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S Jameel

Publications and source records attributed to S Jameel.

At least 19 recordsLinked to original sources
PubMed2001

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.

Amino Acid Sequence↗
PubMed2001

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.

Amino Acids↗
PubMed2001

A yeast two-hybrid study on self-association of the ORF2 protein of hepatitis E virus.

Hepatitis E virus is a human RNA virus containing three open reading frames. Of these, ORF2 encodes the major capsid protein (pORF2) and 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.

Dimerization↗
PubMed2001

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.

Dimerization↗
PubMed2001

Role of chest CT in the follow-up of ovarian adenocarcinoma.

OBJECTIVE: We describe the prevalence of metastatic chest disease in ovarian adenocarcinoma as seen on CT. We sought to determine whether routine chest CT added any pertinent information to the follow-up examination of patients with ovarian adenocarcinoma. MATERIALS AND METHODS: Retrospective review of our tumor registry yielded 96 patients with ovarian adenocarcinoma who had only a single primary malignancy and at least one CT scan of the chest, abdomen, and pelvis. CT scans were reviewed to assess the presence of metastatic chest disease in relation to disease activity in the abdomen and pelvis. Chest CT findings were correlated with the physical examination findings and CA-125 levels and were reviewed in consultation with a gynecologic oncologist to select only those patients with chest abnormalities attributable to metastatic disease. RESULTS: A total of 266 CT scans were obtained. Forty (41.7%) of the 96 patients had abnormalities attributable to metastatic chest disease on one or more scans. In the absence of disease progression in the abdomen and pelvis, chest disease progression was seen in only six (2.7%) of the 226 follow-up CT scans. Five of the six patients had rising CA-125 levels. CONCLUSION: Correlation of the findings of abdominal and pelvic CT with the physical findings and the CA-125 levels serves as effective follow-up in patients with ovarian adenocarcinoma. The contribution of additional chest CT in these patients is small.

Adenocarcinoma↗
PubMed2000

Hepatitis E virus in Cuba.

The hepatitis E virus (HEV) has a global distribution and is known to have caused large waterborne epidemics of icteric hepatitis. The transmission is primarily fecal-oral. Some reports have suggested parenteral transmission of HEV from its association to hepatitis B or hepatitis C infection, or due to the development of hepatitis E after blood transfusion. Though most of the developing countries in Asia and Africa have been shown to be endemic for HEV infection, studies in the Latin American countries have been limited to Mexico, Brazil and Venezuela. We have developed an enzyme immunoassay (EIA) for IgM and IgG antibodies to a recombinant protein containing antigenic epitopes of the ORF3 region of the HEV. This system, as well as a commercial kit that includes ORF2 and ORF3 antigenic epitopes, were used to study the prevalence of anti-HEV antibodies in a sample of Cuban blood donors, acute hepatitis cases and individuals subjected to plasmapheresis. The incidence of anti-HEV IgM was compared with other viral hepatitis markers. Our findings suggest that infections due to HEV are an important viral cause of sporadic hepatitis in Cuba, and that HEV is endemic to this region of the world.

Adult↗
PubMed2000

Augmentation of immune responses to hepatitis E virus ORF2 DNA vaccination by codelivery of cytokine genes.

DNA vaccines encoding a viral structural protein have been shown to induce antiviral immune responses and provide protection against subsequent viral challenge. In the present study we show that DNA immunization with a plasmid expressing the hepatitis E virus ORF2 structural protein (pcDNA-ORF2) induced low levels of long-lasting antibody responses in the murine model. The use of plasmids expressing interleukin-2 (IL-2) and granulocyte-macrophage colony-stimulating-factor (GM-CSF) in conjunction with pcDNA-ORF2 enhanced the antibody responses generated by pORF-2. We further show that the immune responses generated by plasmid pcDNA-ORF2 can be boosted with virus-like particles composed of the ORF2 protein expressed through a baculovirus expression system.

Animals↗
PubMed2000

The in vitro-synthesized RNA from a cDNA clone of hepatitis E virus is infectious.

