Identification of Glyceraldehyde-3-phosphate Dehydrogenase as a Cellular Protein that Binds to the Hepatitis B Virus Posttranscriptional Regulatory Element

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). In the present study, we investigated the antiviral activity of MxA protein against HBV. MxA-expressing HuH7 clones were established and transiently transfected with HBV, and viral replication was then studied. Viral protein secretion was profoundly reduced in MxA-expressing clones by 80% for HBV surface antigen (HBsAg) and 70% for HBV e antigen (HBeAg). The levels of intracytoplasmic HBsAg and HBeAg were reduced by about 80 and 50% in the two MxA-positive clones tested. A nearly complete disappearance of HBV DNA replicative intermediates was observed in MxA-expressing clones. Although the expression of total viral RNAs was not modified, two-to fourfold reductions in HBV cytoplasmic RNAs were found in MxA-expressing clones. This suggests the inhibition of HBV replication at a posttranscriptional level. Indeed, using the well-characterized posttranscriptional regulation element (PRE) reporter system, we were able to demonstrate a marked reduction (three-to eightfold) in the nucleocytoplasmic export of unspliced RNA in MxA-expressing clones. In addition, MxA protein did not interact with HBV nucleocapsid or interfere with HBV nucleocapsid formation. Our results show an antiviral effect of MxA protein on a DNA virus for the first time. MxA protein acts, at least in part, by inhibiting the nucleocytoplasmic export of viral mRNA via the PRE sequence.

Section: Discussionmentioning

confidence: 99%

Inhibition of Hepatitis B Virus Replication by the Interferon-Inducible MxA Protein

Gordien

Rosmorduc

Peltekian

et al. 2001

167

126

“…It was constructed by using PCR to generate the entire human PTB cDNA and inserting this fragment into the XhoI and NotI sites of plasmid pCMV-myc/cyto (Invitrogen). Plasmid pGEM-FIII1, which contains the fragment III region of PRE under the transcriptional control of a T7 promoter, has been previously described (40). Mutants of pGEM-FIII1 were made by two-stage PCR (11).…”

Section: Methodsmentioning

confidence: 99%

“…Two cellular proteins, approximately 35 and 55 kDa in mass, that bind specifically to functionally important regions of the PRE were previously identified (21). The smaller protein was previously shown to be glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (40), whose functional role in PRE export is still unclear. In this report, we present evidence that the larger protein is the polypyrimidine tract binding protein (PTB), or hnRNP I, an RNA binding protein previously postulated to play a role in mRNA export.…”

mentioning

confidence: 99%

Role of Polypyrimidine Tract Binding Protein in the Function of the Hepatitis B Virus Posttranscriptional Regulatory Element

Zang

Huang

et al. 2001

Self Cite

The hepatitis B virus posttranscriptional regulatory element (PRE) is an RNA element that increases the expression of unspliced mRNAs, apparently by facilitating their export from the nucleus. We have identified a cellular protein that binds to the PRE as the polypyrimidine tract binding protein (PTB), which shuttles rapidly between the nucleus and the cytoplasm. Mutants of the PRE with mutations in PTB binding sites show markedly decreased activity, while cells that stably overexpress PTB show increased PRE-dependent gene expression. Export of PTB from the nucleus, like PRE function, is blocked by a mutant form of Ran binding protein 1 but not by leptomycin B. Therefore, PTB is important for PRE activity and appears to function as an export factor for PRE-containing mRNAs.Eucaryotic mRNA transcription takes place in the nucleus, but translation occurs in the cytoplasm, necessitating the export of mRNA through nuclear pores. In mammalian cells, this export is strictly controlled, in that only fully spliced and processed mRNA is exported (22,27). Part of the control is at the level of retention of incompletely spliced mRNA, probably by splicing factors binding to splice sites. However, this retention mechanism cannot explain all of the available data. First, some genes give rise to alternatively spliced transcripts, in which some of the mature mRNAs still contain splice sites. Second, for at least some cellular genes (e.g., the ␤-globin gene), removal of all introns leads to a defect in the export of mRNA to the cytoplasm (4). Therefore, the presence of splice sites does not always preclude RNA export, while the absence of splice sites does not always lead to export. These data imply that at least some mRNAs contain cis-acting elements that can effect export independently of splicing.In recent years, the existence of such RNA export elements has been confirmed. The best-studied element is the Rev response element (RRE) of human immunodeficiency virus (HIV) and related lentiviruses (6,14). Like almost all retroviruses, HIV contains only one promoter that gives rise to a transcript that is alternatively spliced, resulting in the export of completely spliced, partially spliced, and unspliced mRNAs into the cytoplasm. HIV codes for a trans-acting protein product that modulates the relative amounts of completely spliced versus incompletely spliced and unspliced messages. This protein, called Rev, binds to the RRE in the nucleus and strongly enhances the export of RRE-containing, unspliced or incompletely spliced transcripts. It has become clear that Rev contains a leucine-rich nuclear export signal (NES) that allows it to form a trimolecular complex with two cellular proteins, Crm1 (exportin) and Ran (8,9,29,34). This complex, together with its RNA cargo, interacts with components of the nuclear pore in order to migrate into the cytoplasm. Rev is then stripped off the mRNA in the cytoplasm and is recycled back into the nucleus by virtue of a nuclear localization signal.Other retroviruses also contain RNA export elemen...

