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R W Compans

Publications and source records attributed to R W Compans.

At least 127 records · Page 7Linked to original sources
PubMed1991

Assembly and polarized release of Punta Toro virus and effects of brefeldin A.

Punta Toro virus (PTV), a member of the sandfly fever group of bunyaviruses, is assembled by budding at intracellular membranes of the Golgi complex. We have examined PTV glycoprotein transport, assembly, and release and the effects of brefeldin A (BFA) on these processes. Both the G1 and G2 proteins were transported out of the endoplasmic reticulum (ER) and retained in the Golgi complex in a stable structure, either during PTV infection or when expressed from a vaccinia virus recombinant. BFA treatment causes a rapid and dramatic change in the distribution of the G1 and G2 proteins, from a Golgi pattern to an ER pattern. The G1 and G2 proteins were found to be modified by medial but not trans Golgi network enzymes, in the presence or absence of BFA. We found that BFA blocks PTV release from cells but does not interfere with the intracellular assembly of infectious virions. Further, the BFA block of virus release is fully reversible, with high levels of virus release occurring upon removal of the inhibitor. It was also found that the release of PTV virions is polarized, occurring exclusively from the basolateral surfaces of the polarized Vero C1008 epithelial cell line.

Animals↗
PubMed1991

The P gene of human parainfluenza virus type 1 encodes P and C proteins but not a cysteine-rich V protein.

The nucleotide sequence of the P gene of human parainfluenza virus type 1 (PIV1) was determined from cloned cDNA copies of the mRNA. By analogy with the gene organization of Sendai virus, two open reading frames in the mRNA sense of the gene were identified as coding sequences for the P protein (568 amino acids with an estimated molecular weight of 64,655) and the C protein (204 amino acids with an estimated molecular weight of 24,108). Comparison of the deduced amino acid sequences of the P and C proteins of PIV1 with those of Sendai virus showed a high degree of homology. However, a sequence for the cysteine-rich V protein, which was considered a common feature of other paramyxoviruses, was interrupted by the presence of multiple stop codons. The sequence analysis of three P-gene-specific cDNA clones generated from genomic RNA by polymerase chain reaction and one additional clone generated from mRNA confirmed that the coding sequence for the cysteine-rich region is silent in the PIV1 gene and thus is not translated into protein. Two potential editing sites with the consensus sequence 3'UUYUCCC were found in the PIV1 P gene at positions 564 to 570 and 1430 to 1436. However, examination of the PIV1 mRNA population by a primer extension method indicated that neither of these sites is utilized. These results indicate that the PIV1 P gene has a coding strategy different from those of other paramyxovirus P genes.

Amino Acid Sequence↗
PubMed1991

Isolation of an arenavirus from a marmoset with callitrichid hepatitis and its serologic association with disease.

Callitrichid hepatitis (CH) is an acute, often fatal viral infection of New World primates from the family Callitrichidae. The etiologic agent of CH is unknown. We report here the isolation of an arenavirus from a common marmoset (Callithrix jacchus) with CH by using in vitro cultures of marmoset hepatocytes and Vero-E6 cells. Enveloped virions 67 to 133 nm in diameter with ribosomelike internal structures were seen in infected cultures. Immunofluorescence and Western immunoblot analysis using CH-specific antisera (principally from animals exposed to CH during zoo outbreaks) revealed three antigens in cells infected with this CH-associated virus (CHV). These antigens had the same electrophoretic mobilities on sodium dodecyl sulfate-polyacrylamide gels as did the nucleocapsid, GP2, and GPC proteins of lymphocytic choriomeningitis virus (LCMV). Monoclonal antibodies specific for these arenavirus proteins also reacted with the three CHV antigens. Conversely, the CH-specific antisera reacted with the nucleocapsid, GP2, and GPC proteins of LCMV. CHV thus appears to be a close antigenic relative of LCMV. The serologic association of CHV with several CH outbreaks implicate it as the etiologic agent of this disease.

Animals↗
PubMed1990

Expression and characterization of a functional human immunodeficiency virus envelope glycoprotein in insect cells.

