Self-association and mapping of the interaction domain of hepatitis E virus ORF3 protein

Hepatitis E virus (HEV) is a major human pathogen in the developing world. In the absence of an in vitro culture system, very little information on the basic biology of the virus exists. A small protein (approximately 13.5 kDa) of unknown function, pORF3, is encoded by the third open reading frame of HEV. The N-terminal region of pORF3 is associated with the cytoskeleton using one of its hydrophobic domains. The C-terminal half of pORF3 is rich in proline residues and contains a putative src homology 3 (SH3) binding domain and a mitogen-activated protein kinase phosphorylation site. In this study, we demonstrate that pORF3 can homodimerize in vivo, using the yeast two-hybrid system. We have isolated a 43-amino-acid interaction domain of pORF3 which is capable of self-association in vivo and in vitro. The overlap of the dimerization domain with the SH3 binding and phosphorylation domains suggests that pORF3 may have a dimerization-dependent regulatory role to play in the signal transduction pathway.     

The full-length and N-terminal deletion of ORF2 protein of hepatitis E virus can dimerize

Hepatitis E virus is a human RNA virus containing three open reading frames. Of these ORF2 encodes, the major capsid protein (pORF2), may possess regulatory functions, in addition to a structural one. In this study, we have shown using the yeast two-hybrid system and in vitro immobilization experiments that full-length pORF2 is capable of self-association, thus forming a homodimer. Using mutational analysis we have studied dimerization of various truncated versions of the ORF2 capsid protein using the yeast two-hybrid system and supported our findings with in vitro immobilization experiments. Deletions of pORF2 reveal a loss of the dimerization potential for all deletions except an N-terminal 127-amino-acid deletion. Our studies suggest that the dimerization property of pORF2 may not be amino-acid sequence-dependent but instead a complex formation of a specific tertiary structure that imparts pORF2 its property to self-associate.     

The 41-amino-acid C-terminal region of the hepatitis E virus ORF3 protein interacts with bikunin, a kunitz-type serine protease inhibitor

Hepatitis E virus (HEV), a human plus-stranded RNA virus, contains three open reading frames (ORF). Of these, ORF1 encodes the viral nonstructural polyprotein, ORF2 encodes the major capsid protein, and ORF3 codes for a phosphoprotein of undefined function. Recently, using the yeast two-hybrid system to screen a human cDNA liver library, we have isolated and characterized AMBP (alpha1-microglobulin/bikunin precursor), which specifically interacts with the ORF3 protein of HEV. The ORF3 protein expedites the processing and secretion of alpha1-microglobulin. When checked individually for interaction, the second processed protein from AMBP, bikunin, strongly interacted with the full-length ORF3 protein. This protein-protein interaction has been validated by immunoprecipitation in both COS-1 and Huh7 cells and by His6 pull-down assays. In dual-labeling immunofluorescent staining, followed by fluorescence microscopy of transfected human liver cells, ORF3 colocalized with endogenously expressed bikunin. Finally, a 41-amino-acid C-terminal region of ORF3 has been found to be responsible for interacting with bikunin. The importance of this virus-host protein-protein interaction, with reference to the viral life cycle, has been discussed.     

The ORF3 protein of hepatitis E virus is a phosphoprotein that associates with the cytoskeleton.

Hepatitis E virus (HEV) is a major human pathogen in the developing world. In the absence of an in vitro culture system, very little information exists on the basic biology of the virus. A small protein (approximately 13.5 kDa) of unknown function, pORF3, is encoded by the third open reading frame of HEV. We expressed pORF3 in transiently transfected COS-1 and Huh-7 cells and showed that it is a phosphoprotein which is modified at a serine residue(s). Deletion and site-directed mutants were created to establish Ser-80 as the phosphorylation site. This residue is present within a conserved primary sequence that showed consensus sites for phosphorylation by p34cdc2 kinase (cdc2K) and mitogen-activated protein kinase (MAPK). In vitro experiments with hexahistidine-tagged pORF3 expressed either in Escherichia coli or in COS-1 cells showed efficient phosphorylation with exogenously added MAPK. The pORF3 mutants also exhibited an in vitro phosphorylation profile with MAPK which was identical to that observed in vivo. In its primary sequence, pORF3 possesses two highly hydrophobic N-terminal domains. On subcellular fractionation, pORF3 was found to partition with the cytoskeletal fraction, and this association with the cytoskeleton was lost on deletion of hydrophobic domain I (amino acid residues 1 to 32). These results suggest that HEV pORF3 is a cytoskeleton-associated phosphoprotein and are discussed in terms of a possible function for pORF3 within the HEV replicative cycle.     

