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Shahid Jameel

Publications and source records attributed to Shahid Jameel.

18 recordsLinked to original sources
PubMed2006

Expression and processing of the Hepatitis E virus ORF1 nonstructural polyprotein.

BACKGROUND: The ORF1 of hepatitis E virus (HEV) encodes a nonstructural polyprotein of approximately 186 kDa that has putative domains for four enzymes: a methyltransferase, a papain-like cysteine protease, a RNA helicase and a RNA dependent RNA polymerase. In the absence of a culture system for HEV, the ORF1 expressed using bacterial and mammalian expression systems has shown an approximately 186 kDa protein, but no processing of the polyprotein has been observed. Based on these observations, it was proposed that the ORF1 polyprotein does not undergo processing into functional units. We have studied ORF1 polyprotein expression and processing through a baculovirus expression vector system because of the high level expression and post-translational modification abilities of this system. RESULTS: The baculovirus expressed ORF1 polyprotein was processed into smaller fragments that could be detected using antibodies directed against tags engineered at both ends. Processing of this approximately 192 kDa tagged ORF1 polyprotein and accumulation of lower molecular weight species took place in a time-dependent manner. This processing was inhibited by E-64d, a cell-permeable cysteine protease inhibitor. MALDI-TOF analysis of a 35 kDa processed fragment revealed 9 peptide sequences that matched the HEV methyltransferase (MeT), the first putative domain of the ORF1 polyprotein. Antibodies to the MeT region also revealed an ORF1 processing pattern identical to that observed for the N-terminal tag. CONCLUSION: When expressed through baculovirus, the ORF1 polyprotein of HEV was processed into smaller proteins that correlated with their proposed functional domains. Though the involvement of non-cysteine protease(s) could not be be ruled out, this processing mainly depended upon a cysteine protease.

Amino Acid Sequence↗
PubMed2006

Inhibition of hepatitis B virus DNA replicative intermediate forms by recombinant interferon-gamma.

AIM: To evaluate the in vitro anti-HBV activity of recombinant human IFN-gamma, alone and in combination with lamivudine. METHODS: A recombinant baculovirus-HBV/HepG2 culture system was developed which could support productive HBV infection in vitro. Expression of HBsAg and HBeAg in infected HepG2 culture medium was detected by commercial enzyme immunoassays. HBV DNA replication intermediates were detected in infected cells by Southern hybridization and viral DNA load was determined by dot hybridization. RESULTS: IFN-gamma at 0.1 to 5 microg/L efficiently down regulated HBsAg expression in transduced HepG2 cells. At 5 microg/L, IFN-gamma also suppressed HBV DNA replication in these cells. While treatment with a combination of lamivudine and IFN-gamma showed no additive effect, sequential treatment first with lamivudine and then IFN-gamma was found to be promising. In this culture system the best HBV suppression was observed with a pulse of 2 micromol/L lamivudine for two days, followed by 1 microg/L IFN-gamma for another four days. Compared to treatment with lamivudine alone, the sequential use of 0.2 micromol/L lamivudine for two days, followed by 5 microg/L IFN-gamma for six days showed a 72% reduction in HBV cccDNA pool. CONCLUSION: This in vitro study warrants further evaluation of a combination of IFN-gamma and lamivudine, especially in IFN-alpha non-responder chronic hepatitis B patients. A reduced duration of lamivudine treatment would also restrict the emergence of drug-resistant HBV mutants.

Antiviral Agents↗
PubMed2006

Molecular analysis and phylogenetic characterization of HIV in Iran.

The rate of human immunodeficiency virus type 1 (HIV-1) infection in Iran has increased dramatically in the last few years. While the earliest cases were found in hemophiliacs, intravenous drug users are now fueling the outbreak. In this study, both the 122 clones of HIV-1 gag p17 and the 131 clones of env V1-V5 region were obtained from 61 HIV-1 seropositives belonging to these two groups in Iran. HIV-1 subtyping and phylogenetic analysis was done by heteroduplex mobility assays (HMA) and multiple clone sequencing. The result indicated all hemophiliacs are infected with HIV-1 subtype B and all intravenous drug users are infected with HIV-1 subtype A. Since intravenous drug abuse is the major transmission route in Iran, HIV-1 subtype A is likely to be the dominant viral subtype circulating in the country. The analysis of genetic distances showed subtype B viruses in Iran to be twice as heterogeneous as the subtype A viruses. In conclusion, this first molecular study of HIV-1 genotypes in Iran suggests two parallel outbreaks in distinct high-risk populations and may offer clues to the origin and spread of infection in Iran.

