Biomedical subjects

R W Compans

Publications and source records attributed to R W Compans.

At least 19 recordsLinked to original sources

PubMed2005

Prevention of HIV-1 infection by platinum triazines.

To identify and explore the activity of compounds which may act as anti-HIV virucidal agents, we have investigated platinum compounds, especially those containing N-donor aromatic ligands. After screening over 70 related agents, including N-donor aromatic ligands and metal precursors, we have identified a novel class of platinum(II) complexes with 2-pyridyl-1,2,4-triazine derivatives and Pt(II) formulations with these derivatives (ptt compounds) as having the highest anti-HIV activity. The maximum activity was observed when the agents were added immediately post-infection. The ptt agents did not block cell fusion activity of HIV-1 Env proteins in cells bearing CD4X4 or CD4R5 receptors, indicating a lack of interaction with the Env protein. The ptt compounds exhibit low toxicity for human epithelial cells, and are thus promising candidates for use as microbicides or antiviral agents against HIV.

Animals↗

PubMed2003

Sin Nombre virus glycoprotein trafficking.

Sin Nombre virus (SNV) is a major representative of the New World hantaviruses and the most common cause of hantavirus pulmonary syndrome (HPS) with high mortality in North America. Unlike other members of the family Bunyaviridae which mature in the Golgi complex, New World hantaviruses have been previously reported to mature at the cell surface. For family Bunyaviridae viruses, retention of the viral glycoproteins at the Golgi complex is thought to be responsible for their Golgi maturation. In our studies, the majority of SNV glycoproteins, G1 and G2, was localized in the Golgi complex when expressed from a full-length GPC clone or in SNV-infected cells, in agreement with data for other members of the family Bunyaviridae, including the Old World hantaviruses. However, the SNV glycoproteins could also be detected at the cell surface at advanced posttransfection or postinfection time points. G1 expressed in the absence of G2 did not accumulate in the Golgi, but remained predominantly associated with the endoplasmic reticulum (ER). Overexpressed amounts of apparently misfolded G1 were aggregated in a subcellular compartment likely to represent the aggresome. Unexpectedly, an additional major pool of G1 was detected intracellularly in SNV-infected and GPC-expressing transfected cells, by using a SNV G1-specific Fab antibody. This pool of G1 is predominantly localized in late endosomes-lysosomes.

Animals↗

PubMed2003

Mutations in the cytoplasmic domain of a paramyxovirus fusion glycoprotein rescue syncytium formation and eliminate the hemagglutinin-neuraminidase protein requirement for membrane fusion.

SER virus is closely related to the paramyxovirus simian virus 5 (SV5) but is defective in syncytium formation. The SER virus F protein has a long cytoplasmic tail (CT) domain that has been shown to inhibit membrane fusion, and this inhibitory effect could be eliminated by truncation of the C-terminal sequence (S. Tong, M. Li, A. Vincent, R. W. Compans, E. Fritsch, R. Beier, C. Klenk, M. Ohuchi, and H.-D. Klenk, Virology 301:322-333, 2002). To study the sequence requirements for regulation of fusion, codons for SER virus F protein residues spanning amino acids 535 to 542 and 548 were mutated singly to alanines, and the two leucine residues at positions 539 and 548 were mutated doubly to alanines. We found that leu-539 and leu-548 in the CT domain played a critical role in the inhibition of fusion, as mutation of the two leucines singly to alanines partially rescued fusion, and the double mutation L539, 548A completely rescued syncytium formation. Mutation of charged residues to alanines had little effect on the suppression of fusion activity, whereas the mutation of serine residues to alanines enhanced fusion activity significantly. The L539, 548A mutant also showed extensive syncytium formation when expressed without the SER virus HN protein. By constructing a chimeric SV5-SER virus F CT protein, we also found that the inhibitory effect of the long CT of the SER virus F protein could be partially transferred to the SV5 F protein. These results demonstrate that an elongated CT of a paramyxovirus F protein interferes with membrane fusion in a sequence-dependent manner.

Amino Acid Sequence↗

PubMed2003

Activation of fusion by the SER virus F protein: a low-pH-dependent paramyxovirus entry process.

