Ahmed A. Azad

Deletion mapping and expression in Escherichia coli of the large genomic segment of a birnavirus.

The large genomic segment of infectious bursal disease virus encodes a polyprotein in which the viral polypeptides are present in the following order: N-VP2-VP4-VP3-C. Expression in Escherichia coli of the large segment results in the processing of the polyprotein. The expression product reacts with a virus neutralizing and protective monoclonal antibody that recognizes a conformational epitope on the surface of the virus. Different regions of the large genomic segment were deleted at defined restriction sites and the truncated fragments were ligated to various expression vectors for high-level expression in E. coli. The expressed proteins were probed with three different monoclonal antibodies that recognize epitopes encoded by different regions of the large genomic segment. These deletion mapping studies suggest that VP4 is involved in the processing of the precursor polyprotein, and the conformational epitope recognized by the virus neutralizing monoclonal antibody is present within VP2.

Sequence Analysis and Expression of the Host-protective Immunogen VP2 of a Variant Strain of Infectious Bursal Disease Virus Which Can Circumvent Vaccination with Standard Type I Strains

The host-protective antigen VP2 of a variant strain of infectious bursal disease virus (IBDV) which emerged from a vaccinated flock and is able to circumvent vaccination with classic type I strains of IBDV, was cloned and its nucleotide sequence determined. Virus-neutralizing monoclonal antibodies (MAbs) raised against the Australian 002-73 strain of IBDV did not react or reacted only very weakly with the expression product of the variant virus. The deduced amino acid sequence of VP2 from the variant strain differed in 17 residues from that of the Australian strain and in eight positions from a consensus sequence compiled from six type I strains of IBDV. All the amino acid changes mapped within the central, variable region of VP2, which forms the conformational epitope recognized by virus-neutralizing MAbs. Changes in the two hydrophilic regions at either end of this fragment were unique to the variant virus and were crucial for its ability to escape the virus-neutralizing antibodies induced by vaccination with a standard type I vaccine.

Human Immunodeficiency Virus Type 1 Nef Binds to Tumor Suppressor p53 and Protects Cells against p53-Mediated Apoptosis

ABSTRACT The nef gene product of human immunodeficiency virus type 1 (HIV-1) is important for the induction of AIDS, and key to its function is its ability to manipulate T-cell function by targeting cellular signal transduction proteins. We reported that Nef coprecipitates a multiprotein complex from cells which contains tumor suppressor protein p53. We now show that Nef interacts directly with p53. Binding assays showed that an N-terminal, 57-residue fragment of Nef (Nef 1-57) contains the p53-binding domain. Nef also interacted with p53 during HIV-1 infection in vitro. As p53 plays a critical role in the regulation of apoptosis, we hypothesized that Nef may alter this process. Nef inhibited UV light-induced, p53-dependent apoptosis in MOLT-4 cells, with Nef 1-57 being as effective as its full-length counterpart. The inhibition by Nef of p53 apoptotic function is most likely due its observed ability to decrease p53 protein half-life and, consequently, p53 DNA binding activity and transcriptional activation. These data show that HIV-1 Nef may augment HIV replication by prolonging the viability of infected cells by blocking p53-mediated apoptosis.

Sequence of the small double-stranded RNA genomic segment of infectious bursal disease virus and its deduced 90-kDa product

The smaller dsRNA segment of the genome of infectious bursal disease virus (IBDV) encodes a single polypeptide of approximately 90 kDa (VP1). The consensus nucleotide sequence, derived from independent and overlapping cDNA clones, contains a single open reading frame which begins with an exact Kozak sequence and could encode a polypeptide of 878 amino acid residues. It has been suggested that VP1 could be the viral RNA-dependent RNA polymerase. A comparison of the predicted amino acid sequence of this protein with those of other DNA-dependent and ssRNA-dependent RNA polymerases has failed to reveal any homology between VP1 and the conserved regions in these enzymes. It is possible that the polypeptide encoded by the IBDV virus may represent a new class of polymerases which are involved in the replication of double-stranded RNA genomes.

Passive protection against infectious bursal disease virus by viral VP2 expressed in yeast

Infectious bursal disease virus (IBDV), a pathogen of major economic importance to the worlds poultry industries, causes a severe immunodepressive disease in young chickens. Maternal antibodies are able to protect the progeny passively from IBDV infection. The gene encoding the IBDV host-protective antigen (VP2) has been cloned and expressed in yeast resulting in the production of an antigen that very closely resembles native VP2. When injected into specific pathogen free chickens a single dose of microgram quantities of the yeast derived antigen induces high titres of virus neutralizing antibodies that are capable of passively protecting young chickens from infection with IBDV.

