Biomedical subjects
Publications and source records attributed to S Mounir.
Combinatorial chemistry is playing an increasingly prominent role in the process of drug discovery. A nucleic acid-based (NAB) scaffold can be engineered to create functional group and topological diversity in a library. Described herein is the parallel solid-phase synthesis of combinatorial libraries of nucleoside phosphoramidates, and the first evaluation of antiviral activity against hepatitis B virus (HBV).
An in vitro assay based on the expression of Fluci reporter gene under the translational control of HCV IRES was used to evaluate and screen compound libraries. A structure-activity relationship study on a phenazine hit was conducted. Our data suggest that an intact phenazine or phenazine-like core with two distal polar substitutions is crucial for potency.
A number of human xenograft orthotopic models of hepatocellular carcinoma (HCC) have been previously established by growing histologically-intact patient specimens in nude mice. However, the availability of HBV and HCV negative human hepatocellular carcinoma specimens is scarce and the pattern of tumor growth in nude mice varies depending on the tumor type. In the present study, we have established a reproducible xenograft orthotopic model using a human HCC cell line designated HuT7-3 that was derived from two rounds of subcloning of the parental Huh-7 cell line. The tumor growth rate of the HuT7-3 cell line, grown at a primary subcutaneous site, was markedly higher than that of the Huh-7 parental cell line or the human hepatoblastoma Hep-G2 cell line. Furthermore, we have shown that doxorubicin, when administered intravenously, is efficient in inhibiting the development of subcutaneous tumor but leads to the regression of the orthotopic human HCC. Consequently, this novel HCC xenograft orthotopic model can be used for the evaluation of antitumor drugs.
To determine whether caspase-3-induced cleavage of poly(ADP-ribose) polymerase (PARP), a DNA damage-sensitive enzyme, alters the balance between survival and death of the cells following DNA damage, we created stable cell lines that express either caspase-uncleavable mutant or wild type PARP in the background of PARP (-/-) fibroblasts. The survival and apoptotic responses of these cells were compared after exposure to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a DNA-damaging agent that activates PARP, or to tumor necrosis factor-alpha, which causes apoptosis without initial DNA damage. In response to MNNG, the cells with caspase-uncleavable PARP were very resistant to loss of viability or induction of apoptosis. Most significantly, approximately 25% of these cells survived and retained clonogenicity at a level of DNA damage that eliminated the cells with wild type PARP or PARP (-/-) cells. Expression of caspase-uncleavable PARP could not protect the cells from death induced by tumor necrosis factor, although there was a slower progression of apoptotic events in these cells. Therefore, one of the functions for cleavage of PARP during apoptosis induced by alkylating agents is to prevent survival of the extensively damaged cells.
The kinetics of appearance of antibodies directed to the major structural proteins N, M and E of porcine reproductive and respiratory syndrome virus (PRRSV) was followed in pigs naturally- and experimentally-exposed to the virus. Specific IgM antibody titers were first detected by indirect immunofluorescence (IIF) at the end of the first week of PRRSV infection, peaked by day 14 to 21 post-inoculation (p.i.), then rapidly decreased to undetectable levels by day 35 to 42 p.i. On the other hand, specific IgG antibody titers peaked by day 21 to 28 p.i. and remained unchanged to the end of the 6- or 9-week observation period; in addition, a persistent viremia was observed. Virus neutralizing (VN) antibody titers > 8 were not detected until 3 to 4 weeks p.i. Taken together, the results obtained by Western blotting analyses using purified virus and E. coli-expressed ORFs 5 to 7 gene products, suggested that antibodies directed against the envelope E protein appear by day 7 p.i., whereas antibodies directed against the nucleocapsid N and membrane M proteins can only be detected by the end of the second week p.i. No correlation could be demonstrated between VN and IIF antibody titers, viremia, and viral protein specificities of circulating antibodies at various times p.i.