Amino Acid Sequence↗
PubMed2005

The Nef protein of HIV-1 induces loss of cell surface costimulatory molecules CD80 and CD86 in APCs.

The Nef protein of HIV-1 is essential for its pathogenicity and is known to down-regulate MHC expression on infected cell surfaces. We now show that Nef also redistributes the costimulatory molecules CD80 and CD86 away from the cell surface in the human monocytic U937 cell line as well as in mouse macrophages and dendritic cells. Furthermore, HIV-1-infected U937 cells and human blood-derived macrophages show a similar loss of cell surface CD80 and CD86. Nef colocalizes with MHC class I (MHCI), CD80, and CD86 in intracellular compartments, and binds to both mouse and human CD80 and CD86. Some Nef mutants defective in MHCI down-modulation, including one from a clinical isolate, remain capable of down-modulating CD80 and CD86. Nef-mediated loss of surface CD80/CD86 is functionally significant, because it leads to compromised activation of naive T cells. This novel immunomodulatory role of Nef may be of potential importance in explaining the correlations of macrophage-tropism and Nef with HIV-1 pathogenicity and immune evasion.

Amino Acid Sequence↗
PubMed2005

Tumor suppressor SMAR1 activates and stabilizes p53 through its arginine-serine-rich motif.

Various stresses and DNA-damaging agents trigger transcriptional activity of p53 by post-translational modifications, making it a global regulatory switch that controls cell proliferation and apoptosis. Earlier we have shown that the novel MAR-associated protein SMAR1 interacts with p53. Here we delineate the minimal domain of SMAR1 (the arginine-serine-rich domain) that is phosphorylated by protein kinase C family proteins and is responsible for p53 interaction, activation, and stabilization within the nucleus. SMAR1-mediated stabilization of p53 is brought about by inhibiting Mdm2-mediated degradation of p53. We also demonstrate that this arginine-serine (RS)-rich domain triggers the various cell cycle modulating proteins that decide cell fate. Furthermore, phenotypic knock-down experiments using small interfering RNA showed that SMAR1 is required for activation and nuclear retention of p53. The level of phosphorylated p53 was significantly increased in the thymus of SMAR1 transgenic mice, showing in vivo significance of SMAR1 expression. This is the first report that demonstrates the mechanism of action of the MAR-binding protein SMAR1 in modulating the activity of p53, often referred to as the "guardian of the genome."

Amino Acid Motifs↗
PubMed2005

Biology of the HIV Nef protein.

The accessory Nef protein is expressed by all primate lentiviruses--HIV-1,HIV-2 and simian immune deficiency virus (SIV). Its expression in the early stages of the viral life cycle ensures two basic attributes of HIV infection. These are T-cell activation and the establishment of a persistent state of infection. Nef has a positive effect on viral infection and replication by promoting the survival of infected cells. Its role in HIV persistence is based largely on the ability of Nef to downmodulate the surface levels of important molecules at the immune synapse. These include major histocompatibility complex-I (MHC I) and (MHC II) present on antigen-presenting cells (APCs) and target cells, and CD4 and CD28 present on helper T cells. In this review we present these biological properties of Nef from a mechanistic point of view, and relate them to the structural attributes and interactions of the Nef protein. A brief outline of the limited studies on Nef from Indian subtype C HIV-1 isolates is also presented.

Amino Acid Sequence↗
PubMed2004

The SARS coronavirus nucleocapsid protein induces actin reorganization and apoptosis in COS-1 cells in the absence of growth factors.

