P-glycoprotein-overexpressing multidrug-resistant cells are resistant to infection by enveloped viruses that enter via the plasma membrane.
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The multidrug resistance gene product P-glycoprotein confers drug resistance to tumor cells by acting as a transporter that blocks the entry into the cell of a great variety of drugs and hydrophobic peptides. In this study we find that in drug-resistant cells, the insertion of the influenza virus fusion protein (hemagglutinin-2) into the plasma membrane is blocked and that the fusion of the viral envelope with the plasma membrane of these cells is impaired. Multidrug-resistant cells display significant resistance to infection by envelope viruses that invade cells by fusion with the plasma membrane, but not to infection by pH-dependent viruses that penetrate cells by fusion with endocytic vesicles. These observations suggest that multidrug resistance phenomena may protect cells from infection by a large group of disease-causing viruses that includes human immunodeficiency virus, herpes simplex virus, and some cancer-inducing retroviruses. (+info)
Efficiencies of translation in three reading frames of unusual non-ORF sequences isolated from phage display.
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An unusual nucleotide sequence, called H10, was previously isolated by biopanning with a random peptide library on filamentous phage. The sequence encoded a peptide that bound to the growth hormone binding protein. Despite the fact that the H10 sequence can be expressed in Escherichia coli as a fusion to the gene III minor coat protein of the M13 phage, the sequence contained two TGA stop codons in the zero frame. Several mutant derivatives of the H10 sequence carried not only a stop codon, but also showed frameshifts, either +1 or -1 in individual isolates, between the H10 start and the gene III sequences. In this work, we have subcloned the H10 sequence and three of its derivatives (one requiring a +1 reading frameshift for expression, one requiring a -1 reading frameshift, and one open reading frame) in gene fusions to a reporter beta-galactosidase gene. These sequences have been cloned in all three reading frames relative to the reporter. The non-open reading frame constructs gave (surprisingly) high expression of the reporter (10-40% of control vector expression levels) in two out of the three frames. A site-directed mutant of the TGA stop codon (to TTA) in the +1 shifter greatly reduced the frameshift and gave expression primarily in the zero frame. By contrast, a site-directed mutant of the TGA in the -1 shifter had little effect on the pattern of expression, and alteration of the first TGA (of two) in H10 itself paradoxically reduced expression by half. We believe these phenomena to reflect a translational recoding mechanism in which ribosomes switch reading frames or read past stop codons upon encountering a signal encoded in the nucleotide sequence of the mRNA, because both the open reading frame derivative (which has six nucleotide changes from parental H10) and the site-directed mutant of the +1 shifter, primarily expressed the reporter only in the zero frame. (+info)
Fusogenic membrane glycoproteins as a novel class of genes for the local and immune-mediated control of tumor growth.
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We report here the use of viral fusogenic membrane glycoproteins (FMGs) as a new class of therapeutic genes for the control of tumor growth. FMGs kill cells by fusing them into large multinucleated syncytia, which die by sequestration of cell nuclei and subsequent nuclear fusion by a mechanism that is nonapoptotic, as assessed by multiple criteria. Direct and bystander killing of three different FMGs were at least one log more potent than that of herpes simplex virus thymidine kinase or cytosine deaminase suicide genes. Transduction of human tumor xenografts with plasmid DNA prevented tumor outgrowth in vivo, and cytotoxicity could be regulated through transcriptional targeting. Syncytial formation is accompanied by the induction of immunostimulatory heat shock proteins, and tumor-associated FMG expression in immunocompetent animals generated specific antitumor immunity. (+info)
Virus entry is a major determinant of cell tropism of Edmonston and wild-type strains of measles virus as revealed by vesicular stomatitis virus pseudotypes bearing their envelope proteins.
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The Edmonston strain of measles virus (MV) that utilizes the human CD46 as the cellular receptor produced cytopathic effects (CPE) in all of the primate cell lines examined. In contrast, the wild-type MV strains isolated in a marmoset B-cell line B95a (the KA and Ichinose strains) replicated and produced CPE in some but not all of the primate lymphoid cell lines. To determine the mechanism underlying this difference in cell tropism, we used a recently developed recombinant vesicular stomatitis virus (VSV) containing as a reporter the green fluorescent protein gene in lieu of the VSV G protein gene (VSVDeltaG*). MV glycoproteins were efficiently incorporated into VSVDeltaG*, producing the VSV pseudotypes. VSVDeltaG* complemented with VSV G protein efficiently infected all of the cell lines tested. The VSV pseudotype bearing the Edmonston hemagglutinin (H) and fusion (F) protein (VSVDeltaG*-EdHF) infected all cell lines in which the Edmonston strain caused CPE, including the rodent cell lines to which the human CD46 gene was stably transfected. The pseudotype bearing the wild-type KA H protein and Edmonston F protein (VSVDeltaG*-KAHF) infected all lymphoid cell lines in which the wild-type MV strains caused CPE as efficiently as VSVDeltaG*-EdHF, but it did not infect any of the cell lines resistant to infection with the KA strain. The results indicate that the difference in cell tropism between these MV strains was largely determined by virus entry, in which the H proteins of respective MV strains play a decisive role. (+info)
The phage infection process: a functional role for the distal linker region of bacteriophage protein 3.