The 3' end genomic region of a Québec PRRSV reference strain (IAF-exp91), propagated in porcine alveolar macrophages (PAM), was sequenced and compared to the prototype European strain, the Lelystad virus (LV). The sequence, which represents the 3'-terminal 2834 nucleotides, encompassed 5 ORFs corresponding to ORFs 3 to 7 of LV. Extensive genomic variations resulting from an important rate of nucleotide additions, substitutions, and deletions were demonstrated between the two viruses. Indeed, the two corresponding sequences displayed a total of 66% and 63% identity at the nucleotide and amino acid levels, respectively. The predicted products of ORFs 5, 3, and 7, showed the highest rate of amino acid variations with percentages of identity of 52, 54, and 59, respectively. Sequence analysis of an additional Québec strain that could be propagated in a continuous cell line (MARC-145), suggested that Québec PRRSV strains belong to a genotype distinct from that of LV, thus confirming previous serological results which allowed to divide PRRSV isolates into two distinct antigenic subgroups (U.S. and European). Six viral major polypeptides with apparent M(rs) of 14.5K, 15K, 19K, 24.5K, 29K, and 42K could be identified from lysates of viral infected cells, of which the 15, 19 and 24.5K species seemed to be structural. In vitro translation products of ORFs 7 and 6 comigrated with the 15 and 19 K viral proteins, whereas that of ORF 5 may be associated to the 24.5K when translated in presence of microsomes. Consequently, it is likely that ORFs 7 to 5, encode the three major structural proteins.
Porcine reproductive and respiratory syndrome virus (PRRSV) causes abortions and respiratory diseases in pigs. The PRRSV genome is a positive-sense polyadenylated RNA molecule of about 15 kb. Along with the genes for structural proteins (envelope, matrix and nucleocapsid proteins) PRRSV genome contains a number of ORFs potentially encoding nonstructural proteins (ns). To investigate the nature of the PRRSV ORFs 7, 5 and 4 products, we have cloned the envelope (ORF5) nucleocapsid (ORF7) and ns4 (ORF4) protein genes in the bacterial expression vector pMAL(TM)-c2 under control of the "tac" promoter and expressed the proteins in E. coli. The recombinant proteins were recognized by porcine and rabbit hyperimmune serums to PRRSV, suggesting their structural nature.
The cDNA sequence of the 3'-terminal genomic region of the Québec IAF-exp91 strain of porcine reproductive and respiratory syndrome virus (PRRSV) was determined and compared to those of other reference strains from Europe (Lelystad virus) and US (ATCC VR2385, MN-1b). The sequence (2834 nucleotides) which encompassed ORFs 3 to 7 revealed extensive genomic variations between the Québec strain and Lelystad virus (LV), resulting from high number of base substitutions, additions and deletions. The ORFs 5, 3, and 7 seemed to be relatively the most variable; the predicted encoding products of the Québec and LV strains displayed only 52%, 54%, and 59% amino acid identities, respectively. Nevertheless, in vitro translation experiments of the structural genes (ORFs 5, 6, and 7) and radioimmunoprecipitation assays with extracellular virions gave results similar to those previously reported for LV. In contrast, close genomic relationships were demonstrated between Québec and US strains. Taking together, these results indicate that, although structurally similar, North American PRRSV strains belong to a genotype distinct from that of the LV, thus supporting previous findings that allowed to divide PRRSV isolates into two antigenic subgroups (U.S. and European).
The complete nucleotide sequence of the ns2 gene of human coronavirus OC43 (HCV-OC43) was determined. Sequence analysis revealed an open reading frame that could encode a protein of 278 amino acids, with an estimated molecular mass of 32.2 kDa. Six potential phosphorylation sites are present but no sites of N-glycosylation were found. The amino acid sequence of the HCV-OC43 ns2 protein shows 92% identity with that of the Mebus strain of bovine coronavirus (BCV). However, a stretch of nine consecutive amino acids near the C terminus is completely different, causing it to be very hydrophilic, which contrasts with the hydrophobic nature of this region in BCV. As shown by immunofluorescence with a monospecific antiserum, the ns2 protein was expressed in the cytoplasm of HCV-OC43-infected HRT-18 cells.