Hepatitis E virus (HEV) is an important etiological agent of epidemic and sporadic hepatitis, which is endemic to the Indian subcontinent and prevalent in most of the developing parts of the world. The infection is often associated with acute liver failure and high mortality, particularly in pregnant women. In order to develop methods of intervention, it is essential to understand the biology of the virus. This is particularly important as no reliable in vitro culture system is available. We have constructed a cDNA clone encompassing the complete HEV genome from independently characterized subgenomic fragments of an Indian epidemic isolate. Transfection studies were carried out with HepG2 cells using in vitro-transcribed RNA from this full-length HEV cDNA clone. The presence of negative-sense RNA, indicative of viral replication, was demonstrated in the transfected cells by strand-specific reverse transcription-PCR and slot blot hybridization. The viral proteins pORF2 and pORF3 and processed components of the pORF1 polyprotein (putative methyltransferase, helicase, and RNA-dependent RNA polymerase) were identified in the transfected cells by metabolic pulse-labeling with [(35)S]methionine-cysteine, followed by immunoprecipitation with respective antibodies. The expression of viral proteins in the transfected cells was also demonstrated by immunofluorescence microscopy. Viral replication was detected in the transfected cells up to 33 days posttransfection (six passages). The culture supernatant from the transfected cells was able to produce HEV infection in a rhesus monkey (Macaca mulatta) following intravenous injection, indicating the generation of viable HEV particles following transfection of cells with in vitro-synthesized genomic RNA. This transient cell culture model using in vitro-transcribed RNA should facilitate our understanding of HEV biology.

Animals↗
PubMed2000

Cloning, sequencing, and expression of the hepatitis E virus (HEV) nonstructural open reading frame 1 (ORF1).

Hepatitis E virus (HEV) causes enterically transmitted epidemic and sporadic viral hepatitis affecting millions of people in the developing world. Different geographical isolates of HEV show a high degree of homology at the nucleotide and amino acid levels. The approximately 7.2 kb RNA genome has three open reading frames of which ORF1 is predicted to code for the viral nonstructural polyprotein. The expression, processing and properties of the nonstructural ORF1 polyprotein have not been reported so far. In this study, the complete HEV ORF1 was reconstructed from overlapping fragments amplified by polymerase chain reaction (PCR) of total RNA isolated from the bile fluid of a rhesus monkey experimentally infected with HEV isolate from an epidemic. The complete assembled ORF1 was sequenced using HEV specific primers. The ORF1 polyprotein was expressed in E. coli, in a cell free translation system and in HepG2 cells, and was characterized by western blotting and immunoprecipitation using acute phase patient serum as well as polyclonal antibodies raised against defined parts of the ORF1 polyprotein. The nonstructural polyprotein of HEV was expressed as a 186 kDa protein. No processing was observed into discrete units, either in-vitro based on a kinetic analysis, or in HepG2 cells based on immunoprecipitation.

Animals↗
PubMed1999

Molecular biology and pathogenesis of hepatitis E virus.

Hepatitis E virus (HEV) infection results in hepatitis E, an acute and self-limited disease. The virus is transmitted in a faecal-oral manner and is a major cause of viral hepatitis in much of the developing world, where it causes rampant sporadic infections and large epidemics. A curious feature of hepatitis E is the unusually high rates of mortality that are observed in pregnant women, in whom the disease is exacerbated by the development of fulminant liver disease. In the absence of viable in vitro propagation systems, several geographical isolates of HEV have been maintained in vivo in nonhuman primates and, subsequently,the viral genome has been cloned and sequenced. HEV has been classified provisionally into a separate family known as the HEV-like viruses, which has at least four recognised genotypes, but has only a single serotype. The viral genome is a positive-stranded (+)RNA of ~7.5 kb and encodes at least three proteins. Open reading frame 1 ( ORF1) encodes the viral nonstructural polyprotein, which has domains that are homologous to some of the replication and processing enzymes found in other +RNA viruses. The HEV protein itself remains poorly characterised. The protein encoded by open reading frame 2( ORF2) is the major HEV capsid protein, and the protein encoded by open reading frame 3 ( ORF3) appears to be involved in virus-host interactions. Several questions related to the biology, epidemiology and pathogenesis of HEV remain unanswered; the progress of a few of these is reviewed here.

News↗
PubMed1999

Mutational analysis of glycosylation, membrane translocation, and cell surface expression of the hepatitis E virus ORF2 protein.