SER virus, a paramyxovirus closely related to simian virus 5, induces no syncytium formation. The SER virus F protein has a long cytoplasmic tail (CT), and truncation or mutations of the CT result in enhanced syncytium formation (S. Seth, A. Vincent, and R. W. Compans, J. Virol. 77:167-178, 2003; S. Tong, M. Li, A. Vincent, R. W. Compans, E. Fritsch, R. Beier, C. Klenk, M. Ohuchi, and H.-D. Klenk, Virology 301:322-333, 2002). We hypothesized that the presence of the long CT serves to stabilize the metastable conformation of the F protein. We observed that the hemifusion, cytoplasmic content mixing, and syncytium formation ability of the wild-type SER virus F coexpressed with the SER virus hemagglutinin-neuraminidase (HN) protein was enhanced, both qualitatively and quantitatively, at elevated temperatures. We also observed enhanced hemifusion, content mixing, and syncytium formation in SER virus F- and HN-expressing cells at reduced pH conditions ranging between 4.8 and 6.2. We have obtained evidence that in contrast to other paramyxoviruses, entry of SER virus into cells occurs by a low-pH-dependent process, indicating that the conversion to the fusion-active state for SER virus F is triggered by exposure to reduced pH.

Animals↗

PubMed2002

Regulation of fusion activity by the cytoplasmic domain of a paramyxovirus F protein.

SER virus is a member of the family Paramyxoviridae, genus Rubulavirus, which has been isolated from pigs. It is very closely related to SV5 virus serologically, in protein profile, and in nucleotide sequence. However, unlike SV5, SER induces minimal syncytium formation in infected CV-1 or BHK cells. Fluorescence transfer experiments between labeled erythrocytes and infected MDBK cells revealed that SER also induces hemifusion and pore formation with reduced efficiency. The virion polypeptide profiles of SER and SV5 are very similar, except that the SER F1 subunit shows an apparent molecular weight that is about 2 kDa higher than that of SV5. Comparison of the deduced amino acid sequences revealed the SER F (551 aa) to be longer than SV5 F (529 aa) by 22 residues in the cytoplasmic tail (CT) domain. The HN and M gene sequences of the viruses were found to be very similar. The SER F showed minimal fusion activity when coexpressed with either SV5 or SER HN. In contrast, SV5 F was highly fusogenic when coexpressed with either HN protein, indicating that the restricted fusion capacity of SER virus is a property of its F protein. Truncation in the CT of SER F by 22 residues completely rescued its ability to cause syncytium formation, whereas other truncations rescued syncytium formation partially. These results demonstrate that an elongated CT of a paramyxovirus F protein suppresses its membrane fusion activity.

Animals↗

PubMed2001

HIV envelope proteins differentially utilize CXCR4 and CCR5 coreceptors for induction of apoptosis.

The involvement of CXCR4 and CCR5 coreceptors in apoptosis induced by the HIV envelope (Env) proteins has not been well defined. We found that simian human immunodeficiency virus (SHIV) virus-like particles (VLPs) containing HIV Env proteins preferentially induce apoptosis of cells corresponding to their coreceptor usage in a CD4+ T cell line. We also demonstrated that induction of apoptosis by SHIV VLPs is correlated with coreceptor usage in a non-T cell line. We examined the effects of SHIV VLPs containing Env proteins derived from either a T-cell-tropic HIV (BH10) strain or a dual-tropic HIV (89.6) strain on induction of apoptosis in recombinant CD4+ human osteosarcoma (HOS) cells expressing either CXCR4 (HOS-CD4.CXCR4) or CCR5 coreceptors (HOS-CD4.CCR5). HOS-CD4.CXCR4 or HOS-CD4.CCR5 cells were activated with concanavalin A and cocultured with VLPs. By TUNEL (TdT-mediated dUTP-X nick end labeling) fluorescence staining and flow cytometry assays, SHIV BH10 VLPs were found to preferentially induce apoptosis in HOS-CD4.CXCR4 cells but not in HOS-CD4 or HOS-CD4.CCR5 cells. On the other hand, SHIV 89.6 VLPs induced an elevated level of apoptosis in both HOS-CD4.CXCR4 and HOS-CD4.CCR5 cells in a dose-dependent fashion. These data demonstrate that T-cell-tropic BH10 Env preferentially utilizes CXCR4, but not CCR5, for induction of apoptosis, whereas dual-tropic 89.6 Env induces apoptosis in both CXCR4- and CCR5-containing cell lines.