The sequence of the 3'-terminal region of the genome of Québec reference strain IAF-exp91 of porcine reproductive and respiratory syndrome virus (PRRSV) was investigated by analysis of four cDNA clones. The 3'-terminal 530 nucleotides (nt) encompassed a large open reading frame with a coding capacity of 123 amino acids (M(r) 13,649). The predicted protein was extremely basic and hence was considered to correspond to the nucleocapsid (N) protein gene. When compared to the homologous sequences of two reference Netherlands strains (Lelystad and isolate 10) of PRRSV, the IAF-exp91 N protein was found to be five amino acids shorter and displayed a high degree of divergence. Overall, IAF-exp91 strain showed identities of 63% and 59% with both reference European strains at the nucleotide and amino acid level, respectively. Two amino acid stretches, STAPM and SQGAS, present respectively at the N- and C-terminal regions of the N protein of European strains, were missing in the IAF-exp91 N protein sequence. The 3'-terminal non-coding region (151 nt) of the IAF-exp91 strain was 22 nt longer than that of the European strains. The aligned nucleotide sequence of this non-coding region exhibited an overall identity of 59% with that of the European strains. The Québec reference strain of PRRSV appeared to be related more closely to equine arteritis virus and lactate dehydrogenase-elevating virus than are the two European strains of the virus. Preliminary data obtained by reverse transcription-PCR experiments, using specific or common oligonucleotide primers, suggested that this approach could be useful for distinguishing between PRRSV strains from different geographic origins.
Two sets of oligonucleotide primers (1008PS-1009PR and 1010PLS-1011PLR) were designed according to the sequence of the nucleocapsid protein (N) gene of Quebec reference strain IAF-exp91 of porcine reproductive and respiratory syndrome virus (PRRSV). The primers were used in reverse transcription and PCR (RT-PCR) experiments for detection of viral genomic RNA either from infected porcine alveolar macrophages (PAM) or tissues from experimentally infected specific-pathogen-free pigs. Considering the high degree of variation detected between the nucleotide sequences of the N genes of IAF-exp91 and Lelystad virus (LV) strains of PRRSV, the primers 1008PS-1009PR were referred to as the specific primers, since they were chosen in such a manner that they could amplify only sequences from IAF-exp91 RNA and not from LV. On the other hand, the primer pair 1010PLS-1011PLR was common to both strains of PRRSV. When analyzed by agarose gel electrophoresis, the products of RT-PCR from each set of primers were resolved as single band of the predicted size, the specificity of amplified products being confirmed by Southern blotting with a specific IAF-exp91 N gene probe. No amplification was observed when RNA was extracted from uninfected PAM or from other porcine viruses. As expected, only the common primer pair was able to amplify RNA from the Quebec reference strain and two European strains (LV and Weybridge). The resulting bands displayed differences in electrophoretic mobilities due to the absence of 37 nucleotides in both European strains, thus allowing their differentiation from the IAF-exp91 strain. Most of the tissue culture-adapted Quebec isolates were detected with both primer pairs. The sensitivity of the enzymatic amplification method for detection of PRRSV from lung tissues was a 50% tissue culture infective dose of 5. RT-PCR was found to be more sensitive than indirect immunofluorescence assay for detection of PRRSV in tissues from experimentally infected pigs and as sensitive as virus isolation in PAM, especially when combined with Southern blotting with the digoxigenin-labeled N probe and chemiluminescence detection.
