GPR119, a novel G protein-coupled receptor target for the treatment of type 2 diabetes and obesity.

GPR119 is a G protein-coupled receptor expressed predominantly in the pancreas (beta-cells) and gastrointestinal tract (enteroendocrine cells) in humans. De-orphanization of GPR119 has revealed two classes of possible endogenous ligands, viz., phospholipids and fatty acid amides. Of these, oleoylethanolamide (OEA) is one of the most active ligands tested so far. This fatty acid ethanolamide is of particular interest because of its known effects of reducing food intake and body weight gain when administered to rodents. Agonists at the GPR119 receptor cause an increase in intracellular cAMP levels via G(alphas) coupling to adenylate cyclase. In vitro studies have indicated a role for GPR119 in the modulation of insulin release by pancreatic beta-cells and of GLP-1 secretion by gut enteroendocrine cells. The effects of GPR119 agonists in animal models of diabetes and obesity are reviewed, and the potential value of such compounds in future therapies for these conditions is discussed.

The design and synthesis of potent inhibitors of hepatitis C virus NS3-4A proteinase.

Hepatitis C virus (HCV) is the cause of the majority of transfusion-associated hepatitis and a significant proportion of community-acquired hepatitis worldwide. Infection by HCV frequently leads to persistent infections that result in a range of clinical conditions including an asymptomatic carrier state, severe chronic active hepatitis, cirrhosis and, in some cases, hepatocellular carcinoma. The HCV genome consists of a single-stranded, positive sense RNA containing an open reading frame of approximately 9060 nucleotides. This is translated into a single polyprotein of approximately 3020 amino acids (C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B), which in turn is processed by a series of host and viral proteinases into at least 10 cleavage products. The N-terminal portion of the NS3 protein encodes a serine proteinase that is responsible for the cleavage at the NS3-4A, NS4A-4B, NS4B-5A and NS5A-5B junctions. The 54 amino acid NS4A protein is a cofactor that binds to the NS3 protein and enhances its proteolytic activity. This report describes the expression of a recombinant NS3-4A proteinase fusion protein in Escherichia coli and the in vitro characterization of the enzyme activity using synthetic peptide substrates. It then demonstrates how these results were employed to guide the design of potent inhibitors of this enzyme.

Herpes simplex virus type 1 gene UL13 encodes a phosphoprotein that is a component of the virion.

The UL13 open reading frame of herpes simplex virus type 1 (HSV-1) has been expressed in insect cells by a recombinant baculovirus and in Escherichia coli. In the latter case, the UL13 gene was fused to the gene for glutathione S-transferase (GST) to allow high-level expression of an 80-kDa GST-UL13 fusion protein. Antibody raised against the fusion protein reacted specifically with the 55-kDa UL13 gene product expressed by the recombinant baculovirus. This antibody also recognized a late phosphoprotein in HSV-1-infected cell lysates and a component of purified HSV-1 virions, both with the same electrophoretic mobility as the baculovirus-expressed protein. The virion component was efficiently phosphorylated in vitro by a virion-associated protein kinase. Using the same antibody, the probable homolog of the UL13 gene product was identified in HSV-2-infected cells and purified virions.

Effect of two novel inhibitors of the human immunodeficiency virus protease on the maturation of the HIV gag and gag-pol polyproteins.

The ability of two novel synthetic compounds to inhibit the HIV protease-mediated processing of HIV-1 precursor polyproteins was investigated in an in vitro gag-protease mixed lysate assay system and in an assay using recombinant baculoviruses engineered to express the HIV-1 gag and pol genes in cultured insect cells. With the in vitro mixed lysate assay we have shown that both compounds at 1 microM can completely inhibit the HIV-1 and HIV-2 protease-mediated release of p24 from the HIV-1 gag precursor at pH 5.5 and pH 7.0. In the intracellular baculovirus system these compounds were shown to inhibit the protease-mediated maturation of gag and also the excision of the protease moiety from its precursor.

Expression of HIV-1 gp120 and human soluble CD4 by recombinant baculoviruses and their interaction in vitro.

The soluble domains of the envelope glycoprotein of HIV-1 (gp120) and human CD4 (sCD4) have been individually expressed in insect cells using recombinant baculoviruses. Each product is secreted from infected cells and accumulates in the surrounding media to levels of 1-2 mg/liter of 2 x 10(9) cells. Both molecules have full biological activity, and conditioned media from infected cells have been used to establish a simple assay for gp120-sCD4 interaction that is highly specific and amenable to mass screening. The crystallization of sCD4 purified from this source is reported.