In March 2003, a novel coronavirus was isolated from patients exhibiting atypical pneumonia, and was subsequently proven to be the causative agent of the disease now referred to as SARS (severe acute respiratory syndrome). The complete genome of the SARS-CoV (SARS coronavirus) has since been sequenced. The SARS-CoV nucleocapsid (SARS-CoV N) protein shares little homology with other members of the coronavirus family. In the present paper, we show that SARS-CoV N is capable of inducing apoptosis of COS-1 monkey kidney cells in the absence of growth factors by down-regulating ERK (extracellular-signal-regulated kinase), up-regulating JNK (c-Jun N-terminal kinase) and p38 MAPK (mitogen-activated protein kinase) pathways, and affecting their downstream effectors. SARS-CoV N expression also down-regulated phospho-Akt and Bcl-2 levels, and activated caspases 3 and 7. However, apoptosis was independent of the p53 and Fas signalling pathways. Furthermore, activation of the p38 MAPK pathway was found to induce actin reorganization in cells devoid of growth factors. At the cytoskeletal level, SARS-CoV N down-regulated FAK (focal adhesion kinase) activity and also down-regulated fibronectin expression. This is the first report showing the ability of the N protein of SARS-CoV to induce apoptosis and actin reorganization in mammalian cells under stressed conditions.

Actins↗
PubMed2004

The hepatitis E virus open reading frame 3 protein activates ERK through binding and inhibition of the MAPK phosphatase.

The hepatitis E virus causes acute viral hepatitis endemic in much of the developing world and is a serious public health problem. However, due to the lack of an in vitro culture system or a small animal model, its biology and pathogenesis are poorly understood. We have shown earlier that the ORF3 protein (pORF3) of hepatitis E virus activates ERK, a member of the MAPK superfamily. Here we have explored the mechanism of pORF3-mediated ERK activation and demonstrated it to be independent of the Raf/MEK pathway. Using biochemical assays, yeast two-hybrid analysis, and intracellular fluorescence resonance energy transfer we showed that pORF3 binds Pyst1, a prototypic member of the ERK-specific MAPK phosphatase. The binding regions in the two proteins were mapped to the N terminus of pORF3 and a central portion of Pyst1. Expression of pORF3 protected ERK from the inhibitory effects of ectopically expressed Pyst1. This is the first example of a viral protein regulating ERK activation by inhibition of its cognate dual specificity phosphatase.

Animals↗
PubMed2004

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.

Alpha-Globulins↗
PubMed2004

Acidic pH enhances structure and structural stability of the capsid protein of hepatitis E virus.

Hepatitis E virus (HEV) is enterically transmitted and endemic to tropical areas of the world. The major capsid protein of HEV is pORF2 ( approximately 74 kDa), encoded by open reading frame 2 (ORF2). When expressed in insect cells, it is processed into a approximately 55 kDa form (n-pORF2). We also generated a mutant, m-pORF2, lacking a C-terminal hydrophobic region shown earlier to be required for its homo-oligomerization. Circular dichroism was used to measure the secondary structure and stability of these proteins as a function of pH and temperature. With decreasing pH both proteins acquired increasing alpha-helicity and thermal stability in terms of midpoint of denaturation and the Gibbs energy change.

Animals↗
PubMed2004

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.

5' Untranslated Regions↗
PubMed2003

Hepatitis viruses and the MAPK pathway: is this a survival strategy?

The viruses that cause hepatitis comprise of at least five different agents, which share the ability to cause inflammation and necrosis of the liver. The disease spectrum is quite diverse and the outcome of infection by the different hepatitis viruses can be rationalized based on virus-host cell interactions. New insights into the molecular basis of viral hepatitis reveal that three of these agents - the hepatitis B, C and E viruses (HBV, HCV and HEV) modulate the mitogen-activated protein kinase (MAPK) signaling pathway. In this review we briefly describe the structural organization of the MAPK cascade and emphasize its importance as a central pathway in the signaling network. Selected mechanisms through which HBV, HCV and HEV proteins target various steps in the MAPK pathway are discussed and used to propose a pro-survival outcome for the host cell. In addition, we offer an insight into how the common theme of MAPK activation and its downstream effects may be used to rationalize the different outcomes of hepatitis B, C and E.

Animals↗
PubMed2003

Purification and diagnostic utility of a recombinant hepatitis E virus capsid protein expressed in insect larvae.