Cytolytic and noncytolytic strains of the porcine reproductive and respiratory syndrome virus (PRRSV) were isolated in primary cultures of porcine alveolar macrophages (PAM) from lung homogenates of stillborn fetuses or blood samples of dyspneic piglets collected from Quebec pig farms having experienced acute or chronic outbreaks of PRRS. Serological identification of the virus was confirmed by indirect immunofluorescence and indirect protein A-gold immunoelectron microscopy using reference antiserum prepared from experimentally-infected specific pathogen free (SPF) piglets and monoclonal antibodies (MoAbs) directed against the p15 nucleocapsid (N) protein of the reference ATCC-VR2332 isolate. Intracytoplasmic enveloped viral particles that tended to accumulate into cytoplasmic vesicles were observed in the infected PAM; no budding was demonstrated at the level of the cytoplasmic membrane. The extracellular virions appeared as pleomorphic but mostly spherical enveloped particles, 50-72 nm in diameter (averaged diameter of 50 particles was 58.3 nm), with an isometric core about 25-30 nm. Buoyant density of the virus in CsCL density gradients was estimated to 1.18-1.20 g/mL. No hemagglutinating activity was demonstrated. Analysis of semipurified virions of isolate IAF-exp91 by radioimmunoprecipitation (RIPA) and Western immunoblotting experiments, using reference rabbit and porcine hyperimmune sera, revealed four major viral proteins, a predominant 15 kD N protein and three other proteins with predicted M(r_ of 19, 26 and 42 kD. Progeny viral particles produced in PRRSV-infected PAM in the presence of tunicamycin lacked the 42 kD protein, thus confirming its N-glycosylated nature. Immunoprecipitation experiments using the anti-ATCC-VR2332 MoAbs confirmed the close antigenic relationships between Quebec and American reference isolates of PRRSV.
Nucleotide sequences between the spike (S) and membrane (M) protein genes of the OC43 strain of human corona-virus were obtained from PCR-amplified viral mRNAs. Sequence analysis of this region revealed the presence of two ORFs encoding proteins of 12.9 and 9.5 kDa. These two proteins were identified as putatively nonstructural (ns) due to their homology to the corresponding BCV ns gene products. Northern blot analysis indicated that each of these two genes was present on a separate mRNA (5 and 5-1, respectively). In vitro translation analyses demonstrated that the HCV-OC43 9.5-kDa protein, like its BCV counterpart, is poorly translated when situated downstream of the 12.9-kDa ORF, although immunofluorescence studies did confirm its presence in infected cells. Sequence analysis showed that a large portion of the 3'-end of the leader sequence is present within the viral genome, upstream of the 12.9-kDa ORF. In addition, two ORFs encoding potential 4.9- and 4.8-kDa ns proteins in BCV are absent in HCV-OC43, although a corresponding mRNA 4 was found at a very low level. These results demonstrate that these two putative ns proteins are not essential for virus replication, at least in HRT-18 cells.
The nucleotide sequence of the region between the spike (S) and the membrane (M) protein genes, and sequences of the S and ns2 genes of the OC43 strain of human coronavirus (HCV-OC43) were determined. The ns2 gene comprises an open reading frame (ORF) encoding a putative nonstructural (ns) protein of 279 amino acids with a predicted molecular mass of 32-kDa. The S gene comprises an ORF encoding a protein of 1353 amino acid residues, with a predicted molecular weight of 149,918. Sequence comparison between HCV-OC43 and the antigenically related bovine coronavirus (BCV) revealed more sequence divergence in the putative bulbous part of the S protein (S1) than in the stem region (S2). The cysteine residues near the transmembrane domain and the internal predicted protease cleavage site are conserved in the HCV-OC43 S protein. Nucleotide sequence analysis of the region between the S and M gene loci revealed the presence of an unexpected intragenomic partial leader sequence and two ORFs encoding potential proteins of 12.9 and 9.5-kDa. These two proteins were identified as nonstructural by comparison with the homologous BCV genes. In vitro translation analyses demonstrated that the HCV-OC43 9.5-kDa protein, like its BCV counterpart, is poorly translated when situated down-stream of the 12.9-kDa ORF, but is expressed in infected cells, as shown by immunofluorescence. Interestingly, two ORFs, potentially encoding 4.9 and 4.8-kDa ns proteins in BCV are absent in HCV-OC43, indicating that they are not essential for viral replication in HRT-18 cells.
