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Biomedical subjects

R A Lamb

Publications and source records attributed to R A Lamb.

At least 19 recordsLinked to original sources
PubMed2003

Virus replication in engineered human cells that do not respond to interferons.

The V protein of the paramyxovirus simian virus 5 blocks interferon (IFN) signaling by targeting STAT1 for proteasome-mediated degradation. Here we report on the isolation of human cell lines that express the V protein and can no longer respond to IFN. A variety of viruses, particularly slow-growing wild-type viruses and vaccine candidate viruses (which are attenuated due to mutations that affect virus replication, virus spread, or ability to circumvent the IFN response), form bigger plaques and grow to titers that are increased as much as 10- to 4,000-fold in these IFN-nonresponsive cells. We discuss the practical applications of using such cells in vaccine development and manufacture, virus diagnostics and isolation of newly emerging viruses, and studies on host cell tropism and pathogenesis.

Animals↗
PubMed2001

Membrane fusion machines of paramyxoviruses: capture of intermediates of fusion.

Peptides derived from heptad repeat regions adjacent to the fusion peptide and transmembrane domains of many viral fusion proteins form stable helical bundles and inhibit fusion specifically. Paramyxovirus SV5 fusion (F) protein-mediated fusion and its inhibition by the peptides N-1 and C-1 were analyzed. The temperature dependence of fusion by F suggests that thermal energy, destabilizing proline residues and receptor binding by the hemagglutinin-neuraminidase (HN) protein collectively contribute to F activation from a metastable native state. F-mediated fusion was reversibly arrested by low temperature or membrane-incorporated lipids, and the resulting F intermediates were characterized. N-1 inhibited an earlier F intermediate than C-1. Co-expression of HN with F lowered the temperature required to attain the N-1-inhibited intermediate, consistent with HN binding to its receptor stimulating a conformational change in F. C-1 bound and inhibited an intermediate of F that could be detected until a point directly preceding membrane merger. The data are consistent with C-1 binding a pre-hairpin intermediate of F and with helical bundle formation being coupled directly to membrane fusion.

Animals↗
PubMed2001

The V protein of human parainfluenza virus 2 antagonizes type I interferon responses by destabilizing signal transducer and activator of transcription 2.

Type I interferon (IFN) induces antiviral responses through the activation of the ISGF3 transcription factor complex that contains the subunit proteins STAT1, STAT2, and p48/ISGF3 gamma/IRF9. The ability of some human paramyxoviruses to overcome IFN actions by specific proteolysis of STAT proteins has been examined. Infection of cells with type 2, but not type 1 or type 3 human parainfluenza virus (HPIV) leads to a loss of cellular STAT2 protein. Expression of a single HPIV2 protein derived from the V open reading frame blocks IFN-dependent transcriptional responses in the absence of other viral proteins. The loss of IFN response is due to V-protein-induced proteolytic degradation of STAT2. Expression of HPIV2 V causes the normally stable STAT2 protein to be rapidly degraded, and this proteolytic activity can be partially alleviated by proteasome inhibition. No V-protein-specific effects on STAT2 mRNA levels were observed. The results indicate that the V protein of HPIV2 is sufficient to recognize and target a specific cellular transcription factor for destruction by cellular machinery.

Cell Line↗
PubMed2001

Paramyxovirus fusion (F) protein: a conformational change on cleavage activation.

The fusion (F) protein of the paramyxovirus SV5 promotes both virus-cell and cell-cell fusion. Recently, the atomic structure at 1.4 A of an extremely thermostable six-helix bundle core complex consisting of two heptad repeat regions of the F protein has been described (K. A. Baker, R. E. Dutch, R. A. Lamb, and T. S Jardetsky, Mol. Cell 3, 309-319, 1999). To analyze the conformations of the F protein at various stages of the membrane fusion process and to understand further the role of formation of the six-helix bundle core complex in promotion of membrane fusion, antibodies to peptides corresponding to regions of the F protein were obtained. Major changes in F protein antibody recognition were found after cleavage of the precursor protein F(0) to the fusogenically active disulfide-linked heterodimer, F(1) + F(2), and antibodies directed against the heptad repeat regions recognized only the uncleaved form. A monoclonal antibody directed against the F protein showed increased recognition at the cell surface of the cleaved form of the F protein as compared to uncleaved F protein, again indicating changes in conformation between the uncleaved and cleaved forms of the F protein. Anti-peptide antibodies specific for the heptad repeat regions were unable to precipitate a synthetic protein that consisted of the heptad repeat regions separated only by a small spacer, suggesting that the antibodies are unable to recognize their target regions when the heptad repeats are present in the six-helix bundle core complex. Taken together, these data indicate that the six-helix bundle core complex is not present in the precursor molecule F(0) and that significant conformational changes occur subsequent to cleavage of the F protein.

