Department of Microbiology, Immunology and Molecular Genetics, UCLA School of Medicine, University of California at Los Angeles, Los Angeles, California, CA-90095, USA1
Author for correspondence: Asim Dasgupta. Fax +1 310 206 3865. e-mail dasgupta{at}ucla.edu
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Abstract |
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The 2C protein encoded by the P2 region of the polyprotein is highly conserved among picornaviruses (Gorbalenya et al., 1989 ). During PV infection, 2C and its precursor 2BC migrate to the rough endoplasmic reticulum where they induce the formation of smooth membrane vesicles that bud off and become the site of viral RNA synthesis, the replication complex (Bienz et al., 1987
, 1992
, 1990
; Cho et al., 1994
; Teterina et al., 1997
). 2C is a multifunctional protein and some of these functions include ATPase and GTPase (Rodriguez & Carrasco, 1993
; Mirzayan & Wimmer, 1994
; Pfister & Wimmer, 1999
), membrane-binding (Kusov et al., 1998
; Echeverri & Dasgupta, 1995
; Aldabe & Carrasco, 1995
) and RNA-binding activities (Rodriguez & Carrasco, 1995
).
In a previous study, we have shown that PV-encoded 2C and the precursor polypeptide 2BC specifically interact with the 3'-terminal sequences of the negative-strand RNA, but not with the corresponding 5'-terminal sequences of the positive-strand RNA (Banerjee et al., 1997 , 2001
). We have also demonstrated that this interaction requires a stable stemloop structure to be present at the 3' terminus of the negative-strand RNA. Because PV replication occurs in the cytoplasmic membrane and the 2C protein is capable of interacting with both the membrane and the viral negative-strand RNA, it was hypothesized that the 2C protein anchors the negative-strand RNA to the membrane. This anchoring may be crucial for the synthesis of positive-strand RNA from the negative-strand RNA template. Here, we have extended our 2CRNA interaction studies to demonstrate that the purified 2C proteins from both HAV and HRV-14 also interact specifically with the 3'-terminal sequences of the corresponding negative-strand RNAs.
To examine the RNA-binding properties of HAV- and HRV-encoded 2C protein, plasmids containing the 2C-coding sequences were expressed in Escherichia coli. The expressed proteins had a T7 tag at the N terminus and a six residue histidine tag at the C terminus for ease of purification. The recombinant proteins were affinity purified through a Co2+-charged resin, as reported previously (Banerjee et al., 2000 ). As can be seen in Fig. 1
, both HAV- and HRV-encoded 2C polypeptides (molecular mass of approximately 45 kDa) were purified to near homogeneity, as judged by Coomassie staining after SDSPAGE. Both proteins reacted with a monoclonal antibody against the sequence of the T7 tag on Western blot. However, the PV-encoded 2C protein lacking the T7 tag did not react with the antibody, thus confirming the specificity of this antibody (Fig. 1B
, compare lanes 1 and 2).
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The specificity of RNAprotein complex formation was examined by competition gel retardation analyses. The addition of increasing quantities of unlabelled, homologous, negative-strand HAV RNA (3' UTR100) inhibited the formation of labelled RNAprotein complexes to approximately 10% of the control, while the addition of the same concentrations of an unlabelled, heterologous RNA of a similar size did not affect the formation of RNAprotein complexes (Fig. 3A, compare lanes 3 and 4 with lanes 5 and 6). The possibility that the heterologous RNA is less stable than the homologous competitor was ruled out by comparing its half-life with that of the homologous RNA (data not shown). These results suggest that the HAV 2C protein specifically binds to the 3'-terminal sequence of the negative-strand UTR. Similar results were observed for the HRV 2C protein (Fig. 3B
).