Recombinant baculoviruses were used to express the gp160 envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1) and a truncated variant designated gp160(t) which lacks a transmembrane domain. Glycosylation, proteolytic cleavage, secretion, and biological activities of gp160 and gp160(t) 160(t) were studied in Spodoptera frugiperda cells. Both proteins were rapidly glycosylated and initially were found to be totally endo-beta-N-acetyl-D-glucosaminidase H (endo-H) sensitive. However, partial resistance to endo-H was gradually acquired by both molecules. gp160 was found to remain cell-associated, whereas gp160(t) was secreted into the culture medium in large amounts. A fraction of gp160 and gp160(t) appeared to be proteolytically cleaved, and a cleavage product corresponding in size to gp120 was identified in the culture medium. gp160(t) was found to interact specifically with CD4 receptors without any requirement for proteolytic cleavage. The gp160 protein was shown to be expressed on the surface of S. frugiperda cells by direct immunofluorescence. These surface molecules were biologically active, as demonstrated by their ability to induce syncytium formation when cocultivated with HeLa T4 cells.

Acetylglucosaminidase↗
PubMed1990

The human immunodeficiency virus type 1 envelope glycoprotein precursor acquires aberrant intermolecular disulfide bonds that may prevent normal proteolytic processing.

The envelope glycoprotein of human immunodeficiency virus consists of two subunits, designated gp120 and gp41, derived from the cleavage of a precursor polypeptide gp160. When expressed from a recombinant vaccinia virus and analyzed by velocity gradient sedimentation and polyacrylamide gel electrophoresis, a significant proportion of gp160 molecules formed oligomers that were stabilized by intermolecular disulfide bonds. Oligomeric forms of both gp120 and gp41 were also observed, but these oligomers were noncovalently associated. Both the intermolecularly linked oligomers of gp160 and the unlinked oligomeric envelope protein subunits were found to accumulate with time. These results indicate that there are two populations of gp160 precursors, one that is folded and processed correctly into gp120 and gp41 and another that is intermolecularly disulfide bonded and remains uncleaved. We propose that the formation of intermolecular disulfide bonds is not an intermediate step in the maturation of the envelope glycoprotein, but rather a result of misfolding of the gp160 precursor which prevents it from being properly processed.

Animals↗
PubMed1990

Molecular cloning and sequence analysis of the fusion glycoprotein gene of human parainfluenza virus type 2.

A cDNA clone containing a 2.0-kb insert was identified as the human parainfluenza virus type 2 (PI2) fusion glycoprotein gene by hybridizing with a viral RNA probe and a synthetic oligonucleotide derived from a conserved sequence found in other paramyxovirus fusion protein genes. The complete nucleotide sequence of the glycoprotein gene was determined by the dideoxynucleotide sequencing procedure and found to contain a single, large open reading frame encoding a protein of 551 amino acids with a calculated molecular weight of 59,664. Comparison of the P12 fusion protein with those of other paramyxoviruses indicated similarities in overall length, N-terminal signal peptide sequence (amino acids 7 to 25), C-terminal membrane-spanning region (amino acids 486 to 513), and a highly conserved fusion sequence region at the N-terminus of the F1 subunit (amino acids 107 to 132).

Amino Acid Sequence↗
PubMed1990

Antiviral effects of apolipoprotein A-I and its synthetic amphipathic peptide analogs.

Apolipoprotein A-I (apo A-I), the major protein component of serum high density lipoproteins, was found to inhibit herpes simplex virus (HSV)-induced cell fusion at physiological (approximately 1 microM) concentrations. An 18 amino acid-long synthetic amphipathic alpha-helical peptide analog of apo A-I (18A) was also found to inhibit HSV-induced cell fusion at similar concentration (approximately 2 microM). Dimers of 18A connected via a proline (37pA) or an alanine (37aA) residue also inhibited virus-induced cell fusion at similar concentration, suggesting that the presence of a proline turn does not influence the antiviral activity of the amphipathic peptides. However, a peptide analog 18R, in which the distribution of charged residues was reversed, inhibited virus-induced cell fusion only at a higher (approximately 125 microM) concentration, suggesting that the anti-viral activity of the amphipathic peptide is strongly influenced by the nature of the charge distribution at the polar-nonpolar interface. Consistent with their ability to inhibit virus-induced cell fusion, the peptides inhibited the spread of HSV infection as demonstrated by a 10-fold reduction in the virus yield, when virus-infected cells were maintained in the presence of amphipathic peptides. The amphipathic peptides also inhibited penetration of virus into cells, but did not exert any effect on virus adsorption. A nearly complete inhibition of virus penetration was observed when the virus, or both virus and cells, was pretreated with the peptide, suggesting that the peptides may have a direct effect on the virus. The results indicate that amphipathic helices may be useful in designing novel antiviral agents that inhibit penetration and spread of enveloped viruses.