Animals↗
PubMed2001

Deletion of the cytoplasmic tail of the fusion protein of the paramyxovirus simian virus 5 affects fusion pore enlargement.

The fusion (F) protein of the paramxyovirus simian parainfluenza virus 5 (SV5) promotes virus-cell and cell-cell membrane fusion. Previous work had indicated that removal of the SV5 F protein cytoplasmic tail (F Tail- or FDelta19) caused a block in fusion promotion at the hemifusion stage. Further examination has shown that although the F Tail- mutant is severely debilitated in promotion of fusion as measured by using two reporter gene assays and is debilitated in the formation of syncytia relative to the wild-type F protein, the F Tail- mutant is capable of promoting the transfer of small aqueous dyes. These data indicate that F Tail- is fully capable of promoting formation of small fusion pores. However, enlargement of fusion pores is debilitated, suggesting that either the cytoplasmic tail of the F protein plays a direct role in pore expansion or that it interacts with other components which control pore growth.

Animals↗
PubMed2001

Single amino acid substitution in the V protein of simian virus 5 differentiates its ability to block interferon signaling in human and murine cells.

Previous work has demonstrated that the V protein of simian virus 5 (SV5) targets STAT1 for proteasome-mediated degradation (thereby blocking interferon [IFN] signaling) in human but not in murine cells. In murine BF cells, SV5 establishes a low-grade persistent infection in which the virus fluxes between active and repressed states in response to local production of IFN. Upon passage of persistently infected BF cells, virus mutants were selected that were better able to replicate in murine cells than the parental W3 strain of SV5 (wild type [wt]). Viruses with mutations in the Pk region of the N-terminal domain of the V protein came to predominate the population of viruses carried in the persistently infected cell cultures. One of these mutant viruses, termed SV5 mci-2, was isolated. Sequence analysis of the V/P gene of SV5 mci-2 revealed two nucleotide differences compared to wt SV5, only one of which resulted in an amino acid substitution (asparagine [N], residue 100, to aspartic acid [D]) in V. Unlike the protein of wt SV5, the V protein of SV5 mci-2 blocked IFN signaling in murine cells. Since the SV5 mci-2 virus had additional mutations in genes other than the V/P gene, a recombinant virus (termed rSV5-V/P N(100)D) was constructed that contained this substitution alone within the wt SV5 backbone to evaluate what effect the asparagine-to-aspartic-acid substitution in V had on the virus phenotype. In contrast to wt SV5, rSV5-V/P N(100)D blocked IFN signaling in murine cells. Furthermore, rSV5-V/P N(100)D virus protein synthesis in BF cells continued for significantly longer periods than that for wt SV5. However, even in cells infected with rSV5-V/P N(100)D, there was a late, but significant, inhibition in virus protein synthesis. Nevertheless, there was an increase in virus yield from BF cells infected with rSV5-V/P N(100)D compared to wt SV5, demonstrating a clear selective advantage to SV5 in being able to block IFN signaling in these cells.

Animals↗
PubMed2001

The SH integral membrane protein of the paramyxovirus simian virus 5 is required to block apoptosis in MDBK cells.

In some cell types the paramyxovirus simian virus 5 (SV5) causes little cytopathic effect (CPE) and infection continues productively for long periods of time; e.g., SV5 can be produced from MDBK cells for up to 40 days with little CPE. SV5 differs from most paramyxoviruses in that it encodes a small (44-amino-acid) hydrophobic integral membrane protein (SH). When MDBK cells were infected with a recombinant SV5 containing a deletion of the SH gene (rSV5DeltaSH), the MDBK cells exhibited an increase in CPE compared to cells infected with wild-type SV5 (recovered from cDNA; rSV5). The increased CPE correlated with an increase in apoptosis in rSV5DeltaSH-infected cells over mock-infected and rSV5-infected cells when assayed for annexin V binding, DNA content (propidium iodide staining), and DNA fragmentation (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay). In rSV5DeltaSH-infected MDBK cells an increase in caspase-2 and caspase-3 activities was observed. By using peptide inhibitors of individual caspases it was found that caspase-2 and caspase-3 were activated separately in rSV5DeltaSH-infected cells. Expression of caspase-2 and -3 in rSV5DeltaSH-infected MDBK cells appeared not to require STAT1 protein, as STAT1 protein could not be detected in SV5-infected MDBK cells. When mutant mice homologous for a targeted disruption of STAT1 were used as a model animal system and infected with the viruses it was found that rSV5DeltaSH caused less mortality than wild-type rSV5, consistent with the notion of clearance of apoptotic cells in a host species.