The ORF3 protein of hepatitis E virus binds to Src homology 3 domains and activates MAPK

The hepatitis E virus (HEV) is the causative agent of hepatitis E, an acute form of viral hepatitis. The biology and pathogenesis of HEV remain poorly understood. We have used in vitro binding assays to show that the HEV ORF3 protein (pORF3) binds to a number of cellular signal transduction pathway proteins. This includes the protein tyrosine kinases Src, Hck, and Fyn, the p85alpha regulatory subunit of phosphatidylinositol 3-kinase, phospholipase Cgamma, and the adaptor protein Grb2. A yeast two-hybrid assay was used to further confirm the pORF3-Grb2 interaction. The binding involves a proline-rich region in pORF3 and the src homology 3 (SH3) domains in the cellular proteins. Competition assays and computer-assisted modeling was used to evaluate the binding surfaces and interaction energies of the pORF3.SH3 complex. In pORF3-expressing cells, pp60(src) was found to associate with an 80-kDa protein, but no activation of the Src kinase was observed in these cells. However, there was increased activity and nuclear localization of ERK in the pORF3-expressing cells. These studies suggest that pORF3 is a viral regulatory protein involved in the modulation of cell signaling. The ORF3 protein of HEV appears to be the first example of a SH3 domain-binding protein encoded by a virus that causes an acute and primarily self-limited infection.     

A serine-kinase-containing protein complex interacts with the terminal protein domain of polymerase of hepatitis B virus

Polymerase of human hepatitis B virus is required for viral replication and pregenomic RNA encapsidation. Using recombinant GST fusion proteins, we show that the terminal protein domain of polymerase can interact specifically with a protein complex containing kinase activity and a tightly associated 35-kD protein (p35). This kinase is termed terminal-protein-associated kinase (TPAK). The phosphoamino acid analysis of phosphorylated p35 demonstrates that TPAK is a serine kinase. Analysis of deletion mutants shows that amino acids 1–95 of the terminal protein domain are required for the interaction with TPAK/p35 and phosphorylation of p35. TPAK/p35 are found predominantly in the cytoplasm. Furthermore, TPAK can be inhibited by heparin and manganese ions, but is resistant to spermidine, DRB, H89 or H7. These results indicate that TPAK is not protein kinase A or protein kinase C.     

Infectivity determinants of the hepatitis B virus pre-S domain are confined to the N-terminal 75 amino acid residues

The N-terminal pre-S domain of the large hepatitis B virus (HBV) envelope protein plays a pivotal role at the initial step of the viral entry pathway. In the present study, the entire pre-S domain was mapped for infectivity determinants, following a reverse-genetics approach and using in vitro infection assays with hepatitis delta virus (HDV) or HBV particles. The results demonstrate that lesions created within the N-terminal 75 amino acids of the pre-S region abrogate infectivity, whereas mutations between amino acids 76 and 113, overlapping the matrix domain, had no effect. In contrast to the results of a recent study (L. Stoeckl, A. Funk, A. Kopitzki, B. Brandenburg, S. Oess, H. Will, H. Sirma, and E. Hildt, Proc. Natl. Acad. Sci. 103:6730-6734, 2006), the deletion of a cell membrane translocation motif (TLM) located between amino acids 148 and 161 at the C terminus of pre-S2 did not interfere with the infectivity of the resulting HDV or HBV mutants. Furthermore, a series of large deletions overlapping the pre-S2 domain were compatible with infectivity, although the efficiency of infection was reduced when the deletions extended to the pre-S1 domain. Overall, the results demonstrate that the activity of the pre-S domain at viral entry solely depends on the integrity of its first 75 amino acids and thus excludes any function of the matrix domain or TLM.     

The central hydrophobic domain of the bovine papillomavirus E5 transforming protein can be functionally replaced by many hydrophobic amino acid sequences containing a glutamine.