Amino Acid Sequence↗
PubMed1990

Sequence of the hemagglutinin-neuraminidase gene of human parainfluenza virus type 1.

The nucleotide sequence of the gene encoding the hemagglutinin-neuraminidase (HN) glycoprotein of human parainfluenza virus type 1 (PI1) was determined from cDNA clones derived from poly(A)+ RNA extracted from infected cells. A single open reading frame in the sequence was found to encode a putative protein of 575 amino acids with a calculated molecular weight of 63,960. The predicted amino acid sequence contains ten potential sites for N-linked glycosylation and one major hydrophobic region located 35 amino acids from the amino terminus, which appears to be the signal-anchor domain of HN. Comparison of the amino acid sequence with the HN glycoproteins of other paramyxoviruses indicated that the PI1 HN protein is most closely related to the Sendai virus (SV) HN protein.

Amino Acid Sequence↗
PubMed1990

Host cell-dependent lateral mobility of viral glycoproteins.

The lateral mobility of viral envelope proteins on the plasma membranes of infected cells is an important factor in both virus assembly and pathogenesis. The envelope glycoproteins of measles and human parainfluenza virus are mobile on the surfaces of infected HeLa cells and undergo lateral redistribution in the presence of specific antibody, forming unipolar caps. In contrast, no such redistribution was observed with influenza virus hemagglutinin (HA) or vesicular stomatitis virus (VSV) G glycoproteins on infected HeLa cell surfaces. However, the HA and G glycoproteins were both found to be mobile in the plasma membrane of CV-1 cells, or human or murine peritoneal macrophages. These results indicate that host cell-dependent as well as virus-specific factors are involved in determining viral glycoprotein mobility. No significant differences in the patterns of synthesis of influenza or VSV viral proteins were found in the various cell types examined. The HA and G proteins, when expressed from vaccinia virus recombinants, were each found to be immobile in HeLa cells and mobile in CV-1 cells, thus indicating that the host cell-dependent differences in mobility are an intrinsic property of each viral glycoprotein molecule and not the result of interaction with other viral components. It is suggested that the association of viral glycoproteins with either the cytoskeleton or membrane-associated cellular proteins may be related to the observed differences in lateral mobility.

Animals↗
PubMed1990

Oligopeptide inhibitors of HIV-induced syncytium formation.

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein is essential for virus entry and the formation of multinucleated giant cells by cell fusion, one of the major virus-induced cytopathic effects. To study the effects of potential fusion inhibitors, a vaccinia virus recombinant expressing the envelope glycoprotein was generated and used to infect HeLa CD4+ cells. Syncytium induction was observed as early as 4 h postinfection and continued until the entire monolayer was fused. The N-terminus of the gp41 subunit of the HIV envelope protein is very hydrophobic, and appears to be involved in virus-induced membrane fusion. We synthesized several oligopeptide analogs of the N-terminal region of gp41 and determined their ability to inhibit HIV-induced cell fusion in CD4+ HeLa cells. A hexapeptide which was identical in amino acid sequence to the N-terminus of gp41 was found to completely inhibit cell fusion, whereas peptides with altered sequences showed reduced inhibitory activity. These peptides had no effect on protein synthesis, processing, or transport to the cell surface, and showed no signs of toxicity to cells even at very high concentrations. These results indicate that oligopeptides which are homologous to the fusion peptide of HIV inhibit virus-induced cytopathology, and should be evaluated further as potential antiviral agents.

Amino Acid Sequence↗
PubMed1990

The env protein of an infectious noncytopathic HIV-2 is deficient in syncytium formation.