Apoptosis↗

PubMed2001

An infectious clone of the West Nile flavivirus.

West Nile (WN) virus is the most widespread among flaviviruses, but until recently it was not known on the American continent. We describe here design of a subgenomic replicon, as well as a full-length infectious clone of the lineage II WN strain, which appeared surprisingly stable compared to other flavivirus infectious clones. This infectious clone was used to investigate effects of 5'- and 3'-nonrelated sequences on virus replication and infectivity of synthetic RNA. While a long nonrelated sequence at the 3'-end delayed but did not prevent establishment of the productive infectious cycle, a much shorter extra sequence at the 5'-end completely abrogated virus replication. Replacement of the conserved 5'-adenosine residue substantially delayed, but did not prevent, establishment of virus infection. In all cases, the recovered virus had restored its authentic 5'- and 3'-end genome sequences. However, the presence of extensive nonrelated sequences at both 5'- and 3'-ends could not be repaired.

3' Untranslated Regions↗

PubMed2001

Three membrane-proximal amino acids in the human parainfluenza type 2 (HPIV 2) F protein are critical for fusogenic activity.

In this study, we investigated the role of the membrane-proximal region of the human parainfluenza virus type 2 (HPIV2) F protein by mutational analysis, including deletion, insertion, and substitution. Deletion or replacement of the entire 12 amino acid region (aa 474-485) of the HPIV2 F protein completely abolished its fusion activity when coexpressed with the HPIV2 HN protein. Deletion of groups of four of aa 478-485, single alanine, or other amino acid substitutions among aa 478-485 had minimal or limited effects on HPIV2 F/HN-induced cell fusion. However, a significant reduction in, or complete inhibition of, fusion activity was observed when aa 474-477 were deleted, or the N475, F476, or F477 residues were singly substituted with alanine. In addition, insertions of four amino acids at this region or deletion of eight or more amino acids significantly reduced F protein fusion activity. The oligomerization patterns and levels of cell surface expression of the mutant F proteins were compared to those of the wild-type HPIV2 F protein. The mutant HPIV2 F proteins defective in fusion were also found to be unable to initiate hemifusion, indicating that there is a specific requirement for three specific amino acids as well as the spacing in this region for initiating lipid mixing.

Amino Acids↗

PubMed2001

Hantavirus nucleocapsid protein is expressed as a membrane-associated protein in the perinuclear region.

Black Creek Canal virus (BCCV) is a New World hantavirus which is associated with hantavirus pulmonary syndrome. We have examined the site of expression of the BCCV nucleocapsid protein (NBCCV) in the absence of BCCV glycoproteins and found that the majority of the protein is localized to the Golgi region. Immunofluorescence analysis of BHK21 cells expressing the NBCCV and La Crosse virus nucleocapsid protein (NLACV) showed different intracellular localization patterns of these proteins within the same cell: NLACV is cytoplasmic, whereas NBCCV is perinuclear. NBCCV was found to be colocalized with alpha-mannosidase II, a marker for the Golgi complex. Also, NBCCV was found to be associated with microsomal membranes following cell fractionation. Sedimentation analysis in density gradients revealed that the membrane association of NBCCV is sensitive to treatments with high-salt and high-pH solutions, which indicates that NBCCV is a peripheral membrane protein. Analysis of NBCCV truncation mutants revealed that the 141-amino-acid C-terminal portion of this protein was capable of targeting green fluorescent protein to the perinuclear region. The difference in the intracellular localization between the NBCCV and NLACV proteins suggests that the mechanisms involved in the morphogenesis of New World hantaviruses are distinct from that documented for other members of the Bunyaviridae family.

Animals↗

PubMed2001

Mutations in the cytoplasmic tail of murine leukemia virus envelope protein suppress fusion inhibition by R peptide.

