Department of Medicine, Gastroenterology Section (111-GI), VA Palo Alto Health Care System, 3801 Miranda Avenue, Palo Alto, CA 94304, USA1
Division of Gastroenterology and Hepatology, Stanford University School of Medicine, Stanford, CA, USA2
Author for correspondence: Suzanne Matsui (at VA Palo Alto Health Care System). Fax +1 650 852 3259. e-mail sumatsui{at}Stanford.edu
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Abstract |
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The capsid protein with a calculated molecular mass 87 kDa appears to be processed into at least three proteins which are found on the mature, trypsin-activated viral particles. Depending on the serotype and the isolation method used, these proteins range in size from 24 to 26 kDa, 29 to 31·5 kDa and 32 to 34 kDa (Willcocks et al., 1990 ; Monroe et al., 1991
; Sánchez-Fauquier et al., 1994
; Belliot et al., 1997
; Bass & Qiu, 2000
). In one study (Sánchez-Fauquier et al., 1994
), N-terminal sequencing of the 26 and 29 kDa fragments indicated processing after aa 361 and 394 of the capsid protein, respectively. In a subsequent study (Bass & Qiu, 2000
), this cleavage was shown to occur extracellularly on the viral particle after addition of trypsin only. In addition, Bass & Qiu (2000)
proposed that intracellular processing of the capsid protein at aa 70/71 in the capsid protein is a prerequisite for virus assembly. The fate of the N-terminal 70 aa after cleavage was not determined because this region of the capsid protein could not be detected by the antibodies used in the study.
The high concentration of basic amino acids in the N-terminal region of the astroviral capsid protein is reminiscent of the capsid or coat proteins of other icosahedral RNA viruses, where a highly basic N-terminal region has been shown to be involved in packaging of the viral genomic RNA into the virion (Geigenmüller-Gnirke et al., 1993 ; Baer et al., 1994
; Rao & Grantham, 1996
; Schmitz & Rao, 1998
). It is therefore likely that the arginine- and lysine-rich sequence from aa 18 through aa 62 of the astroviral capsid protein also plays a crucial role in binding of the viral genomic RNA, and knowledge of its fate during and after capsid protein processing is critical for understanding astroviral assembly. However, tracking of the N-terminal portion of the capsid protein has been hampered by the fact that none of the available capsid protein-specific antibodies (Herrmann et al., 1991
; Sánchez-Fauquier et al., 1994
; Bass & Upadhyayula, 1997
) are directed at epitopes close to the N terminus.
Here we describe the construction of an infectious mutant of HAstV-1 expressing an antigenic epitope tag within the N-terminal region of the capsid protein which enables tracking of the proposed N-terminal processing product of the capsid protein with a commercially available antibody. In a control construct, a different tag was fused at the C terminus of the capsid protein, where no intracellular processing is expected. Using immunological reagents against the N-proximal and C-terminal tags, respectively, as well as antibodies against internal capsid protein-specific epitopes, we analysed processing of wild-type and epitope-tagged capsid protein in infected Caco-2 cells. In contrast to published results (Bass & Qiu, 2000 ), no intracellular processing of the capsid protein could be detected, while assembled viral particles were readily observed in infected cells.
Based on its proposed role in packaging of the viral RNA, the N-terminal region of the capsid protein is likely to be exposed on the inner surface of the capsid shell. It therefore seemed probable that the capsid protein would be able to tolerate deletions and insertions near its N terminus without losing the ability to form infectious viral particles. From studies of astroviral reporter constructs that encode capsid-reporter fusion proteins (Matsui et al., 2001 ), we knew that the 5' 30 nucleotides (nt) of ORF2, but not the 5' 6 nt, were sufficient to ensure efficient expression of ORF2. Deletions were introduced downstream of the 5' 30 nt of ORF2, preserving the N-terminal 10 aa of the capsid protein. Specifically, sequences encoding aa 1130 or aa 3150 of the capsid protein were deleted from the infectious astroviral cDNA clone, pAVIC (Geigenmüller et al., 1997
), and replaced by a 24 base-pair sequence containing three unique restriction sites (SmaI, PmlI, StuI), generating pAVIC
1130 and pAVIC
3150, respectively (Table 1
). To determine the infectious phenotype of these astroviral mutants, RNA was transcribed in vitro from pAVIC
1130, pAVIC
3150 and pAVIC with T7 DNA-dependent RNA polymerase (Promega), and used in Lipofectin-mediated transfection of BHK cells according to the manufacturers instructions (Invitrogen). After overnight incubation, cells plus supernatant were harvested, and the titre of viral particles generated by the transfected BHK cells was determined by infection of Caco-2 cells as described previously (Geigenmüller et al., 1997
). Successfully infected Caco-2 cells were identified by immunostaining with a capsid-protein specific monoclonal antibody, mAb 8E7 (Herrmann et al., 1991
), and a secondary goat anti-mouse Texas Red- or FITC-conjugated IgG, and observed under an immunofluorescence microscope. Both mutant RNAs, encoding capsid proteins lacking aa 1130 or 3150, gave rise to infectious viral particles. Compared to AVIC RNA, the titre of infectious units derived from AVIC
3150 RNA was reduced dramatically by a factor of 1000 (Table 1
). In contrast, infectivity of AVIC
1130 RNA was only 2-fold lower than that of wild-type AVIC RNA. Thus, the region of the capsid protein encompassing aa 1130 shows a high tolerance for deletions and insertions in the context of the viral particle. This finding supports the initial hypothesis that the N-terminal part of the capsid protein remains exposed within the virion and is involved in packaging of the viral RNA.