“…Moreover, certain isoforms of GAPDH have been shown to be involved in specific functions (17,33). Recently, GAPDH has been shown to interact with the cis-acting RNAs of several viruses, including HPIV3 (11), hepatitis A virus (43), hepatitis B virus (54), rabies virus (A. K. Gupta et al, unpublished data), and measles virus (B. P. De et al, unpublished data). These findings suggest that GAPDH, besides being involved in normal cellular function, may also play a role in the regulation of gene expression of several viruses.…”

mentioning

confidence: 99%

Specific Phosphorylated Forms of Glyceraldehyde 3-Phosphate Dehydrogenase Associate with Human Parainfluenza Virus Type 3 and Inhibit Viral Transcription In Vitro

Choudhary

Banerjee

2000

). To gain further insight into these molecular interactions, we analyzed the virion-associated GAPDH and La protein using two-dimensional gel electrophoresis and immunoblotting. The GAPDH was resolved into two major and one minor molecular species migrating in the pI range of 7.6 to 8.3, while the La protein was resolved into five molecular species in the pI range of 6.8 to 7.5. The GAPDH isoforms present in the virions were also detected in the cytoplasmic fraction of CV-1 cell extract, albeit as minor species. On the other hand, the multiple molecular forms of La protein as seen within the virions were readily detected in the total CV-1 cell extract. Further analysis of virion-associated GAPDH by in vivo labeling with [ 32 P]orthophosphate revealed the presence of multiple phosphorylated species. The phosphorylated species were able to bind specifically to the viral cis-acting 3 genome sense RNA but failed to bind to the leader sense RNA, as determined by gel mobility shift assay. In contrast, the La protein isoforms present within the virions were not phosphorylated and bound to the viral cis-acting RNAs in a phosphorylation-independent manner. The GAPDH isoforms purified from the CV-1 cell cytoplasmic fraction inhibited viral transcription in vitro. Consistent with this, flag-tagged recombinant GAPDH synthesized by using the vaccinia virus expression system also inhibited viral transcription. Together, these data indicate that specific phosphorylated forms of GAPDH associate with HPIV3 and are involved in the regulation of virus gene expression.Human parainfluenza virus type 3 (HPIV3), a paramyxovirus, is second only to respiratory syncytial virus in causing severe respiratory tract infections in children and young adults (6). The single-stranded RNA genome of HPIV3, 15,461 nucleotides (nt) long, is contained within a helical nucleocapsid (15). Three virus-encoded proteins, the nucleocapsid protein N (68 kDa), the phosphoprotein P (90 kDa), and the RNA polymerase L (257 kDa), are associated with the nucleocapsid to form a transcribing ribonucleoprotein (RNP) complex (1, 15). The N protein enwraps the genome RNA, while the L and P proteins together constitute the RNA-dependent RNA polymerase complex that transcribes and replicates the N-bound genome RNA. During transcription, the RNA polymerase synthesizes a short leader RNA, followed by six capped and polyadenylated mRNAs. During replication, on the other hand, a full-length plus-strand copy of the genome RNA is synthesized which, in turn, serves as the template for synthesis of the minus-strand genome RNA (1, 6, 15). Studies from our laboratory, as well as others, indicate that cellular cytoskeletal proteins such as actin and tubulin are involved in the activation of transcription of several paramyxoviruses (4,9,12,22,34,35). Similarly, cellular RNA binding proteins that interact with the viral cis-acting elements are believed to be involved in the regulation of transcription and replication (25,26,28).The minus-sense genomic 3Ј end and its complementary l...