Expression of glycoprotein D of herpes simplex virus type 1 in a recombinant baculovirus: protective responses and T cell recognition of the recombinant-infected cell extracts.

Recombinant baculoviruses expressing glycoprotein D (gD) of herpes simplex virus type 1 (HSV-1) have been generated. The proteins expressed from the recombinants have been characterized using monoclonal antibodies on Western blots and by immunoprecipitation. Partially glycosylated 48K polypeptides have been identified as products of the gD gene. Polyclonal sera from H-2k mice infected with HSV-1 recognized the same polypeptides. Furthermore, draining lymph node cells from H-2k mice infected with HSV-1 proliferated in vitro in response to recombinant-infected cell extracts. Immunization with such extracts generated high titre complement-dependent and -independent neutralizing antibody and the mice were protected against a challenge with HSV-1.

The protease and gag gene products of the human immunodeficiency virus: authentic cleavage and post-translational modification in an insect cell expression system.

Three recombinant baculoviruses which are capable of expressing human immunodeficiency virus (HIV) protease, p55gag, and both products simultaneously in insect cell culture have been constructed. Upon co-infection of cells with the protease and p55gag-expressing viruses, authentic processing of the gag precursor is observed to take place. This processing could be reproduced in vitro using mixtures of cellular lysates containing the expressed proteins. When expressed alone, uncleaved p55gag precursor appears to form retroviral core-like particles within the cytoplasm of infected cells. Metabolic labeling studies of the baculovirus-expressed gag products have demonstrated that p17 is myristylated at its amino terminus, and that p24 is phosphorylated. In these respects, the insect cell system is evidently capable of carrying out post-translational processing resembling that which occurs in authentic HIV-1 replication.

Baculovirus expression vectors: the requirements for high level expression of proteins, including glycoproteins.

The requirements for high level expression of three foreign proteins using the polyhedrin gene promoter of Autographa californica nuclear polyhedrosis virus (AcNPV, Baculoviridae) have been investigated. In Spodoptera frugiperda cells infected with the appropriate recombinant baculoviruses, the synthesis of the two S RNA coded genes of lymphocytic choriomeningitis virus (LCMV; i.e. the nucleoprotein, N, and glycoprotein precursor, GPC), or the haemagglutinin gene of influenza A virus, appears to be related to the degree of integrity of the 5' upstream sequence of the polyhedrin gene. No effect on the level of N protein expression was detected when all the polyhedrin gene coding sequences or some of the immediate 3' downstream sequences were deleted. Using the most efficient expression viruses derived from a new transfer vector, pAcYM1, it has been estimated that LCMV N protein represented approximately 50% of the total cellular protein, an observation consistent with the presence of numerous inclusion bodies in the cytoplasm of infected cells. For recombinant viruses derived from the pAcYM1 transfer vector containing the LCMV GPC gene, the level of synthesis of the arenavirus glycoprotein was equivalent to approximately 20% of the cellular protein. Thin sections of cells infected with the GPC recombinant revealed a highly vacuolated cytoplasm.

Identification of the N and NSS proteins coded by the ambisense S RNA of Punta Toro phlebovirus using monospecific antisera raised to baculovirus expressed N and NSS proteins.

An essentially complete DNA copy of the ambisense S RNA species of Punta Toro (PT) phlebovirus (T. Ihara, H. Akashi, and D.H.L. Bishop, 1984, Virology 136, 293-306) has been inserted in either orientation into Autographa californica nuclear polyhedrosis baculovirus (AcNPV) in lieu of the 5' coding region of the AcNPV polyhedrin gene (G.E. Smith, M.D. Summers, and M.J. Fraser, 1983, Mol. Cell. Biol. 3, 2156-2165). The two types of recombinant viruses were used to infect Spodoptera frugiperda cells and the expressed PT viral proteins characterized. Recombinant AcNPV having the S DNA in one orientation expressed PT virus N protein in amounts estimated to represent some 50% of the infected cell extracts, whereas recombinants with the S DNA in the other orientation expressed the putative PT virus NSS protein in lower quantities. Antisera that were monospecific with respect to each of the two PT proteins virus were raised in mice using the corresponding S. frugiperda infected cell extracts and were employed to identify N and NSS proteins in PT virus-infected Vero cells.

Molecular studies of the differential replication at pyrexial temperatures of two influenza viruses differing in virulence for ferrets.

