The CDC has identified as a priority the need for 'simple detection methods that are more sensitive and more specific than the current EIA' (Glass et al 1996). Such methods would permit larger scale epidemiological studies and improve understanding of astrovirus infection and immunity. To enhance detection of astrovirus, we propose development of a reporter cell line. Such cell lines have been described for several other viruses (Rocancourt et al 1990, Kimpton & Emerman 1992, Stabell & Olivo 1992, Olivo et al 1994), and their design depends on the replication strategy of the virus to be detected.
The replication and transcription scheme postulated for astrovirus is inferred from the well characterized life cycle of alphaviruses such as Sindbis virus. Astroviruses have a similar genome organization (5'-non-structural—structural-3') and structural protein translation strategy (subgenomic RNA) to alphaviruses (Schlesinger & Schlesinger 1996). Upon infection, the non-structural proteins are translated from the input positive strand viral genome. The non-structural proteins then participate in transcribing a full-length negative strand RNA which subsequently serves as the template for the transcription of new genomic and subgenomic RNAs. The capsid precursor protein is finally produced by translation of the subgenomic RNA. Detection of newly synthesized capsid protein indicates viral replication has occurred.
On the basis of this strategy, development of a reporter cell line for astrovirus requires, first, assembly of a reporter construct, a cDNA copy of a defective astroviral genome that is deficient in some transreplication function (Fig. 5). The construct carries the reporter gene for green fluorescent protein (GFP) inserted inframe into ORF2. Subsequently, the construct is stably inserted into the genome of Caco-2 cells, under the control of a Rous sarcoma virus promoter which should give rise to constitutive expression of a replication- and transcription-deficient astroviral reporter construct in the uninfected cell. In this state, no subgenomic RNA is expected to be transcribed due to the defect in a critical non-structural protein. In addition, it is unlikely that any GFP reporter protein will be translated directly from the defective genomic RNA since that would require internal ribosomal initiation. When the reporter cells are infected with a wildtype astrovirus, functional non-structural proteins translated from the infecting virus should complement in trans the defective replication and transcription of the reporter construct, leading to transcription ofa subgenomic RNA and, ultimately, expression of GFP. The infected reporter cells should be detectable by their green fluorescence when observed with an optical fluorescence microscope.
This assay has several potential applications, including the determination of whether a clinical or field sample contains infectious astrovirus. In the laboratory, this assay could be used in place ofthe plaque assay to quantify the infectious titre of a viral sample, purify astrovirus or isolate neutralization escape mutants.
Our preliminary reporter construct is based on the ability to complement ORF1b function (U. Geigenmuller & S. M. Matsui, unpublished data). The construct, pAT3329-10GFP (shown in Fig. 5), displays two important features. First, the deletion of a single nucleotide at position 3329 introduces a premature stop codon in the ORF1b product that results in production of a non-functional RNA-dependent RNA polymerase and impairment of RNA transcription. This defect can be complemented in trans by intact ORF1b product supplied by wildtype astrovirus. Second, ORF2 is modified to encode a capsid—GFP fusion protein in which the first 10 N-terminal amino acids of the astrovirus capsid are preserved and fused in-frame with GFP. This capsid—GFP fusion construct (10GFP) was selected among several others which were designed with longer stretches of retained astrovirus capsid sequence, because of the consistently strong green fluorescence demonstrated by this construct. Transcription of the subgenomic RNA, and hence expression of the capsid—GFP fusion protein, is dependent on the provision of functional ORF1b product by wild-type virus.
cDNA insert ^^ into pBK-RSV
ORF-1b RNA Pol II
FIG. 5. Defective human astro virus serotype 1 cDNA (pAT3329) with GFP reporter gene insert, and replication of its transcribed RNA. Black boxes at both ends indicate important plasmid features that surround the defective H-Astl/reporter gene construct. Astro virus sequence is shown in white, with the defect in ORFlb indicated by the black arrowhead and the premature stop codonit introduces indicated by (*). The GFP reporter gene is indicated by the box with the speckled pattern. The astrovirus subgenomic promoter, which is functional only in the negative strand and preserved in this construct, is denoted under the symbol (§).
A line of Caco-2 cells that has been stably transformed with pAT3329-lOGFP has been developed. No expression of GFP was detectable in these cells when uninfected. This cell line was then incubated with a cell culture-adapted strain of human astrovirus serotype l, and successfully infected cells were detected with astrovirus group-specific monoclonal antibody 8E7 (Herrmann et al l988) and Texas red. Initial examination by confocal microscopy revealed only a few cells with green fluorescence. By adjusting the brightness over these cells to a higher level, GFP expression was observed in every infected cell. Green fluorescence colocalized in the cytoplasm with expressed astrovirus capsid antigen (red). Green signal was also detected in the nucleus and nucleoli of infected cells since the 28.2 kDa capsid—GFP fusion protein is sufficiently small to diffuse freely between the nuclear and cytoplasmic compartments.
The most significant findings of our reporter cell line study were that GFP is expressed in every infected cell, lending support to the principle of complementation in trans upon which this assay is based, and that expression of GFP from the reporter construct is strictly dependent on trans-complementation. This pilot study is encouraging for the development of a simple, rapid and accurate detection assay for astrovirus infection, although many aspects of this system require further optimization. Of utmost importance is the need to improve sensitivity, if this test is to have practical applications for clinical and epidemiological studies.
In conclusion, the development of the astrovirus genomic clone pAVIC has enabled systematic exploration of many aspects of the molecular biology of astrovirus, by allowing the targeted introduction of mutations into the viral genome. At the level of the non-structural astroviral proteins, we have been able to show that the protease, encoded by ORFla, is involved in the processing of the ORFla translation product itself. We have also demonstrated that the function of the RNA-dependent RNA polymerase, encoded by ORFlb, can be complemented in trans. With regard to the structural viral proteins, we found that most of ORF2 can be replaced by a foreign gene, such as the GFP reporter gene, and display efficient expression of GFP in transfected cells. These findings suggest the use of astrovirus as a human expression vector and demonstrate the feasibility of establishing a reporter cell line for astrovirus. Finally, by introducing mutations into ORF2, we are studying requirements for viral assembly and formation of infectious viral particles.
This work was supported by a Department of Veterans Affairs Merit Review grant and National Institutes of Health grant R21 AI43513 (to S.M.M.). The Molecular and Cellular Biology Core Facilities of the Stanford Digestive Disease Center (National Institutes of Health grant DK 38707) were used. D.K. is supported by National Institutes of Health grant 1F32 DK09829.
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