Animals↗
PubMed2000

Permeation and activation of the M2 ion channel of influenza A virus.

The M(2) ion channel protein of influenza A virus is essential for mediating protein-protein dissociation during the virus uncoating process that occurs when the virus is in the acidic environment of the lumen of the secondary endosome. The difficulty of determining the ion selectivity of this minimalistic ion channel is due in part to the fact that the channel activity is so great that it causes local acidification in the expressing cells and a consequent alteration of reversal voltage, V(rev). We have confirmed the high proton selectivity of the channel (1.5-2.0 x 10(6)) in both oocytes and mammalian cells by using four methods as follows: 1) comparison of V(rev) with proton equilibrium potential; 2) measurement of pH(in) and V(rev) while Na(+)(out) was replaced; 3) measurements with limiting external buffer concentration to limit proton currents specifically; and 4) comparison of measurements of M(2)-expressing cells with cells exposed to a protonophore. Increased currents at low pH(out) are due to true activation and not merely increased [H(+)](out) because increased pH(out) stops the outward current of acidified cells. Although the proton conductance is the biologically relevant conductance in an influenza virus-infected cell, experiments employing methods 1-3 show that the channel is also capable of conducting NH(4)(+), probably by a different mechanism from H(+).

Animals↗
PubMed2000

Fusion protein of the paramyxovirus SV5: destabilizing and stabilizing mutants of fusion activation.

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.

Amino Acid Substitution↗
PubMed2000

The cytoplasmic tails of the influenza virus spike glycoproteins are required for normal genome packaging.

Deletion of the cytoplasmic tails of the influenza A virus spike glycoproteins, hemagglutinin (HA) and neuraminidase (NA), has previously been shown to result in markedly defective virion morphogenesis (Jin et al., 1997, EMBO J. 16, 1236-1247). We have found that influenza A virus preparations lacking the HA and NA cytoplasmic tails (HAt-/NAt-) have a reduced vRNA to protein content, contain an increase in cellular RNA contaminants, and exhibit increased resistance to ultraviolet (UV) inactivation. There is also a direct correlation between abnormal virion morphology and reduced infectivity. The data suggest that the HAt-/NAt- virion population contains a broader range of number of packaged RNA segments than wild-type (wt) virus. Sucrose gradient centrifugation analysis indicated the presence of a subpopulation of virions with pronounced deformation in virion morphology and reduced infectivity. The role of the HA and NA cytoplasmic tails was examined further by using a trans-complementation assay and it was found that expression of wt HA and NA from cDNAs followed by HAt-/NAt- virus infection caused the formation of a pseudotype virus with wt sedimentation properties. Taken together the data indicate that the HA and NA cytoplasmic tails affect not only virion morphology but also proper genome packaging.

Animals↗
PubMed2000

Mechanism for proton conduction of the M(2) ion channel of influenza A virus.

The M(2) integral membrane protein of influenza A virus forms a proton-selective ion channel. We investigated the mechanism for proton transport of the M(2) protein in Xenopus oocytes using a two-electrode voltage clamp and in CV-1 cells using the whole cell patch clamp technique. Membrane currents were recorded while manipulating the external solution to alter either the total or free proton concentration or the solvent itself. Membrane conductance decreased by approximately 50% when D(2)O replaced H(2)O as the solvent. From this, we conclude that hydrogen ions do not pass through M(2) as hydronium ions, but instead must interact with titratable groups that line the pore of the channel. M(2) currents measured in solutions of low buffer concentration (<15 mM in oocytes and <0.15 mM in CV-1 cells) were smaller than those studied in solutions of high buffer concentration. Furthermore, the reversal voltage measured in low buffer was shifted to a more negative voltage than in high buffer. Also, at a given pH, M(2) current amplitude in 15 mM buffer decreased when pH-pK(a) was increased by changing the buffer pK(a). Collectively, these results demonstrate that M(2) currents can be limited by external buffer capacity. The data presented in this study were also used to estimate the maximum single channel current of the M(2) ion channel, which was calculated to be on the order of 1-10 fA.