Replication of a virulent clone (7a) of the reassortant influenza virus A/Puerto Rico/8/34-A/England/939/69 (H3N2) in ferret nasal turbinate tissue is less affected than that of an attenuated clone (64d) by temperatures which occur during pyrexia in ferrets. This is a factor which contributes to the difference in virulence of the two clones. The differential replication of the two clones at pyrexial temperatures has been reproduced in allantois-on-shell (egg-bit) cultures, and the synthesis of viral polypeptides and RNA species examined. This virus-host system was chosen because it was more convenient to use than organ cultures but, like the latter, might provide information relevant to the in vivo situation. With this system it was not possible to achieve single cycle replication: the observed effects are cumulative over several (2 to 3) cycles of replication (24 h) and therefore conclusions from them may not be as definitive as those from single cycle conditions. However, in cells infected with clone 64d both A(+) cRNA and polypeptide synthesis were little affected at 40 degrees C but levels were decreased by about 70-80% at 41 degrees C; A(+) cRNA and polypeptide levels were unaffected even at 41 degrees C with clone 7a. These reductions seem insufficient to account for the 10-fold reduction in infectious yields of clone 64d at 40 degrees C or the 100-fold and 10-fold reductions in yields of clones 64d and 7a respectively at 41 degrees C. There was no evidence of increased production of non-infectious virus at elevated temperatures by either clone. Levels of vRNA were considerably reduced at 40 and 41 degrees C for both clones, but the levels were considerably greater at all temperatures in clone 7a-infected cells than in those infected with clone 64d; vRNA levels were higher for clone 7a at 41 degrees C than for clone 64d at 37 degrees C. The different levels of vRNA do not reflect differences in the availability of template A(-) cRNAs since levels of these were similar for both clones at 37 and 40 degrees C and only reduced for clone 64d at 41 degrees C. Although the interpretation of these data is complicated by multiple cycles of replication it appears that limited availability of vRNA could be an important constraint on the ability of clone 64d to replicate at pyrexial temperatures.

Severity of fever in influenza: studies on the relation between viral surface antigens, pyrexia, level of nasal virus and inflammatory response in the ferret.

Previous work has shown that fever in influenza of ferrets occurs following release of endogenous pyrogen from virus-phagocyte interaction in the upper respiratory tract (URT), and suggested that the poor inflammatory response and correspondingly low fever elicited by A/Puerto Rico/8/34 (H1N1), compared with H3N2 reassortant clones of A/Puerto Rico/8/34-A/England/939/69, were related to its H1 and N1 surface antigens. Nasal virus levels, inflammatory and pyrexial responses produced in ferrets by clones 31 (H3N1) and 64b (H1N2) of the same reassortant system suggested a connection between the H1 antigen and low inflammatory response, but results were not conclusive. Unlike A/Puerto Rico/8/34, two recent H1N1 isolates, A/USSR/90/77 and A/Fiji/15899/83, produced a high inflammatory response yet low fever despite large amounts of virus in the URT, suggesting that either no connection exists between the acquisition of the H1 antigen and production of a low inflammatory response, or the H1 antigen of recent isolates, whilst antigenically related to that of A/Puerto Rico/8/34, is biologically different.

Further studies of the reasons for the lack of alveolar infection during influenza in ferrets.

Intratracheal inoculation of influenza virus in the ferret was followed by a more severe airway infection than that produced by nasal infection and was mainly bronchiolar rather than bronchial. Also, virus isolation from the alveolar zone of the lung together with immunofluorescence and immunoperoxidase techniques showed that some virus reached the alveoli. Nevertheless, there was no subsequent alveolitis suggesting the existence of a clearance phenomenon. Alveolar macrophages were shown to have phagocytosed virus in vivo and phagocytosis studies in vitro showed that two mechanisms could operate to eradicate the virus. First, a rapid destruction of virus and second an abortive cycle of replication which produced virus antigen but not infectious virus. Experiments with large doses of virus indicated that after intranasal inoculation little virus reached the alveoli so it would probably be quickly cleared by the macrophages.

Recent H1N1 viruses (A/USSR/90/77, A/Fiji/15899/83, A/Firenze/13/83) replicate poorly in ferret bronchial epithelium. Brief report.

Three recent wild-type H1N1 influenza virus isolates (A/USSR/90/77, A/Fiji/15899/83 and A/Firenze/13/83) replicated poorly in organ cultures of ferret bronchial tissue compared with the replication of an H3N2 wild-type virus (A/England/939/69). All four viruses replicated well in nasal turbinate tissue. Examination of one H1N1 virus (A/USSR/90/77) in vivo showed heavy infection in the upper respiratory tract of ferrets but little in the lower respiratory tract. These results raise the possibility that the mildness of human influenza arising from the H1N1 strains may be due to lack of capacity to attack the lower respiratory tract as well as the presence of antibody in previously exposed persons.