The 44-amino-acid E5 transforming protein of bovine papillomavirus can induce growth transformation of cultured rodent fibroblast cell lines. Previous studies revealed that efficient transformation of mouse C127 cells by the E5 protein required a central core of hydrophobic amino acids and several specific carboxyl-terminal amino acids. Although a randomly derived sequence of hydrophobic amino acids could functionally replace the wild-type hydrophobic core, most such sequences could not. We show here that the conserved glutamine at position 17 in the hydrophobic domain is also important for transformation and that insertion of the glutamine can rescue the transforming activity of many but not all otherwise defective mutants containing random hydrophobic sequences. However, a class of mutants was identified that transform efficiently even in the absence of glutamine, demonstrating that the presence of this amino acid is not absolutely required for efficient transformation. E5 proteins containing the glutamine appear to display increased homodimer formation compared with mutant proteins lacking the glutamine, but this amino acid has no apparent effect on protein stability.     

The N-terminal domain of colicin E3 interacts with TolB which is involved in the colicin translocation step

Colicins use two envelope multiprotein systems to reach their cellular target in susceptible cells of Escherichia coli : the Tol system for group A colicins and the TonB system for group B colicins. The N-terminal domain of colicins is involved in the translocation step. To determine whether it interacts in vivo with proteins of the translocation system, constructs were designed to produce and export to the cell periplasm the N-terminal domains of colicin E3 (group A) and colicin B (group B). Producing cells became specifically tolerant to entire extracellular colicins of the same group. The periplasmic N-terminal domains therefore compete with entire colicins for proteins of the translocation system and thus interact in situ with these proteins on the inner side of the outer membrane. In vivo cross-linking and co-immunoprecipitation experiments in cells producing the colicin E3 N-terminal domain demonstrated the existence of a 120 kDa complex containing the colicin domain and TolB. After in vitro cross-linking experiments with these two purified proteins, a 120 kDa complex was also obtained. This suggests that the complex obtained in vivo contains exclusively TolB and the colicin E3 domain. The N-terminal domain of a translocation-defective colicin E3 mutant was found to no longer interact with TolB. Hence, this interaction must play an important role in colicin E3 translocation.     

The hepatitis E virus ORF3 protein regulates the expression of liver-specific genes by modulating localization of hepatocyte nuclear factor 4

The hepatitis E virus (HEV) is a small RNA virus and the cause of acute viral hepatitis E. The open reading frame 3 protein (pORF3) of HEV appears to be a pleiotropic regulatory protein that helps in the establishment, propagation and progression of viral infection. However, the global cellular effects of this protein remain to be explored. In the absence of traditional in vitro viral infection systems or efficient replicon systems, we made an adenovirus based ORF3 protein expression system to study its effects on host cell gene expression. We infected Huh7 hepatoma cells with recombinant adenoviruses expressing pORF3 and performed microarray-based gene expression analyses. Several genes down regulated in pORF3-expressing cells were found to be under regulation of the liver-enriched hepatocyte nuclear factor 4 (HNF4), which regulates hepatocyte-specific gene expression. While HNF4 localizes to the nucleus, its phosphorylation results in impaired nuclear localization of HNF4. Here we report that pORF3 increases HNF4 phosphorylation through the ERK and Akt kinases, which results in impaired nuclear translocation of HNF4 and subsequently the down modulation of HNF4-responsive genes in pORF3-expressing cells. We propose that modulation of several hepatocyte specific genes by pORF3 will create an environment favorable for viral replication and pathogenesis.     

The ORF3 protein of hepatitis E virus interacts with liver-specific alpha1-microglobulin and its precursor alpha1-microglobulin/bikunin precursor (AMBP) and expedites their export from the hepatocyte

Hepatitis E virus (HEV), a plus-stranded RNA virus contains three open reading frames. Of these, ORF1 encodes the viral nonstructural polyprotein; ORF2 encodes the major capsid protein and ORF3 codes for a phosphoprotein of undefined function. Using the yeast two-hybrid system to screen a human cDNA liver library we have isolated, an N-terminal deleted protein, alpha(1) -microglobulin/bikunin precursor (AMBP) that specifically interacts with the ORF3 protein of HEV. Independently cloned, full-length AMBP was obtained and tested positive for interaction with ORF3 using a variety of in vivo and in vitro techniques. AMBP, a liver-specific precursor protein codes for two different unrelated proteins alpha(1)-microglobulin (alpha(1)m) and bikunin. alpha(1) m individually interacted with ORF3. The above findings were validated by COS-1 cell immunoprecipitation, His(6) pull-down experiments, and co-localization experiments followed by fluorescence resonance energy transfer analysis. Human liver cells showing co-localization of ORF3 with endogenously expressing alpha(1) m showed a distinct disappearance of the protein from the Golgi compartment, suggesting that ORF3 enhances the secretion of alpha(1)m out of the hepatocyte. Using drugs to block the secretory pathway, we showed that alpha m was not degraded in the presence of ORF3. Finally, (1)pulse labeling of alpha(1)m showed that its secretion was expedited out of the liver cell at faster rates in the presence of the ORF3 protein. Hence, ORF3 has a direct biological role in enhancing alpha(1)m export from the hepatocyte.     

