Hepatitis C virus (HCV) chronically infects approximately 170 million people worldwide, with an increased risk of developing cirrhosis and hepatocellular carcinoma. The study of HCV replication and pathogenesis has been hampered by the lack of an efficient stable cell culture system and small-animal models of HCV infection and propagation. In an effort to develop a robust HCV infection system, we constructed stable human hepatoma cell lines that contain a chromosomally integrated genotype 2a HCV cDNA and constitutively produce infectious virus. Transcriptional expression of the full-length HCV RNA genome is under the control of a cellular Pol II polymerase promoter at the 5 end and a hepatitis delta virus ribozyme at the 3 end. The resulting HCV RNA was expressed and replicated efficiently, as shown by the presence of high levels of HCV proteins as well as both positive-and negative-strand RNAs in the stable Huh7 cell lines. Stable cell lines robustly produce HCV virions with up to 10 8 copies of HCV viral RNA per milliliter (ml) of the culture medium. Subsequent infection of naïve Huh7.5 cells with HCV released from the stable cell lines resulted in high levels of HCV proteins and RNAs. Additionally, HCV infection was inhibited by monoclonal antibodies specific to CD81 and the HCV envelope glycoproteins E1 and E2, and HCV replication was suppressed by alpha interferon. Collectively, these results demonstrate the establishment of a stable HCV culture system that robustly produces infectious virus, which will allow the study of each aspect of the entire HCV life cycle.Discovered in 1989 by molecular cloning (10), hepatitis C virus (HCV) has been recognized as a major cause of viral hepatitis in humans. HCV infection is characterized by the establishment of chronic infection in the majority (up to 85%) of individuals exposed to HCV. It is estimated that approximately 4 million people in the United States and 170 million people worldwide are persistently infected (9, 38). The chronic HCV infection carries an increased risk of developing fatal liver diseases such as cirrhosis, liver failure, and hepatocellular carcinoma. HCV is a single-stranded positive-sense RNA virus belonging to the Hepacivirus genus of the family Flaviviridae (30). The 9.6-kb RNA genome encodes a single polyprotein that is cleaved by cellular and viral proteases into at least 10 structural (C, E1, E2, and probably p7) and nonstructural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins that play important roles in virus entry, replication, assembly, and pathogenesis (24, 29). The sequence and structures of the untranslated regions (UTR) at both the 5Ј and 3Ј ends of the HCV RNA genome, which contain cis-acting RNA elements required for HCV RNA translation and replication, are highly conserved (7,13,20,24,25,35,40,41).A great deal of progress has been made with respect to the HCV genome organization, properties and roles of viral proteins and conserved RNA sequence/structures, virus-host interactions, and mechanisms of HCV replication since the dis...
The IR2 protein (IR2P) is a truncated form of the immediate-early protein (IEP) lacking the essential acidic transcriptional activation domain (TAD) and serine-rich tract and yet retaining binding domains for DNA and TFIIB and nuclear localization signal (NLS). Analysis of the IR2 promoter indicated that the IR2 promoter was upregulated by the EICP0P. The IR2P was first detected in the nucleus at 5 h postinfection in equine herpesvirus 1 (EHV-1)-infected HeLa and equine NBL6 cells. Transient-transfection assays revealed that (i) the IR2P by itself downregulated EHV-1 early promoters (EICP0, TK, EICP22, and EICP27) in a dosedependent manner; (ii) the IR2P abrogated the IEP and the EICP27P (UL5) mediated transactivation of viral promoters in a dose-dependent manner; and (iii) the IR2P, like the IEP itself, also downregulated the IE promoter, indicating that the IEP TAD is not necessary to downregulate the IE promoter. In vitro interaction assays revealed that the IR2P interacts with TATA box-binding protein (TBP). The essential domain(s) of the IR2P that mediate negative regulation were mapped to amino acid residues 1 to 706, indicating that the DNA-binding domain and the NLS of the IR2P may be important for the downregulation. In transienttransfection and virus growth assays, the IR2P reduced EHV-1 production by 23-fold compared to virus titers achieved in cells transfected with the empty vector. Overall, these studies suggest that the IR2P downregulates viral gene expression by acting as a dominant-negative protein that blocks IEP-binding to viral promoters and/or squelching the limited supplies of TFIIB and TBP.The equine herpesvirus 1 (EHV-1) genome is comprised of 78 genes that are coordinately regulated and temporally expressed as immediate-early (IE), early (E), and late (L) ␥1 and true late ␥2 genes (10,16,17). The coordinated transcription of EHV-1 genes is regulated by six regulatory proteins that are expressed as one IE protein (IEP), four early proteins (EICP22P, EICP27P, EICP0P, and IR2P), and the late protein ETIF (5-7, 10, 13, 16-18, 20, 22, 24-26, 34-37). In addition, our ongoing work indicated that the IR3 transcript that is antisense to a portion of the IE mRNA negatively regulates IE gene expression (21; B. C. Ahn, S. K. Kim, and D. J. O'Callaghan, unpublished data).During a lytic infection, two transcripts arise from the IE open reading frame: a single, spliced 6.0-kb IE mRNA and a 3Ј-coterminal 4.4-kb early IR2 mRNA (20). The IEP (1,487 amino acids [aa]) is the major regulatory protein of EHV-1 and mediates the activation of transcription from E and some L viral promoters (34, 35) and is, thus, essential for replication (15). At the same time, the IEP downregulates its own promoter (34) and represses transcription of the true late glycoprotein K (gK) gene by binding to the transcription initiation site of the gK promoter (24). The IEP binds to the consensus binding sequence 5Ј-ATCGT-3Ј that overlaps the transcription initiation site of the IE promoter and to degenerate versions of this cognate...