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To analyse processing of both the wild-type capsid protein and the tagged versions encoded by pAVIC-His and pAVIC-Strep, Caco-2 cells were either infected with wild-type HAstV-1 at an m.o.i. of 0·5, or with viral particles generated by BHK cells that had been transfected with AVIC-RNA, AVIC-His RNA or AVIC-Strep RNA. After overnight incubation in the absence of trypsin, the infected Caco-2 cells were placed in methionine/cysteine-free medium for 30 min, and then metabolically labelled with [35S]methionine and [35S]cysteine (100 µCi Tran35S-label per well, ICN Biomedicals) for 30 min. This labelling pulse was followed by a 3 h chase with medium containing unlabelled methionine and cysteine, following the conditions described by Bass & Qiu (2000) . Subsequently cells were lysed, and the lysates subjected to immunoprecipitation with a panel of capsid protein- or tag-specific antibodies (Gilbert & Greenberg, 1997
). Capsid protein containing the C-terminal Strep-tag was detected by direct precipitation with streptavidinagarose according to the suppliers instructions (Gibco-BRL). All precipitates were separated by SDSPAGE (Laemmli, 1970
).
Three antibodies were used for immunoprecipitation. While the monoclonal antibody mAb His (Qiagen) is directed against the N-proximal epitope expressed only by AVIC-His, the polyclonal antibody pAb 14 and the monoclonal antibody mAb 8E7 both recognize capsid protein-specific epitopes. Specifically, pAb 14 was produced in mice inoculated with a bacterially expressed dihydrofolate reductase fusion protein containing aa 395488 of the capsid protein. This peptide sequence was previously identified as an immunoreactive epitope on mature viral particles (Matsui et al., 1993
), and is contained within the 29 kDa and 26 kDa capsid protein cleavage products. The epitope for mAb 8E7, which was raised against complete astrovirus (Herrmann et al., 1991
), was narrowed down to aa 71260 of the capsid protein by deletion analysis (U. Geigenmüller, unpublished data).
Surprisingly, all of the reagents used for immunoprecipitation detected only one major capsid protein-derived band of about 98 kDa in infected Caco-2 cells (Fig. 1A). The reactivity of the 98 kDa fragment with all of the immunological reagents, directed against the N terminus (mAb
His), the C terminus (streptavidin) and two central regions of the capsid protein (mAb 8E7, pAb 14), unambiguously identified it as the full-length capsid protein. There was no indication of any intracellular processing of the capsid protein close to the N terminus. Two fainter bands of about 82 kDa and 75 kDa, that were also seen in Fig. 1(A)
, were detected with both mAb 8E7 and mAb
His, and therefore cannot represent products of cleavage at or near the N terminus, which would remove the His-tag from any C-terminal cleavage product. Instead, these fainter bands could result from either non-specific degradation or specific cleavage of a small portion of the full-length capsid protein near the C terminus. The lack of processing of the 98 kDa fragment close to the N terminus is unlikely to be due to an inhibitory effect of the N-proximal located His-tag, because no processing of the 98 kDa band could be detected in Caco-2 cells expressing wild-type capsid protein, either. Detection of ordered aggregates of viral particles in those cells by electron microscopy (Fig. 2
) furthermore indicates that the unprocessed capsid protein can assemble into viral particles. At present there is no obvious explanation for the discrepancy between the data presented here and those published by Bass & Qiu (2000)
. However, studies on the processing of the capsid protein of HAstV-8 have also failed to detect cleavage close to the N terminus (E. Méndez, personal communication).
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In summary, we describe the construction of two infectious mutants of HAstV-1 which code for capsid proteins carrying either an N-proximal His-tag or a C-terminal Strep-tag. The region of the capsid protein encompassing aa 1130 was found to be highly tolerant of deletions and insertions, and an antigenic His-tag inserted in between aa 10 and 31 was readily accessible to antibody recognition on capsid protein present in cell lysates. Similarly, a 9 aa Strep-tag replacing aa 783787 of the capsid protein could be detected by binding to streptavidin. When Caco-2 cells were infected with wild-type HAstV-1 or the HAstV-1 mutants encoding tagged capsid proteins, no intracellular processing of the astroviral capsid protein could be detected. Since ordered arrays of assembled viral particles were clearly visible in infected cells, virus assembly does not seem to depend on prior processing of the capsid protein.
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Acknowledgments |
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We are indebted to Lyndon Oshiro for his help with the electron microscopy studies.
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References |
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Received 3 October 2001;
accepted 18 February 2002.