The 299E prototype strain of human coronavirus (HCV-229E) has so far been mainly associated with infections of the respiratory tract. In the present study, we show evidence for infection of the central nervous system (CNS) by HCV-229E, both in vitro and in vivo. Various human cell lines of CNS origin were tested for their susceptibility to infection by HCV-229E. Production of viral antigens was monitored by indirect immunofluorescence with monoclonal antibodies and infectious progeny virions by plaque assay on the L132 human embryonic lung cell line. The SK-N-SH neuroblastoma and H4 neuroglioma cell lines were highly susceptible to infection. The U-87 MG and U-373 MG astrocytoma cell lines were also infectable by HCV-229E. We could also demonstrate infection of the MO3.13 cell line, which was established by fusion of human oligodendrocytes with a thioguanine-resistant mutant of the TE671 (RD) human rhabdomyosarcoma cell line. An apparently more extensive infection of the MO3.13 cells, when compared to the parental cells, supports the notion that human oligodendrocytes are differentially susceptible to infection by this virus. We also tested for HCV-229E gene expression in pathological brain specimens. For that purpose, we developed a reverse transcription-polymerase chain reaction (RT-PCR) assay to amplify a portion of the mRNA encoding the viral nucleocapsid protein. Using stringent laboratory conditions, viral RNA was detectable in brain tissue of 4 of 11 multiple sclerosis patients and none of 6 neurological and 5 normal controls.(ABSTRACT TRUNCATED AT 250 WORDS)
The gene encoding the spike protein of the OC43 strain of human coronavirus (HCV-OC43) was cloned and sequenced. The complete nucleotide sequence revealed an open reading frame of 4062 nucleotides encoding a protein of 1353 amino acids with a predicted M(r) of 150,078. Structural features include 22 N-glycosylation sites, an N-terminal hydrophobic signal sequence of 17 amino acids, an hydrophilic cysteine-rich sequence of 35 amino acids near the C terminus, and a potential proteolytic cleavage site (RRSR) between amino acid residues 758 and 759, yielding S1 and S2 segments of 84,730 and 65,366 M(r), respectively. The predicted amino acid sequence of the spike protein of HCV-OC43 has 91% identity with that of the Mebus strain of bovine coronavirus, revealing more sequence divergence in the putative bulbous part (S1) than in the predicted stem region (S2).
Total RNA extracted from both white and gray matter of brain tissue from multiple sclerosis (MS) patients and controls was analyzed using a reverse transcription-polymerase chain reaction for the presence of the nucleic acid of human coronavirus (HCV) 229E and OC43, the two strains characterized to date and associated with respiratory infections. HCV-229E viral RNA was detectable in the central nervous system tissue of 4 of 11 MS patients and in none of 6 neurological and 5 normal controls. No HCV-OC43 nucleic acid was detected in any of the specimens. These results suggest a neurotropism on the part of the 229E strain of human coronavirus and underline the importance of further studies on its tissue distribution. The fact that it was detected only in tissue from MS patients illustrates the need for continued studies on the possible role of coronaviruses in the etiology of MS.
Human coronaviruses (HCV) are important pathogens responsible for respiratory, gastrointestinal and possibly neurological disorders. To better understand the molecular biology of the prototype HCV-229E strain, the nucleotide sequence of the 5'-unique regions of mRNAs 4 and 5 were determined from cloned cDNAs. Sequence analysis of the cDNAs synthesized from mRNA 4 revealed a major difference with previously published results. However, polymerase chain reaction amplification of this region showed that the sequenced cDNAs were produced from minor RNA species, an indication of possible genetic polymorphism in this region of the viral genome. The mutated messenger RNA 4 contains two ORFs: (1) ORF4a consisting of 132 nucleotides which potentially encodes a 44-amino acid polypeptide of 4653 Da; this coding sequence is preceded by a consensus transcriptional initiation sequence, CUAAACU, similar to the ones found upstream of the N and M genes; (2) ORF4b of 249 nucleotides potentially encoding an 83-amino acid basic and leucine-rich polypeptide of 9550 Da. On the other hand, mRNA 5 contains one single ORF of 231 nucleotides which could encode a 77-amino acid basic and leucine-rich polypeptide of 9046 Da. This putative protein presents a significant degree of amino acid homology (33%) with its counterpart found in transmissible gastroenteritis coronavirus (TGEV). The proteins in the two different viruses exhibit similar molecular weights and are extremely hydrophobic. Interestingly, a sequence homology of five amino acids was found between the protein encoded by ORF4b of HCV-229E and an immunologically important region of human myelin basic protein.