The IR3 transcript of equine herpesvirus-1 (EHV-1) harbors 117 nts antisense to the immediate-early (IE) mRNA, suggesting it plays a regulatory role. Here, we show that the IR3 transcript downregulates IE gene expression and that the absence of IR3 expression altered EHV-1 biological properties and virulence in mice. Reporter assays revealed that the IR3/IE overlapping sequences [IR3(+226/+342)] and an additional IR3(+343/+433) region are necessary for the IR3 RNA to downregulate IE expression. Experiments with the ΔIR3 EHV-1 showed that the IR3 gene is dispensable for EHV-1 replication. Protein expression of the IE and representative EHV-1 genes was increased in cells infected with ΔIR3 EHV-1 as compared to that of cells infected with wt EHV-1. The ΔIR3 EHV-1 exhibited increased virulence in mice as compared to the parent virus. The finding that the IR3 transcript affects IE gene expression extends the role of RNA as a regulatory molecule in alphaherpesvirus infection.
The unique IR3 gene of equine herpesvirus 1 (EHV-1) is expressed as a late 1.0-kb transcript. Previous studies confirmed the IR3 transcription initiation site and tentatively identified other cis-acting elements specific to IR3 such as a TATA box, a 443 base pair 5'untranslated region (UTR), a 285 base pair open reading frame (ORF), and a poly adenylation (A) signal [Holden, V.R., Harty, R.N., Yalamanchili, R.R., O'Callaghan, D.J., 1992. The IR3 gene of equine herpesvirus type 1: a unique gene regulated by sequences within the intron of the immediate-early gene. DNA Seq. 3, 143-152]. Transient transfection assays revealed that the IR3 promoter is strongly trans-activated by the IE protein (IEP) and that coexpression of the IEP with the early EICP0 and IR4 regulatory proteins results in maximal trans-activation of the IR3 promoter. Gel shift assays revealed that the IEP directly binds to the IR3 promoter region. Western blot analysis showed that the IR3 protein produced in E. coli was detected by antibodies to IR3 synthetic peptides; however, the IR3 protein was not detected in EHV-1 infected cell extracts by these same anti-IR3 antibodies, even though the IR3 transcript was detected by northern blot. These findings suggest that the IR3 may not be expressed to a protein. Expression of an IR3/GFP fusion gene was not observed, but expression of a GFP/IR3 fusion gene was detected by fluorescent microscopy. In further attempts to detect the IR3/GFP fusion protein using anti-GFP antibody, western blot analysis showed that the IR3/GFP fusion protein was not detected in vivo. Interestingly, a truncated form of the GFP/IR3 protein was synthesized from the GFP/IR3 fusion gene. However, GFP/IR3 and IR3/GFP fusion proteins of the predicted sizes were synthesized by in vitro coupled transcription and translation of the fusion genes, suggesting poor expression of the IR3 protein in vivo. The possible role of the IR3 transcript in EHV-1 infection is discussed.
The equine herpesvirus 1 (EHV-1) negative regulatory IR2 protein (IR2P), an early 1,165-amino acid (aa) truncated form of the 1,487-aa immediate-early protein (IEP), lacks the trans-activation domain essential for IEP activation functions but retains domains for binding DNA, TFIIB, and TBP and the nuclear localization signal. IR2P mutants of the N-terminal region which lack either DNA-binding activity or TFIIB-binding activity were unable to down-regulate EHV-1 promoters. In EHV-1-infected cells expressing full-length IR2P, transcription and protein expression of viral regulatory IE, early EICP0, IR4, and UL5, and late ETIF genes were dramatically inhibited. Viral DNA levels were reduced to 2.1% of control infected cells, but were vey weakly affected in cells that express the N-terminal 706 residues of IR2P. These results suggest that IR2P function requires the two N-terminal domains for binding DNA and TFIIB as well as the C-terminal residues 707 to 1116 containing the TBP-binding domain.
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