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The filamentous bacteriophage infects Escherichia coli by interaction with the F pilus and the TolQRA complex. The virus-encoded protein initiating this process is the gene 3 protein (g3p). The g3p molecule can be divided into three different domains separated by two glycine-rich linker regions. Though there has been extensive evaluation of the importance of the diverse domains of g3p, no proper function has so far been assigned to these linker regions. Through the design of mutated variants of g3p that were displayed on the surface of bacteriophage, we were able to elucidate a possible role for the distal glycine-rich linker region. A phage that displayed a g3p comprised of only the N1 domain, the first linker region, and the C-terminal domain was able to infect cells at almost the same frequency as the wild-type phage. This infection was proven to be dependent on the motif between amino acid residues 68 and 86 (i.e., the first glycine-rich linker region of g3p) and on F-pilus expression. (+info)
Protective immunity in macaques vaccinated with a modified vaccinia virus Ankara-based measles virus vaccine in the presence of passively acquired antibodies.
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Recombinant modified vaccinia virus Ankara (MVA), encoding the measles virus (MV) fusion (F) and hemagglutinin (H) (MVA-FH) glycoproteins, was evaluated in an MV vaccination-challenge model with macaques. Animals were vaccinated twice in the absence or presence of passively transferred MV-neutralizing macaque antibodies and challenged 1 year later intratracheally with wild-type MV. After the second vaccination with MVA-FH, all the animals developed MV-neutralizing antibodies and MV-specific T-cell responses. Although MVA-FH was slightly less effective in inducing MV-neutralizing antibodies in the absence of passively transferred antibodies than the currently used live attenuated vaccine, it proved to be more effective in the presence of such antibodies. All vaccinated animals were effectively protected from the challenge infection. These data suggest that MVA-FH should be further tested as an alternative to the current vaccine for infants with maternally acquired MV-neutralizing antibodies and for adults with waning vaccine-induced immunity. (+info)
DNA vaccination with both the haemagglutinin and fusion proteins but not the nucleocapsid protein protects against experimental measles virus infection.
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Plasmids that expressed the nucleocapsid, haemagglutinin and fusion proteins of measles virus (MV) were used to immunize cotton rats (Sigmodon hispidus) against intranasal MV infection. After immunization with all three plasmids, T cell responses and MV-specific antibodies were induced. A reduction in virus titre was observed in lung tissue from animals immunized with plasmids expressing the viral glycoproteins. Histologically, however, a moderate peribronchitis was observed after immunization with the plasmid expressing the fusion protein whereas, after immunization with plasmids expressing haemagglutinin or both glycoproteins, only mild or focal peribronchitis was seen. Immunization with the nucleocapsid did not reduce virus titres, probably because of the failure to induce neutralizing antibodies. A disadvantage of plasmid immunization was its inefficacy in the presence of MV-specific 'maternal' antibodies. This indicates that genetic immunization has to be improved to be a useful alternative vaccine against measles. (+info)
Fusion protein of the paramyxovirus SV5: destabilizing and stabilizing mutants of fusion activation.
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The fusion (F) protein of the paramyxovirus SV5 strain W3A causes syncytium formation without coexpression of the SV5 hemagglutinin-neuraminidase (HN) glycoprotein, whereas the F protein of the SV5 strain WR requires coexpression of HN for fusion activity. SV5 strains W3A and WR differ by three amino acid residues at positions 22, 443, and 516. The W3A F protein residues P22, S443, and V516 were changed to amino acids found in the WR F protein (L22, P443, and A516, respectively). Three single-mutants, three double-mutants, and the triple-mutant were constructed, expressed, and assayed for fusion using three different assays. Mutant P22L did not cause fusion under physiological conditions, but fusion was activated at elevated temperatures. Compared with the W3A F protein, mutant S443P enhanced the fusion kinetics with a faster rate and greater extent, and had a lower activation temperature. Mutant V516A had little effect on F protein-mediated fusion. The double-mutant P22L,S443P was capable of causing fusion, suggesting that the two mutations have opposing effects on fusion activation. The WR F protein requires coexpression of HN to cause fusion at 37 degrees C, and does not cause fusion at 37 degrees C when coexpressed with influenza virus hemagglutinin (HA); however, at elevated temperatures coexpression of WR F protein with HA resulted in fusion activation. In the crystal structure of the core trimer of the SV5 F protein (Baker, K. A., Dutch, R. E., Lamb, R.A., and Jardetzky, T. S. (1999). Mol. Cell 3, 309-319), S443 is the last residue (with interpretable electron density) in an extended chain region and the temperature factor for S443 is high, suggesting conformational flexibility at this point. Thus, the presence of prolines at residues 22 and 443 may destabilize the F protein and thereby decrease the energy required to trigger the presumptive conformational change to the fusion-active state. (+info)