proteins from a cytomegalovirus immediate early promoter: full-length p100 (CMV3-p100FL; Leverson et al., 1998), nsp1 (pBKnsp1; Tijms et al., 2002
), N- or C-terminal fusions of nsp1 to GFP (nsp1NnGFP and nsp1cGFP, respectively; Tijms et al., 2002
), or GFP.
As shown in Fig. 2(A), nsp1 and two nsp1GFP fusion proteins co-immunoprecipitated with p100. Nsp1 was also co-immunoprecipitated by the p100 antiserum in the absence of p100 expression, but this can be readily explained as co-immunoprecipitation with endogenous p100. The co-expression of p100 and nsp1 resulted in substantial death and loss of double-transfected cells, as observed by (immunofluorescence) microscopy, explaining the relatively strong nsp1 signal in cells transfected with the nsp1 expression vector only (Fig. 2A
; rightmost lane). Furthermore, the signal from co-precipitating nsp1 or nsp1GFP fusion proteins may have been enhanced by nsp1 multimerization (see above).
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Based on the presence of p100 in both nucleus and cytoplasm, the presence of the nucleic acid-binding tudor domain and its interactions with c-Myb, TFIIE ( and
subunits) and EBNA2, Tong et al. (1995)
postulated p100 to couple transcription to DNA replication in mammalian cells. Intriguingly, EAV nsp1 has also been proposed to couple genome replication and transcription (Tijms et al., 2001
). It is tempting to speculate that the nsp1p100 interaction documented here might be important for viral sg RNA synthesis, either directly or by recruiting a p100-binding protein to the viral RdRp complex. Alternatively, nsp1 might modulate transcription in the infected cell, explaining why the protein is targeted to the nucleus (Tijms et al., 2002
). Remarkably, three of the other proteins that were found to interact with nsp1 in our yeast two-hybrid screen (TFIIE
, Skip and BRG1; Table 2
) also interact with p100 and are involved in c-Myb-directed regulation of RNA polymerase II activity (Dahl et al., 1998
; Holstege et al., 1996
; Khavari et al., 1993
; Ness, 1999
; Tong et al., 1995
; Zhou et al., 2000
; Zhu & Kuziora, 1996
). Although the latter interactions need to be confirmed in an independent assay, the interaction of nsp1 with multiple c-Myb regulatory proteins would present an interesting analogy with EpsteinBarr virus EBNA2, which interacts with p100, Skip and TFIIE
and -
to establish transformation of the host cell (Ness, 1999
; Tong et al., 1995
). Future studies will be aimed at understanding the physiological importance of the interactions between EAV replicase subunit nsp1 and a set of proteins involved in transcriptional regulation of the host cell.
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ACKNOWLEDGEMENTS |
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Received 16 April 2003;
accepted 12 June 2003.