Hepatitis E virus (HEV) is the etiological agent for viral hepatitis type E, which is a major problem in the developing world. Because HEV cannot be cultured in vitro, very little information exists on the mechanisms of HEV gene expression and genome replication. HEV is a positive-strand RNA virus with three potential open reading frames (ORFs), one of which (ORF2) is postulated to encode the major viral capsid protein (pORF2). We earlier showed (S. Jameel, M. Zafrullah, M. H. Ozdener, and S. K. Panda, J. Virol. 70:207-216, 1996) pORF2 to be a approximately 88-kDa glycoprotein, carrying N-linked glycans and a potential endoplasmic reticulum (ER)-directing signal at its N terminus. Treatment with the drugs brefeldin A and monensin suggest that the protein may accumulate within the ER. Based on mutational analysis, we demonstrate Asn-310 to be the major site of N-glycan addition. In COS-1 cell expression and in vitro translation experiments, we confirm the ER-translocating nature of the pORF2 N-terminal hydrophobic sequence and show that the protein is cotranslationally, but not posttranslationally, translocated across the ER membrane. Earlier, we had also demonstrated cell surface localization of a fraction of the COS-1 cell-expressed pORF2. Using glycosylation- and translocation-defective mutants of pORF2, we now show that while transit of pORF2 into the ER is necessary for its cell surface expression, glycosylation of the protein is not required for such localization. These results may offer clues to the mechanisms of gene expression and capsid assembly in HEV.

Amino Acid Sequence↗
PubMed1997

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.

Cloning, Molecular↗
PubMed1997

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.

Animals↗
PubMed1996

Genotype determination of hepatitis C virus from northern India: identification of a new subtype.

Hepatitis C virus (HCV) shows substantial nucleotide sequence diversity distributed throughout the viral genome, with many variants showing only 68-79% overall sequence homology. This has led to problems in diagnosis of HCV using commercial immunoassays. Based on clustering of homologous sequences, various genotypes and subtypes of HCV have been described from different geographical regions. In the present study, 11 isolates from India were genotyped using sequence comparison for part of the non-structural (NS5) and structural (core) regions. Parts of the genome covering 451 bp (nt 9-459) of the core gene and a 249 bp fragment (nt 7959-8207) of the NS5 gene were reverse transcribed and amplified using nested polymerase chain reaction (RT-PCR). The amplified fragments were cloned and sequenced. The classification into genotypes was done on the basis of phylogenetic analysis. Four isolates showed sequence homology to type 1b. Two of the isolates were classified as type 3a. One isolate was classified as type 3b and the remaining four isolates were found to be variants of type 3 but did not belong to any designated subtype. On the basis of phylogenetic analysis two of the unclassified isolates were put into a new subtype of 3 named as 3g. In one of these variants, parts of a 5'-noncoding (5' NCR; 204 bp), envelope-E1 (435 bp), and NS3 (502 bp) regions were also amplified, cloned, and sequenced. This study demonstrates the type 3 variants including a new subtype (3g) to be the major cause of HCV infection in India.

Amino Acid Sequence↗
PubMed1996

Expression in animal cells and characterization of the hepatitis E virus structural proteins.

Hepatitis E virus (HEV) is a major human pathogen in much of the developing world. It is a positive-strand RNA virus with a 7.5-kb polyadenylated genome consisting of three open reading frames (ORFs). In the absence of an in vitro culture system, the replication and expression strategy of HEV and the nature of its encoded polypeptides are not well understood. We have expressed the two ORFs constituting the structural portion of the HEV genome in COS-1 cells by using simian virus 40-based expression vectors and in vitro by using a coupled transcription-translation system. We show here that the major capsid protein, encoded by ORF2, is an 88-kDa glycoprotein which is expressed intracellularly as well as on the cell surface and has the potential to form noncovalent homodimers. It is synthesized as a precursor (ppORF2) which is processed through signal sequence cleavage into the mature protein (pORF2), which is then glycosylated (gpORF2). The minor protein, pORF3, encoded by ORF3 is a 13.5-kDa nonglycosylated protein expressed intracellularly and does not show any major processing. pORF3 interacts with a cellular protein of about 18 kDa which we call 3IP, the pORF3-interacting protein. The significance of these findings are discussed in light of an existing model of HEV genome replication and expression.

Animals↗
PubMed1995

Vertical transmission of hepatitis E virus.

Little is known about vertical transmission of hepatitis E virus from infected mothers to their infants. We studied eight babies born to mothers infected with hepatitis E in third trimester. One baby was icteric at birth with elevated transaminases and four babies had anicteric hepatitis. Two babies were born with hypothermia and hypoglycaemia and died within 24 h; one had massive hepatic necrosis. Hepatitis E virus RNA was detected by PCR in cord or birth blood samples of five infants. Six infants had evidence of hepatitis E infection. We conclude that hepatitis E virus is commonly transmitted from infected mothers to their babies with significant perinatal morbidity and mortality.

Female↗