The preS1 protein of hepatitis B virus is acylated at its amino terminus with myristic acid.

The preS/S coding region of hepatitis B virus encodes two polypeptides (preS1 and preS2) that are larger in size but less abundant than the major viral surface antigen (S) protein. Unlike the preS2 and S proteins, the preS1 protein is preferentially localized on circulating virus particles but is not efficiently secreted from mammalian cells in culture. To search for differences in protein processing that might relate to these properties, we determined whether any of the hepatitis B virus surface proteins are acylated with long-chain fatty acids. Transfected COS cells expressing all three proteins were incubated with 3H-palmitate or 3H-myristate, and the cell extracts were examined by immunoprecipitation. While none of these proteins was labeled with 3H-palmitate, the preS1 protein but not the preS2 or S protein incorporated 3H-myristate via a hydroxylamine-resistant amide linkage. Comparison of the N-terminal amino acid sequences of hepadnaviral preS1 proteins with those of known myristylated proteins suggests that this unusual modification may be a common feature of all hepadnaviral preS1 proteins.     

The C-terminal hydrophobic domain of hepatitis C virus RNA polymerase NS5B can be replaced with a heterologous domain of poliovirus protein 3A

Replication of the plus-stranded RNA genome of hepatitis C virus (HCV) occurs in a membrane-bound replication complex consisting of viral and cellular proteins and viral RNA. NS5B, the RNA polymerase of HCV, is anchored to the membranes via a C-terminal 20-amino-acid-long hydrophobic domain, which is flanked on each side by a highly conserved positively charged arginine. Using a genotype 1b subgenomic replicon (V. Lohmann, F. Korner, J. O. Koch, U. Herian, L. Theilmann, and R. Bartensclager, Science 285:110-113, 1999), we determined the effect of mutations of some highly conserved residues in this domain. The replacement of arginine 570 with alanine completely abolished the colony-forming ability by the replicon, while a R591A change was found to be highly detrimental to replication, viability, and membrane binding by the mutant NS5B protein. Mutations of two other highly conserved amino acids (L588A and P589A) reduced but did not eliminate colony formation. It was of interest, if specific amino acid residues play a role in membrane anchoring of NS5B and replication, to determine whether a complete exchange of the NS5B hydrophobic domain with a domain totally unrelated to NS5B would ablate replication. We selected the 22-amino-acid-long hydrophobic domain of poliovirus polypeptide 3A that is known to adopt a transmembrane configuration, thereby anchoring 3A to membranes. Surprisingly, either partial or full replacement of the NS5B hydrophobic domain with the anchor sequences of poliovirus polypeptide 3A resulted in the replication of replicons whose colony-forming abilities were reduced compared to that of the wild-type replicon. Upon continued passage of the replicon in Huh-7 cells in the presence of neomycin, the replication efficiency of the replicon increased. However, the sequence of the poliovirus polypeptide 3A hydrophobic domain, in the context of the subgenomic HCV replicon, was stably maintained throughout 40 passages. Our results suggest that anchoring NS5B to membranes is necessary but that the amino acid sequence of the anchor per se does not require HCV origin. This suggests that specific interactions between the NS5B hydrophobic domain and other membrane-bound factors may not play a decisive role in HCV replication.     