Alkanesulfonic Acids↗
PubMed2000

Virus membrane fusion proteins: biological machines that undergo a metamorphosis.

Fusion proteins from a group of widely disparate viruses, including the paramyxovirus F protein, the HIV and SIV gp160 proteins, the retroviral Env protein, the Ebola virus Gp, and the influenza virus haemagglutinin, share a number of common features. All contain multiple glycosylation sites, and must be trimeric and undergo proteolytic cleavage to be fusogenically active. Subsequent to proteolytic cleavage, the subunit containing the transmembrane domain in each case has an extremely hydrophobic region, termed the fusion peptide, or at near its newly generated N-terminus. In addition, all of these viral fusion proteins have 4-3 heptad repeat sequences near both the fusion peptide and the transmembrane domain. These regions have been demonstrated from a tight complex, in which the N-terminal heptad repeat forms a trimeric-coiled coil, with the C-terminal heptad repeat forming helical regions that buttress the coiled-coil in an anti-parallel manner. The significance of each of these structural elements in the processing and function of these viral fusion proteins is discussed.

Membrane Fusion↗
PubMed2000

Influenza virus assembly and lipid raft microdomains: a role for the cytoplasmic tails of the spike glycoproteins.

Influenza viruses encoding hemagglutinin (HA) and neuraminidase (NA) glycoproteins with deletions in one or both cytoplasmic tails (HAt- or NAt-) have a reduced association with detergent-insoluble glycolipids (DIGs). Mutations which eliminated various combinations of the three palmitoylation sites in HA exhibited reduced amounts of DIG-associated HA in virus-infected cells. The influenza virus matrix (M(1)) protein was also found to be associated with DIGs, but this association was decreased in cells infected with HAt- or NAt- virus. Regardless of the amount of DIG-associated protein, the HA and NA glycoproteins were targeted primarily to the apical surface of virus-infected, polarized cells. The uncoupling of DIG association and apical transport was augmented by the observation that the influenza A virus M(2) protein as well as the influenza C virus HA-esterase-fusion glycoprotein were not associated with DIGs but were apically targeted. The reduced DIG association of HAt- and NAt- is an intrinsic property of the glycoproteins, as similar reductions in DIG association were observed when the proteins were expressed from cDNA. Examination of purified virions indicated reduced amounts of DIG-associated lipids in the envelope of HAt- and NAt- viruses. The data indicate that deletion of both the HA and NA cytoplasmic tails results in reduced DIG association and changes in both virus polypeptide and lipid composition.

Animals↗
PubMed2000

Analysis of the pore structure of the influenza A virus M(2) ion channel by the substituted-cysteine accessibility method.

The M(2) ion channel of influenza A virus is a small integral membrane protein whose active form is a homotetramer with each polypeptide chain containing 96-amino-acid residues. To identify residues of the transmembrane (TM) domain that line the presumed central ion-conducting pore, a set of mutants was generated in which each residue of the TM domain (residues 25 to 44) was replaced by cysteine. The accessibility of the cysteine mutants to modification by the sulfhydryl-specific reagents methane thiosulfonate ethylammonium (MTSEA) and MTS tetraethylammonium (MTSET) was tested. Extracellular application of MTSEA evoked decreases in the conductances measured from two mutants, M(2)-A30C and M(2)-G34C. The changes observed were not reversible on washout, indicative of a covalent modification. Inhibition by MTSEA, or by the larger reagent MTSET, was not detected for residues closer to the extracellular end of the channel than Ala-30, indicating the pore may be wider near the extracellular opening. To investigate the accessibility of the cysteine mutants to reagents applied intracellularly, oocytes were microinjected directly with reagents during recordings. The conductance of the M(2)-W41C mutant was decreased by intracellular injection of a concentrated MTSET solution. However, intracellular application of MTSET caused no change in the conductance of the M(2)-G34C mutant, a result in contrast to that obtained when the reagent was applied extracellularly. These data suggest that a constriction in the pore exists between residues 34 and 41 which prevents passage of the MTS reagent. These findings are consistent with the proposed role for His-37 as the selectivity filter. Taken together, these data confirm our earlier model that Ala-30, Gly-34, His-37, and Trp-41 line the channel pore (L. H. Pinto, G. R. Dieckmann, C. S. Gandhi, C. G. Papworth, J. Braman, M. A. Shaughnessy, J. D. Lear, R. A. Lamb, and W. F. DeGrado, Proc. Natl. Acad. Sci. USA 94:11301-11306, 1997).

