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PubMed2002

The intracellular loops of the GB2 subunit are crucial for G-protein coupling of the heteromeric gamma-aminobutyrate B receptor.

The gamma-aminobutyrate B (GABA(B)) receptor is the first discovered G-protein-coupled receptor (GPCR) that needs two subunits, GB1 and GB2, to form a functional receptor. The GB1 extracellular domain (ECD) binds GABA, and GB2 contains enough molecular determinants for G-protein activation. The precise role of the two subunits in G-protein coupling is investigated. GB1 and GB2 are structurally related to the metabotropic glutamate, Ca(2+)-sensing and other family 3 GPCRs in which the second (i2) as well as the third (i3) intracellular loop play important roles in G-protein coupling. Here, the role of the i2 loops of GB1 and GB2 in the GABA(B) receptor ability to activate G(alpha)-proteins is investigated. To that aim, the i2 loops were swapped between GB1 and GB2 heptahelical domains (HDs), either in the wild-type subunits or in the chimeric subunits GB1/2 that contain the ECD of GB1 and the HD of GB2. The effect of an additional mutation within the i3 loop of GB2 that prevents coupling of the heteromeric receptor was also examined. Combinations of interest were found to be correctly addressed at the cell surface and to assemble into heteromers. Taken together our data revealed the following new information on the G-protein coupling of the heteromeric GABA(B) receptor: 1) the i2 loop of GB2 within the GB2 HD is required for the heteromeric GABA(B) receptor to couple to G-proteins, whereas the i2 loop of GB1 is not; 2) the presence of the i2 loop of GB2 within the GB1 HD is not sufficient to allow coupling of GB1; 3) the GB2 HD activates the Gqi9 protein whether it is associated with the GB2 or GB1 ECD; 4) in the combination with two GB2 HDs, each is able to couple to G-proteins; and finally, 5) the use of mutations in i2, i3, or both within the GB2 HD brings evidence for the absence of domain swapping enabling the exchange of region including i2 and i3 between the subunits.

Amino Acid Sequence↗
PubMed1983

Response of mouse intestinal loop to botulinum C2 toxin: enterotoxic activity induced by cooperation of nonlinked protein components.

Botulinum C2 toxin, which is composed of two nonlinked protein components, components I and II, induced fluid accumulation in mouse intestinal loops. The secretory response to C2 toxin was initiated after a lag period of 1 to 2 h and increased gradually for at least 10 h. The activity of C2 toxin was enhanced by treatment with trypsin and abolished by neutralization with anti-component I or anti-component II sera. Neither component I nor component II alone induced the fluid accumulation in intestinal loops. The intestinal loop activity was demonstrated with the culture supernatants of strains of Clostridium botulinum types C and D that produced C2 toxin, but not with culture supernatants of strains that did not. None of the botulinum type A through F neurotoxins induced fluid accumulation in mouse intestinal loops. The results indicate that, in addition to lethal and vascular permeability activities, C2 toxin has an enterotoxic activity for which the cooperation of components I and II is necessary. The fluid accumulation in intestinal loops inoculated with C2 toxin was not diminished by removal of the toxin from the loops. Moreover, the secretory response was positive when intestinal lumina were exposed to component II followed by the removal of the component and inoculation with component I, but it was negative when the intestinal lumina were exposed to component I followed by the removal of the component and inoculation with component II. These results suggest that the secretory response of mouse intestinal loops to C2 toxin is induced by the binding of component II to the epithelial cell surfaces of the intestines and the consequent binding or penetration of component I into the cells.

Animals↗
PubMed1994

Enterotoxic activity of Klebsiella oxytoca cytotoxin in rabbit intestinal loops.