UV cross-linking studies were performed to confirm the results obtained by gel retardation analysis. The nucleoprotein complex that was formed between the HRV 2C protein and the 3' negative- or 5' positive-strand RNA probes was subjected to UV irradiation and the resulting complex was analysed by SDSPAGE, as detailed earlier (Banerjee et al., 2000 ). As can be seen in Fig. 3(C)
, a distinct UV cross-linked band was detected with the 3' negative-strand RNA probe (3' UTR100) when the binding reaction contained the 2C protein (Fig. 3C
, compare lanes 4 and 3). A very faint proteinnucleotidyl complex was detected when the protein was incubated with the 5' positive-strand RNA probe (Fig. 3C
, lane 2). To examine the stability of the RNA probes, labelled nucleoprotein complexes formed after incubation were deproteinized and analysed by denaturing gel electrophoresis. As shown in Fig. 3(C
, lower panel), both the positive- and negative-strand RNA probes were equally stable in either the absence or the presence of 2C. UV cross-linking analysis with the purified HAV 2C protein using the viral UTR sequence from the positive- as well as the negative-strand RNA also showed that HAV 2C interacted with the 3' negative-strand UTR more efficiently than with the 5' positive-strand UTR (Fig. 3D
). To prove that the polypeptide bound to the 3' negative-strand UTR probe during gel retardation analysis was indeed 2C, the HRV 2C complex was excised from the native gel (Fig. 2B
) and resolved by second dimension SDSPAGE, followed by silver-staining and simultaneous Western blot analysis using an antibody against the N-terminal T7 tag epitope. As can be seen in Fig. 3(E)
, the RNAprotein complex was resolved into a single polypeptide that reacted to the antibody directed against the T7 tag epitope and also migrated to the range of the expected molecular mass.
The results presented in this communication suggest strongly that, like the PV 2C protein, both HRV- and HAV-encoded 2C polypeptides are capable of forming specific complexes with the 3'-terminal negative-strand RNA. Very little, if any, complex was detected when HRV and HAV 2C protein was incubated with the complementary positive-strand RNA.
Previous results from our laboratory have shown that N-terminal sequences of PV 2C encompassing amino acids 2154 and containing the putative amphipathic helix appear to play an important role in membrane binding both in vitro and in vivo (Echeverri & Dasgupta, 1995 ; Echeverri et al., 1998
). The inherent capacity of this protein to bind both the membrane and the 3' end of viral negative-strand RNA suggests that 2C may be involved in anchoring negative-strand RNA to the virus-induced membranous complex. Such an anchoring mechanism may be essential to keep the 3' end of the negative-strand RNA immobilized so that other viral and host proteins complexed to the 5' positive-strand cloverleaf structure (3CD3ABp36) may be transferred to the 3' end of the negative-strand RNA (Andino et al., 1993
; Xiang et al., 1995
; Harris et al., 1994
; Parsley et al., 1997
). In turn, this would facilitate the initiation of positive-strand RNA synthesis from the membrane-anchored negative-strand RNA. Results published recently have shown that the HAV 2C/2BC protein induces membrane rearrangement and is capable of binding eukaryotic membranes and, also, that this membrane-binding ability resides within the N-terminal amphipathic helix of 2C (Kusov et al., 1998
; Monika et al., 1988
; Teterina et al., 1992
, 1997
). Thus, like its PV counterpart, HAV 2C is capable also of interacting with both 3'-terminal negative-strand RNA sequences (this report) and cellular membranes. These observations, therefore, reinforce our view that the 2C protein may be involved in anchoring negative-strand RNA into the intracellular membrane structures (membranous replication complexes) and aid in the initiation of positive-strand RNA synthesis. In contrast to our findings, a recent report has shown that the 2C protein from echovirus type 9, also a picornavirus, exhibits nonspecific binding when an RNA sequence (371 bases) was used in the assay (Klein et al., 2000
). The precise reason for this discrepancy is not known, but it could be due to the fact that no nonspecific RNA was used in the binding reaction.
The results presented here do not rule out the possibility that the interaction of other viral or host cell proteins with the 5'- and 3'-terminal sequences of viral positive- and/or negative-strand RNA is important for viral genome replication. In fact, interaction of HAV 3AB and 3ABC with the 5'- and 3'-termini of the HAV RNA as well as binding of host cell proteins to rhinovirus 3'-terminal sequences have been reported (Kusov et al., 1997 ; Todd et al., 1995
; Mellits et al., 1998
). Future studies will be directed towards understanding the precise role of the 2C protein in virus replication and mapping the sequence contributing towards the specificity.
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References |
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Received 11 April 2001;
accepted 31 July 2001.