The ORF2 protein of hepatitis E virus binds the 5' region of viral RNA

Hepatitis E virus (HEV) is a major human pathogen in much of the developing world. It is a plus-strand RNA virus with a 7.2-kb polyadenylated genome consisting of three open reading frames, ORF1, ORF2, and ORF3. Of these, ORF2 encodes the major capsid protein of the virus and ORF3 encodes a small protein of unknown function. Using the yeast three-hybrid system and traditional biochemical techniques, we have studied the RNA binding activities of ORF2 and ORF3, two proteins encoded in the 3' structural part of the genome. Since the genomic RNA from HEV has been postulated to contain secondary structures at the 5' and 3' ends, we used these two terminal regions, besides other regions within the genome, in this study. Experiments were designed to test for interactions between the genomic RNA fusion constructs with ORF2 and ORF3 hybrid proteins in a yeast cellular environment. We show here that the ORF2 protein contains RNA binding activity. The ORF2 protein specifically bound the 5' end of the HEV genome. Deletion analysis of this protein showed that its RNA binding activity was lost when deletions were made beyond the N-terminal 111 amino acids. Finer mapping of the interacting RNA revealed that a 76-nucleotide (nt) region at the 5' end of the HEV genome was responsible for binding the ORF2 protein. This 76-nt region included the 51-nt HEV sequence, conserved across alphaviruses. Our results support the requirement of this conserved sequence for interaction with ORF2 and also indicate an increase in the strength of the RNA-protein interaction when an additional 44 bases downstream of this 76-nt region were included. Secondary-structure predictions and the location of the ORF2 binding region within the HEV genome indicate that this interaction may play a role in viral encapsidation.     

The N-terminal transactivation domain of rat estrogen receptor is localized in a hydrophobic domain of eighty amino acids

In order to determine the transactivation domain(s) of the rat estrogen receptor (rat ER), we made a number of rat ER deletion mutants and transfected the mutant plasmids into COS7 cells together with an estrogen-responsive reporter plasmid, ERE-tk(197)-CAT, which contained the estrogen response element of Xenopus vitellogenin A2 gene. We have identified and localized the N-terminal transactivation domain of the rat ER to a hydrophobic region extending from Ala 59 to Glu 140.     

A bicistronic subgenomic mRNA encodes both the ORF2 and ORF3 proteins of hepatitis E virus

Hepatitis E virus replicons containing the neomycin resistance gene expressed from open reading frames (ORFs) 2 and 3 were transfected into Huh-7 cells, and stable cell lines containing functional replicons were selected by constant exposure to G418 sulfate. Northern blot analyses detected full-length replicon RNA and a single subgenomic RNA. This subgenomic RNA, which was capped, initiated at nucleotide 5122 downstream of the first two methionine codons in ORF3 and was bicistronic; two closely spaced methionine codons in different reading frames were used for the initiation of ORF3 and ORF2 translation.     

The 3a accessory protein of SARS coronavirus specifically interacts with the 5'UTR of its genomic RNA, Using a unique 75 amino acid interaction domain

More than four years have passed since the outbreak of the severe acute respiratory syndrome (SARS) epidemic, and still very little is known about the molecular biology and pathogenesis of this deadly virus. Among the accessory proteins of the SARS coronavirus (SARS-CoV), the 3a protein has been shown to interact with the spike, envelope, and membrane glycoprotein and has recently been established to be a structural component of capsid. Recent studies suggest that the 3a protein may function as an ion channel and may promote virus release. In order to further characterize the functional properties of this protein, we initiated studies to check its RNA binding activity. Using the yeast three-hybrid system, electrophoretic mobility shift assay (EMSA), and ultraviolet (UV) cross-linking techniques, we have shown that the 3a protein is capable of binding specifically to the 5' untranslated region (5'UTR) of the SARS virus genomic RNA. Further, we have mapped the interaction domain of the 3a protein responsible for this RNA-protein interaction using a series of deletion mutants and defined it to the central 75 amino acid region. This RNA binding motif of 3a does not share homology with any other known RNA binding protein and may have an important role in viral capsid assembly and pathogenesis.     

Expression and characterization of the hepatitis E virus ORF3 protein in the methylotrophic yeast,Pichia pastoris

We have used the methylotrophic yeast, Pichia pastoris, to express the open reading frame 3 (ORF3) of the hepatitis E virus (HEV). The ORF3 gene codes for a 123-amino-acid protein that contains highly immunodominant epitopes and is a potentially useful diagnostic and immunoprophylactic antigen. The expressed protein showed positive on immunoblots probed against antibodies raised in rabbit and infected human patient sera. In order to optimize the ORF3 protein expression, we have examined the regulated expression of this protein and characterized it. Unlike its expression in E. coli, the ORF3 protein was present in both the soluble and insoluble fractions of the cell lysate. The expressed protein is not glycosylated and does not undergo any major processing in the host strain.