A C-terminal domain of HIV-1 accessory protein Vpr is involved in penetration, mitochondrial dysfunction and apoptosis of human CD4+ lymphocytes

We have previously shown that expression of HIV-1 vpr in yeast results in cell growth arrest and structural defects, and identified a C-terminal domain of Vpr as being responsible for these effects in yeast.1 In this report we show that recombinant Vpr and C-terminal peptides of Vpr containing the conserved sequence HFRIGCRHSRIG caused permeabilization of CD4+ T lymphocytes, a dramatic reduction of mitochondrial membrane potential and finally cell death. Vpr and Vpr peptides containing the conserved sequence rapidly penetrated cells, co-localized with the DNA, and caused increased granularity and formation of dense apoptotic bodies. The above results suggest that Vpr treated cells undergo apoptosis and this was confirmed by demonstration of DNA fragmentation by the highly sensitive TUNEL assay. Our results, together with the demonstration of extracellular Vpr in HIV infected individuals,2,3 suggest the possibility that extracellular Vpr could contribute to the apoptotic death and depletion of bystander cells in lymphoid tissues4,5 during HIV infection.

Expression and analysis of the NS2 protein of influenza A virus

SummaryInfluenza NS2 protein was expressed inSaccharomyces cerevisiae using a copper-inducible promoter. The protein produced had a molecular weight of 13 kDa, was reactive with anti-NS2 antiserum and was localised to the yeast cell nucleus. Two-hybrid analysis identified a direct protein-protein interaction between NS2 and the M2 protein of the virus, involving the C-terminal 163 residues of M1. A filter-binding assay localised the M1 binding region to the C-terminal 70 amino acids of NS2.

HIV-1 protein Vpr causes gross mitochondrial dysfunction in the yeast Saccharomyces cerevisiae

The biological effects of the HIV‐1 accessory protein, Vpr, have been studied in yeast expression systems. In our previous study [1] , employing the pCUP1‐vpr copper‐inducible expression cassette, Vpr was shown to cause growth arrest and structural defects. In this study yeast constitutively expressing vpr, through elevated copy number and/or elevated transcription levels, displayed no growth arrest in fermentative growth conditions while Vpr was produced at much lower levels than in the inducible expression system. However, such cells were respiratory deficient and unable to utilise ethanol or glycerol as the sole carbon source. They exhibited gross mitochondrial dysfunction displayed in the loss of respiratory chain complex I, II, III, IV and citrate synthase activities. The effects on mitochondria required a C‐terminal domain of Vpr that contains a conserved amino acid sequence motif HFRIGCRHSRIG. These results suggest that the widely observed phenomenon of ‘Vpr‐induced growth arrest’ in human cells could be due to mitochondrial dysfunction.

Extracellular addition of a domain of HIV‐1 Vpr containing the amino acid sequence motif H(S/F)RIG causes cell membrane permeabilization and death

Vpr is a virion‐associated protein of human immuno‐deficiency virus type 1 (HIV‐1) whose function in acquired immune deficiency syndrome (AIDS) has been uncertain. We previously employed yeast as a model to examine the effects of Vpr on basic cellular functions; intracellular Vpr was shown to cause cell‐growth arrest and structural defects, and these effects were caused by a region of Vpr containing the sequence HFRIGCRHSRIG. Here we show that peptides containing the H(S/F)RIG amino acid sequence motif cause death when added externally to a variety of yeast including Saccharomyces cerevisiae, Kluyveromyces lactis, Candida glabrata, Candida albicans and Schizosaccharomyces pombe. Such peptides rapldly entered the cell from the time of addition, resulting in cell death. Elevated levels of ions, particularly magnesium and calcium ions, abrogated the cytotoxic effect by preventing the Vpr peptides from entering the cells. Extracellular Vpr found in the serum, or breakdown products of extracellular Vpr, may have similar effects to the Vpr peptides described here and could explain the death of uninfected by‐stander cells during AIDS.

Expression in Escherichia coli of cDNA fragments encoding the gene for the host-protective antigen of infectious bursal disease virus

The larger segment of the IBDV genome codes for a 32-kDa host-protective antigen. Inserts from a cDNA library in pBR 322, containing overlapping cDNA fragments of varying sizes and covering the entire large segment of the IBDV genome, were subcloned into a mixture of expression vectors pUR 290, 291, and 292. Clones expressing the host-protective antigen, or parts of it, were identified by an immunoblot assay and the fusion proteins were further characterized by Western blot analysis using a monoclonal antibody specific for the 32-kDa polypeptide. Hybridization of inserts from expressing clones to the original cDNA library led to the identification of the region of the IBDV genome that codes for the 32-kDa host-protective antigen. Clone D1 which encodes approximately 50% and clone D6 which encodes the entire 32-kDa protein were selected for further studies. The fusion proteins from clones D1 and D6 were affinity purified and tested for their immunogenicity in chickens. Both fusion proteins induced the synthesis of antibodies in both primed and unprimed chickens that reacted specifically with denatured 32-kDa viral protein, but less well with intact virus. It was concluded that the response to the fusion proteins was to linear rather than conformational epitopes on the 32-kDa viral protein.