During viral maturation, the cytoplasmic tail of the murine leukemia virus (MuLV) envelope (Env) protein undergoes proteolytic cleavage by the viral protease to release the 16-amino-acid R peptide, and this cleavage event activates the Env protein's fusion activity. We introduced Gly and/or Ser residues at different positions upstream of the R peptide in the cytoplasmic tail of the Friend MuLV Env protein and investigated their effects on fusion activity. Expression in HeLa T4 cells of a mutant Env protein with a single Gly insertion after I619, five amino acids upstream from the R peptide, induced syncytium formation with overlaid XC cells. Env proteins containing single or double Gly-Ser insertions after F614, 10 amino acids upstream from the R peptide, induced syncytium formation, and mutant proteins with multiple Gly insertions induced various levels of syncytium formation between HeLa T4 and XC cells. Immunoprecipitation and surface biotinylation assays showed that most of the mutants had surface expression levels comparable to those of the wild-type or R peptide-truncated Env proteins. Fluorescence dye redistribution assays also showed no hemifusion in the Env proteins which did not induce fusion. Our results indicate that insertion mutations in the cytoplasmic tail of the MuLV Env protein can suppress the inhibitory effect of the R peptide on membrane fusion and that there are differences in the effects of insertions in two regions in the cytoplasmic tail upstream of the R peptide.

Amino Acid Sequence↗

PubMed2000

The envelope glycoprotein of simian immunodeficiency virus contains an enterotoxin domain.

By the use of a mouse model, the enteropathic effects of the simian immunodeficiency virus (SIV) surface unit (SU) envelope glycoprotein were explored. Purified SU (0.01-0.45 nmol) was administered intraperitoneally to 6- to 8-day-old mouse pups and induced a dose-dependent diarrheal response. Surgical introduction of SU into adult mouse intestinal loops revealed fluid accumulation without histological alterations and SU-treated unstripped intestinal mucosa induced chloride (Cl(-)) secretory currents in Ussing chambers. Similarly to rotavirus NSP4, the first described viral enterotoxin, SU induced a transient increase in intracellular calcium levels and increased inositol 1,4,5-triphosphate (IP(3)) levels in HT-29 cells. These data indicate the calcium response is mediated by IP(3). The presence of diarrhea and fluid accumulation within intestinal loops in the absence of histological alterations and induction of Cl(-) secretory currents demonstrate that SIV contains an enterotoxic domain localized within SU and is the second viral enterotoxin described.

Animals↗

PubMed2000

Oligomerization, secretion, and biological function of an anchor-free parainfluenza virus type 2 (PI2) fusion protein.

A number of studies indicate that the transmembrane domain, the cytoplasmic domain, or both regions of viral surface glycoproteins are involved in quaternary structure formation. In this report, the transmembrane domain and cytoplasmic tail coding sequence of the fusion (F) glycoprotein gene from parainfluenza type 2 virus was truncated by PCR and the resulting gene (PI2F') was expressed in HeLa-T4 cells by using the vaccinia virus-T7 transient expression system. Pulse-chase experiments indicated that the anchor-free PI2F' was expressed and processed into F(1) and F(2) subunits. Both the processed and the unprocessed anchor-free PI2F' proteins were found to be efficiently secreted into the culture medium. Examination of the oligomeric form of the anchor-free PI2F' by chemical cross-linking demonstrated that it assembles posttranslationally into dimers and trimers with a pattern similar to that of the wild-type PI2F protein. In an effort to better understand the biological properties of the truncated form of PI2F', we anchored PI2F' by a glycosyl-phosphatidylinositol (GPI) linkage. The GPI-anchored PI2F' protein, when coexpressed with PI2HN, did not induce cell fusion seen as syncytium formation, but was found to initiate lipid mixing (hemifusion) as observed by transfer of R-18 rhodamine from red blood cells to the GPI-PI2F'/PI2HN cotransfected cells. The results therefore indicate that the extracellular domain of the PI2 fusion protein contains not only the structural information sufficient to direct assembly into higher oligomers, but also is competent to initiate membrane fusion, suggesting that the anchor-free PI2F' may be useful for further structural studies.

Mutation↗

PubMed2000

Production and characterization of simian--human immunodeficiency virus-like particles.