A recent isolate of human immunodeficiency virus type 2 (HIV-2) designated HIV-2ST is deficient in its ability to cause the typical cytopathic effects of HIV infection. The pathogenic potential of HIV-2 in inducing human disease may be less than that of HIV-1, and it is of particular interest to establish the basis for the reduced cytopathogenicity of this isolate in vitro. Utilizing recombinant vaccinia viruses (rVV) carrying the envelope genes (env) of HIV-2ST or those of fully cytopathic HIV-1 or HIV-2 isolates, we have investigated envelope glycoprotein expression, processing, transport, and biological function. Radioimmunoprecipitation and polyacrylamide gel electrophoresis (RIP-PAGE) of rVV-infected cell lysates indicated that the proteins expressed by each recombinant were synthesized, processed, and recognized by specific antisera. Immunofluorescence studies showed that the recombinant env gene products of HIV-2ST and HIV-2ROD reach the cell surface and are retained there in similar amounts. Whereas cells expressing the HIV-1 or HIV-2ROD env gene products were found to undergo fusion with uninfected CD4+ cells, no syncytium formation was observed with three CD4+ cell lines exposed to the cells expressing the envelope glycoproteins of HIV-2ST on their surfaces; one CD4+ lymphoid cell line (SupT1) exhibited few very small syncytia in the presence of recombinant HIV-2ST envelope glycoproteins. The failure of the HIV-2ST envelope glycoprotein to induce cell fusion was not the result of an inhibition by cell-associated CD4, since fusion was also not observed when rVVST-infected CD4- cells were cocultured with CD4+ cells. Thus, the HIV-2ST envelope protein itself is defective in its ability to induce cell fusion. Furthermore, the expression, processing, transport, and surface stability of env products of HIV-2ST are unlikely to be responsible for its attenuation, suggesting that the molecular interactions between its env products and target cell membranes are significantly altered.

CD4 Antigens↗
PubMed1990

Human parainfluenza virus induces a type-specific protective immune response.

Induction of type-specific and cross-protective immune responses against human parainfluenza viruses have been investigated. The envelope glycoproteins HN (76 kDa) and F0 (62 kDa) from parainfluenza type 2 virus were selectively solubilized with octylglucoside. Detergent-soluble envelope glycoproteins were used as vaccine antigens for intranasal immunization of hamsters. The immunized animals showed complete protection from challenge infection with prototype live virus but failed to demonstrate a significant level of protection against either human parainfluenza type 1 or type 3 virus. The sera and bronchial lavages of immunized animals also showed type-specific neutralizing antibodies. A similar type-specific protective immune response was also noted after primary infection with live virus. The results indicate that a multivalent parainfluenza virus vaccine is probably required for protection against natural infection.

Animals↗
PubMed1990

Apolipoprotein A-I and its amphipathic helix peptide analogues inhibit human immunodeficiency virus-induced syncytium formation.

The envelope (membrane) glycoprotein of HIV is essential for virus attachment and entry into host cells. Additionally, when expressed on the plasma membrane of infected cells, the envelope protein is responsible for mediating cell-cell fusion which leads to the formation of multinucleated giant cells, one of the major cytopathic effects of HIV infections. The envelope glycoproteins of HIV contain regions that can fold into amphipathic alpha-helixes, and these regions have been suggested to play a role in subunit associations and in virus-induced cell fusion and cytopathic effects of HIV. We therefore tested the possibility that amphipathic helix-containing peptides and proteins may interfere with the HIV amphipathic peptides and inhibit those steps of HIV infection involving membrane fusion. Apolipoprotein A-I, the major protein component of high density lipoprotein, and its amphipathic peptide analogue were found to inhibit cell fusion, both in HIV-1-infected T cells and in recombinant vaccinia-virus-infected CD4+ HeLa cells expressing HIV envelope protein on their surfaces. The amphipathic peptides inhibited the infectivity of HIV-1. The inhibitory effects were manifest when the virus, but not cells, was pretreated with the peptides. Also, a reduction in HIV-induced cell killing was observed when virus-infected cell cultures were maintained in presence of amphipathic peptides. These results have potential implications for HIV biology and therapy.

Amino Acid Sequence↗
PubMed1989

The spleen focus-forming virus envelope glycoprotein is defective in oligomerization.