We report here the expression and purification of a truncated form of the hepatitis E virus ORF2 protein (ORF2delta111/deltaTM), from the fat bodies of Spodoptera litura larvae infected with a recombinant baculovirus. The purified protein migrated as a doublet of approximately 56 kDa on SDS-PAGE and was found to be glycosylated by staining with concanavalin A-linked horseradish peroxidase. The protein was used in a sensitive and specific enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to HEV. The results showed complete concordance with those obtained using a commercial kit for the detection of anti-HEV antibodies. Antigen expression in the insect larvae system presents a rapid and low-cost method that obviates the need for expensive tissue culture scale-ups or special equipment.

Animals↗
PubMed2002

The phosphorylated form of the ORF3 protein of hepatitis E virus interacts with its non-glycosylated form of the major capsid protein, ORF2.

Hepatitis E virus (HEV) is a human RNA virus containing three open reading frames. Of these, ORF1 encodes the viral nonstructural polyprotein; ORF2 encodes the major capsid protein, which exists in a glycosylated and non-glycosylated form; and ORF3 codes for a phosphoprotein of undefined function. Using fluorescence-based colocalization, yeast two-hybrid experiments, transiently transfected COS-1 cell co-immunoprecipitation, and cell-free coupled transcription-translation techniques, we have shown that the ORF3 protein interacts with the ORF2 protein. The domains involved in this ORF2-ORF3 association have been identified and mapped. Our deletion analysis showed that a 25-amino acid region (residues 57-81) of the ORF3 protein is required for this interaction. Using a Mexican HEV isolate, site-directed mutagenesis of ORF3, and a phosphatase digestion assay, we showed that the ORF2-ORF3 interaction is dependent upon the phosphorylation at Ser(80) of ORF3. Finally, using COS-1 cell immunoprecipitation experiments, we found that the phosphorylated ORF3 protein preferentially interacts with the non-glycosylated ORF2 protein. These findings were confirmed using tunicamycin inhibition, point mutants, and deletion mutants expressing only non-glycosylated ORF2. ORF3 maps in the structural region of the HEV genome and now interacts with the major capsid protein, ORF2, in a post-translational modification-dependent manner. Such an interaction of ORF2 with ORF3 suggests a possible well regulated role for ORF3 in HEV structural assembly.

Amino Acid Sequence↗
PubMed2002

Reevaluation of a North India isolate of hepatitis E virus based on the full-length genomic sequence obtained following long RT-PCR.

The genomic cloning and sequence of hepatitis E virus (HEV) from an epidemic in North India is reported. We describe here a simple method wherein the viral RNA was reverse transcribed and then amplified in a single step using an extra long polymerase chain reaction procedure. The full genome nucleotide sequence of this HEV isolate (called Yam-67) was made up of 7191 nucleotides, excepting the poly(A) tail and had three open reading frames: ORF1 coding for 1693 amino acids (aa), ORF2 coding for 659 aa and ORF3 coding for 122 aa. This North Indian isolate of HEV showed close sequence homology to other HEV isolates from India and Asia, but was distant from the Chinese genotype 4, Japanese, Mexican and US isolates. There is no indication from sequence analysis that this may be an atypical strain of HEV, as reported earlier.

Genome, Viral↗
PubMed2001

A C-Terminal Hydrophobic Region is Required for Homo-Oligomerization of the Hepatitis E Virus Capsid (ORF2) Protein.

Hepatitis E virus (HEV) is the causative agent of hepatitis E, an acute form of viral hepatitis. The open reading frame 2 (ORF2) of HEV encodes the viral capsid protein, which can self-oligomerize into virus-like particles. To understand the domains within this protein important for capsid biogenesis, we have carried out in vitro analyses of association and folding patterns of wild type and mutant ORF2 proteins. When expressed in vitro or in transfected cells, the ORF2 protein assembled as dimers, trimers and higher order forms.While N-terminal deletions upto 111 amino acids had no effect, the deletion of amino acids 585-610 led to reduced homo-oligomerization. This deletion also resulted in aberrant folding of the protein, as determined by its sensitivity to trypsin. This study suggests that a C-terminal hydrophobic region encompassing amino acids 585-610 of the ORF2 protein might be critical for capsid biogenesis.

Journal Article↗