Amino Acid Substitution↗
PubMed2000

The paramyxovirus simian virus 5 V protein slows progression of the cell cycle.

Infection of cells by many viruses affects the cell division cycle of the host cell to favor viral replication. We examined the ability of the paramyxovirus simian parainfluenza virus 5 (SV5) to affect cell cycle progression, and we found that SV5 slows the rate of proliferation of HeLa T4 cells. The SV5-infected cells had a delayed transition from G(1) to S phase and prolonged progression through S phase, and some of the infected cells were arrested in G(2) or M phase. The levels of p53 and p21(CIP1) were not increased in SV5-infected cells compared to mock-infected cells, suggesting that the changes in the cell cycle occur through a p53-independent mechanism. However, the phosphorylation of the retinoblastoma protein (pRB) was delayed and prolonged in SV5-infected cells. The changes in the cell cycle were also observed in cells expressing the SV5 V protein but not in the cells expressing the SV5 P protein or the V protein lacking its unique C terminus (VDeltaC). The unique C terminus of the V protein of SV5 was shown previously to interact with DDB1, which is the 127-kDa subunit of the multifunctional damage-specific DNA-binding protein (DDB) heterodimer. The coexpression of DDB1 with V can partially restore the changes in the cell cycle caused by expression of the V protein.

Cell Cycle↗
PubMed2000

Identification of a membrane targeting and degradation signal in the p42 protein of influenza C virus.

Two mRNA species are derived from the influenza C virus RNA segment six, (i) a colinear transcript containing a 374-amino-acid residue open reading frame (referred to herein as the seg 6 ORF) which is translated to yield the p42 protein, and (ii) a spliced mRNA which encodes the influenza C virus matrix (CM1) protein consisting of the first 242 amino acids of p42. The p42 protein undergoes proteolytic cleavage at a consensus signal peptidase cleavage site after residue 259, yielding the p31 and CM2 proteins. Translocation of p42 into the endoplasmic reticulum membrane occurs cotranslationally and requires the hydrophobic internal signal peptide (residues 239 to 259), as well as the predicted transmembrane domain of CM2 (residues 285 to 308). The p31 protein was found to undergo rapid degradation after cleavage from p42. Addition of the 26S proteasome inhibitor lactacystin to influenza C virus-infected or seg 6 ORF cDNA-transfected cells drastically reduced p31 degradation. Transfer of the 17-residue C-terminal region of p31 to heterologous proteins resulted in their rapid turnover. The hydrophobic nature, but not the specific amino acid sequence of the 17-amino-acid C terminus of p31 appears to act as the signal for targeting the protein to membranes and for degradation.

Amino Acid Sequence↗
PubMed1999

The signal for clathrin-mediated endocytosis of the paramyxovirus SV5 HN protein resides at the transmembrane domain-ectodomain boundary region.

The hemagglutinin-neuraminidase (HN) glycoprotein of the paramyxovirus SV5 is internalized from the cell surface via clathrin-coated pits. However, the cytoplasmic domain of SV5 HN does not contain a previously characterized internalization motif. A cell-surface-expressed chimeric protein (APK), consisting of the cytoplasmic tail, transmembrane (TM) domain, and 12 residues of the ectodomain of HN joined to the cytoplasmic protein pyruvate kinase is internalized, indicating that the N-terminal region of HN contains an internalization signal. Although SV5 HN is internalized at a rate similar to that of influenza virus hemagglutinin (HA) mutant Y543, which contains a degenerate tyrosine-based signal in its cytoplasmic tail, the elimination of the majority of the HN cytoplasmic tail, or substitution of the HN TM domain with leucine residues, did not affect the rate of HN internalization. The HN protein of the closely related virus, Newcastle disease virus (NDV), is not internalized from the cell surface. Working under the usual convention that the TM domain consists of the hydrophobic residues bounded by two charged residues, analysis of internalization of mutant and chimeric NDV HN molecules indicates that the first seven SV5 HN ectodomain residues are critical for internalization of HN. A glutamic acid residue (E37) that abuts this presumptive HN TM domain/ectodomain boundary is important for SV5 HN internalization.

Amino Acid Sequence↗