We have produced and characterized, in a baculovirus expression system, simian-human immunodeficiency virus-like particles (SHIV VLPs) containing SIV Gag and HIV envelope (Env) proteins. Recombinant SIV gag (SIVmac239) and full-length or cytoplasmic domain-truncated HIV env from either HIV BH10 or HIV 89.6 virus were coexpressed in insect cells and Env incorporation into released SHIV VLPs was characterized. The expression level of the Env protein was found to be about 20-50% higher in both strains producing the truncated Env. Cell surface expression of the truncated Env proteins was found to be about eightfold higher than that of the full-length Env proteins. Furthermore, the truncated Env proteins exhibited higher levels of cleavage into gp120 and gp41 compared with the full-length Env. The SHIV VLPs produced by the coexpression of SIV gag and truncated HIV env contained both precursor (gp160) and gp120, while predominantly gp160 was found in the VLPs containing full-length Env. Coinfection of a recombinant virus expressing the protease furin also resulted in more efficient cleavage of gp160 to gp120. Both full-length and truncated Env were found to induce CD4+ cell fusion. Analysis of VLPs by immunoelectron microscopy demonstrated the incorporation of both full-length and truncated Env on the surface of VLPs. Truncated Env also was incorporated at higher levels on the surfaces of VLPs than full-length Env. The assembly of VLPs containing biologically active Env proteins may be useful in vaccine development and in functional studies of the HIV envelope protein.

Animals↗

PubMed2000

Filamentous particle formation by human parainfluenza virus type 2.

Some paramyxoviruses form long filamentous virus particles: however, the determinants of filament formation and the role of such particles in virus transmission and pathogenicity are not clearly defined. By using conventional immunofluorescence microscopy, we found that human parainfluenza virus type 2 (HPIV2) forms filamentous particles ranging from 5 to 15 microm in length in virus-infected, polarized epithelial cells. The formation of filamentous particles was found to be virus type-specific and was not observed when the same cell types were infected with parainfluenza virus type 3 or Sendai virus, suggesting that different paramyxovirus genera exhibit distinct morphological properties. HPIV2 filamentous particle formation was found to be inhibited by cytochalasin D (CD) or jasplakinolide treatment in a dose-dependent manner. In the presence of 4 microg/ml CD or 1 microM jasplakinolide, the formation of filamentous particles was completely abolished, although similar haemagglutination and p.f.u. titres of virus were found to be released into the culture medium at 24 h post-infection. These observations indicate that host cell components, including the actin microfilament network, are important determinants of the morphology of parainfluenza viruses. The predominance of filamentous particles in polarized epithelial cells may reflect specific pathogenic roles of these particles in infection of human epithelial tissues.

Actins↗

PubMed2000

Induction of CD4(+) T-cell-independent immunoglobulin responses by inactivated influenza virus.

Through cognate interaction between antigen-specific B-cell and CD4(+) alphabeta T cells, the CD4(+) alphabeta T cells secrete cytokines that initiate immunoglobulin (Ig) class switching from IgM to IgG. In this study, we show that formalin-inactivated influenza PR8 virus induces virus-specific IgM and IgG responses in the absence of CD4(+) T cells and that all four subclasses of IgG are produced. The immunized CD4-deficient mice were also found to be completely protected against lethal infection with live, pathogenic influenza virus. The ability of CD4(+) T-cell-deficient mice to generate these IgG responses was not found to be impaired when these mice were depleted of CD8(+) T cells with an anti-CD8 monoclonal antibody. In contrast, alphabeta T-cell-deficient mice (TCRbeta(-/-)) were not found to produce significant amounts of IgG upon immunization with formalin-inactivated PR8 virus. These results suggest that CD4(-) CD8(-) double-negative alphabeta T cells are playing a role in regulating Ig class switching in the absence of CD4(+) T cells.

Age Factors↗

PubMed2000

Coreceptor-dependent inhibition of the cell fusion activity of simian immunodeficiency virus Env proteins.