The gp52 envelope glycoprotein of Friend spleen focus-forming virus (SFFV) is a recombinant molecule derived from Friend murine leukemia virus (MuLV) by various deletions, insertions, and substitutions. The SFFV gp52 glycoprotein, unlike MuLV envelope glycoproteins, is defective in transport to the cell surface. Only 3-5% of gp52 eventually reaches the cell surface as a processed form (gp65). Although gp52 lacks cytoplasmic tail residues found in MuLV glycoproteins, we have previously shown that this deletion is not responsible for its defective transport. In order to investigate the basis for the defective transport of gp52, we have examined the folding and assembly of gp52 molecules into oligomeric molecules. CV-1 cells infected with vaccinia virus recombinants expressing SFFV gp52 were pulse labeled and the cell extracts were fractionated by velocity centrifugation through sucrose gradients. Immediately after a 10-min pulse, gp52 was detected as a monomer in the upper part of the sucrose gradient (fractions 12 and 14) and it remained as such after a 2-h chase period. However, the processed form, gp65, was found in a lower part of the gradient (fraction 8) after a 2-h chase. The position of gp65 was found to correspond to the position of trimeric influenza hemagglutinin which was analyzed on a parallel sucrose gradient, suggesting that gp65 also exists as a trimer in this fraction. These results indicate that changes in the external domain of gp52 result in improper folding of the glycoprotein molecule, and suggest that this lack of oligomerization is responsible for the defective transport of the molecules. Only those molecules that do form oligomeric structures are transported to the Golgi complex and undergo further oligosaccharide processing, and transport to the cell surface.

Animals↗
PubMed1989

Immune responses to influenza virus in orally and systemically immunized mice.

In our studies on the induction of an immune response by oral immunization, we have explored the potential of a novel approach for antigen delivery by microencapsulation. This procedure preserved the immunogenicity of the influenza virus introduced by either systemic or oral routes. Furthermore, the levels of specific antibodies in serum and in saliva were enhanced and lasted longer (up to 4 months) in animals immunized with of antigens in microencapsulated form than in animals immunized with equal doses of free suspension. Preliminary challenge experiments showed a correlation between levels of antibodies and protection. All mice systemically immunized were protected against the virus, while mice orally immunized with lower doses of microencapsulated antigen had better survival rates than those immunized with higher doses. Additional experiments suggested that low doses of immunogen were able to generate better protective immunity than high doses, which may instead be tolerogenic. Further experiments with a well characterized microencapsulated antigen (size of microcapsules, time of release of antigen, as well as its dose and form) will be necessary to establish conditions for optimal immunization protocols applicable for the oral or systemic routes.

Administration, Oral↗
PubMed1989

Nucleotide sequence analysis of the capsid genes and the right-hand terminal palindrome of porcine parvovirus, strain NADL-2.

The genome of the porcine parvovirus, strain NADL-2, has been cloned and the sequence of map units 28 to 100, which is 3670 bp in length and contains the capsid coding regions and the right-hand terminal palindrome, has been determined. The sequence shows extensive homology with other parvoviruses such as MVM, H-1, CPV, and FPV in the capsid coding region. Given the information available from these sequences, the regulatory and capsid coding regions for PPV have been proposed and the amino acid sequences of capsid proteins compared. The right end, which has a 188-nucleotide-long imperfect palindromic sequence, has an organization similar to that of other parvoviruses and the homologies in this region with other parvoviruses correspond to the homologies observed in the rest of the respective genomes.

Amino Acid Sequence↗
PubMed1989

Expression of the fusion glycoprotein of human parainfluenza type 3 virus in insect cells by a recombinant baculovirus and analysis of its immunogenic property.

The fusion (F) glycoprotein of human parainfluenza type 3 (PI3) virus was produced in insect cells using a baculovirus expression vector (pAcYM1). The recombinant glycoprotein was identified by its reactivity with specific monoclonal and polyclonal antibodies and showed an apparent molecular mass of 70 kDa. Although the fusion protein was found on the infected cell surface, it did not appear to be proteolytically cleaved to F1 and F2 subunits. Immunization of hamsters with the recombinant protein elicited antibody which neutralized infectivity and blocked fusion of virus-infected cells. The protective response to challenge infection of immunized hamsters was similar to that observed with affinity purified F from PI3 virus (Ray et al., J. Virol. 62, 783-787, 1988).

Animals↗