The cytoplasmic tail (R peptide) sequence is able to regulate the fusion activity of the murine leukemia virus (MuLV) envelope (Env) protein. We have previously shown that this sequence exerts a profound inhibitory effect on the fusion activity of simian immunodeficiency virus (SIV)-MuLV chimeric Env proteins which contain the extracellular and transmembrane domains of the SIV Env protein. Recent studies have shown that SIV can utilize several alternative cellular coreceptors for its fusion and entry into the cell. We have investigated the fusion activity of SIV and SIV-MuLV chimeric Env proteins using cells that express different coreceptors. HeLa cells were transfected with plasmid constructs that carry the SIV or SIV-MuLV chimeric Env protein genes and were overlaid with either CEMx174 cells or Ghost Gpr15 cells, which express the Gpr15 coreceptor for SIV, or Ghost CCR5 cells, which express CCR5, an alternate coreceptor for SIV. The R-peptide sequence in the SIV-MuLV chimeric proteins was found to inhibit the fusion with CEMx174 cells or Ghost Gpr15 cells. However, a significant level of fusion was still observed when HeLa cells expressing the chimeric Env proteins were cocultivated with Ghost CCR5 cells. These results show that the R-peptide sequence exerts differential effects on the fusion activity of SIV Env proteins using target cells that express alternative coreceptors.

Animals↗

PubMed2000

Effect of the cytoplasmic domain of the simian immunodeficiency virus envelope protein on incorporation of heterologous envelope proteins and sensitivity to neutralization.

In addition to the viral envelope (Env) proteins, host cell-derived proteins have been reported to be present in human immunodeficiency virus and simian immunodeficiency virus (SIV) envelopes, and it has been postulated that they may play a role in infection. We investigated whether the incorporation of host cell proteins is affected by the structure and level of incorporation of viral Env proteins. To compare the cellular components incorporated into SIV particles and how this is influenced by the structure of the cytoplasmic domain, we compared SIV virions with full-length and truncated Env proteins. The levels of HLA-I and HLA-II molecules were found to be significantly (15- to 25-fold) higher in virions with full-length Env than in those with a truncated Env. Virions with a truncated Env were also found to be less susceptible to neutralization by specific antibodies against HLA-I or HLA-II proteins. We also compared the level of incorporation into SIV virions of a coexpressed heterologous viral glycoprotein, the influenza virus hemagglutinin (HA) protein. We found that SIV infection of cells expressing influenza virus HA resulted in the production of phenotypically mixed SIV virions containing influenza virus HA as well as SIV envelope proteins. The HA proteins were more effectively incorporated into virions with full-length Env than in virions with truncated Env. The phenotypically mixed particles with full-length Env, containing higher levels of HA, were sensitive to neutralization with anti-HA antibody, whereas virions with truncated Env proteins and containing lower levels of HA were more resistant to neutralization by anti-HA antibody. In contrast, SIV virions with truncated Env proteins were found to be highly sensitive to neutralization by antisera to SIV, whereas virions with full-length Env proteins were relatively resistant to neutralization. These results indicate that the cytoplasmic domain of SIV Env affects the incorporation of cellular as well as heterologous viral membrane proteins into the SIV envelope and may be an important determinant of the sensitivity of the virus to neutralizing antibodies.

Antibody Specificity↗

PubMed2000

Alpha/beta interferons potentiate virus-induced apoptosis through activation of the FADD/Caspase-8 death signaling pathway.

Interferon (IFN) mediates its antiviral effects by inducing a number of responsive genes, including the double-stranded RNA (dsRNA)-dependent protein kinase, PKR. Here we report that inducible overexpression of functional PKR in murine fibroblasts sensitized cells to apoptosis induced by influenza virus, while in contrast, cells expressing a dominant-negative variant of PKR were completely resistant. We determined that the mechanism of influenza virus-induced apoptosis involved death signaling through FADD/caspase-8 activation, while other viruses such as vesicular stomatitis virus (VSV) and Sindbis virus (SNV) did not significantly provoke PKR-mediated apoptosis but did induce cytolysis of fibroblasts via activation of caspase-9. Significantly, treatment with IFN-alpha/beta greatly sensitized the fibroblasts to FADD-dependent apoptosis in response to dsRNA treatment or influenza virus infection but completely protected the cells against VSV and SNV replication in the absence of any cellular destruction. The mechanism by which IFN increases the cells' susceptibility to lysis by dsRNA or certain virus infection is by priming cells to FADD-dependent apoptosis, possibly by regulating the activity of the death-induced signaling complex (DISC). Conversely, IFN is also able to prevent the replication of viruses such as VSV that avoid triggering FADD-mediated DISC activity, by noncytopathic mechanisms, thus preventing destruction of the cell.

Adaptor Proteins, Signal Transducing↗