We examined the enterotoxicity of a Klebsiella oxytoca cytotoxin which is produced by K. oxytoca OK-1, a strain from a patient with antibiotic-associated hemorrhagic colitis. Injection of the cytotoxin into ligated ileal and colonic loops in rabbits caused the accumulation of fluid in the loops. The fluid was bloody in the ileal loops but not in the colonic ones. Histological examination revealed intense mucosal hemorrhage with erosion in the ileum, whereas no microscopic change was noted in the colon. The fluid accumulation was shown to be a dose-dependent response in both ileal and colonic loops. The amounts of the cytotoxin required for maximal fluid accumulation in ileal and colonic loops were 60 and 10 micrograms, respectively. Fluid accumulation was first noticeable in ileal loops 12 h and in colonic ones 5 h after the injection of these doses of the cytotoxin and then proceeded with time. When K. oxytoca OK-1, a cytotoxin-producing strain, was inoculated into the loops at doses of 1 x 10(8) and 5 x 10(9) CFU, similar fluid accumulation was observed. However, inoculation of K. oxytoca ATCC 13182, a non-cytotoxin-producing strain, at the same doses did not cause any change. These results suggest that the cytotoxin-producing strain of K. oxytoca is the causative organism of antibiotic-associated hemorrhagic colitis and that the toxin is the factor responsible for pathogenesis.

Animals↗
PubMed2001

Antibodies to loop 6 of the P2 porin protein of nontypeable Haemophilus influenzae are bactericidal against multiple strains.

The P2 porin protein is the most abundant protein in the outer membrane of nontypeable Haemophilus influenzae (NTHI). Analysis of sequences of P2 from different strains reveals the presence of both heterogeneous and conserved surface-exposed loops of the P2 molecule among strains. The present study was undertaken to test the hypothesis that antibodies to a conserved surface-exposed loop are bactericidal for multiple strains of NTHI and could thus form the basis of vaccines to prevent infection due to NTHI. Polyclonal antiserum to a peptide corresponding to loop 6 was raised and was immunopurified over a loop 6 peptide column. Analysis of the antibodies to whole organisms and peptides corresponding to each of the eight loops of P2 by immunoassays revealed that the antibodies were highly specific for loop 6 of P2. The immunopurified antibodies bound to P2 of 14 of 15 strains in immunoblot assays. These antibodies to loop 6 demonstrated complement-mediated bactericidal killing of 8 of 15 strains. These results support the concept of using conserved regions of the P2 protein as a vaccine antigen.

Amino Acid Sequence↗
PubMed1997

Role of conserved residues in hydrophilic loop 8-9 of the lactose permease.

A peptide motif, GXXX(D/E)(R/K)XG(R/K)(R/K), has been conserved in a large group of evolutionarily related membrane proteins that transport small molecules across the membrane. Within the superfamily, this motif is located in two cytoplasmic loops that connect transmembrane segments 2 and 3 and transmembrane segments 8 and 9. In a previous study concerning the loop 2-3 motif of the lactose permease (A. E. Jessen-Marshall, N. J. Paul, and R. J. Brooker, J. Biol. Chem. 270:16251-16257, 1995), it was shown that the first-position glycine and the fifth-position aspartate are critical for transport activity since a variety of site-directed mutations greatly diminished the rate of transport. In the current study, a similar approach was used to investigate the functional significance of the conserved residues in the loop 8-9 motif. In the wild-type lactose permease, however, this motif has been evolutionarily modified so that the first-position glycine (an alpha-helix breaker) has been changed to proline (also a helix breaker); the fifth position has been changed to an asparagine; and one of the basic residues has been altered. In this investigation, we made a total of 28 single and 7 double mutants within the loop 8-9 motif to explore the functional importance of this loop. With regard to transport activity, amino acid substitutions within the loop 8-9 motif tend to be fairly well tolerated. Most substitutions produced permeases with normal or mildly defective transport activities. However, three substitutions at the first position (i.e., position 280) resulted in defective lactose transport. Kinetic analysis of position 280 mutants indicated that the defect decreased the Vmax for lactose uptake. Besides substitutions at position 280, a Gly-288-to-Thr mutant had the interesting property that the kinetic parameters for lactose uptake were normal yet the rates of lactose efflux and exchange were approximately 10-fold faster than wild-type rates. The results of this study suggest that loop 8-9 may facilitate conformational changes that translocate lactose.

Amino Acid Sequence↗
PubMed2000

trp RNA-binding attenuation protein-5' stem-loop RNA interaction is required for proper transcription attenuation control of the Bacillus subtilis trpEDCFBA operon.

The trp RNA-binding attenuation protein (TRAP) regulates expression of the Bacillus subtilis trpEDCFBA operon by a novel transcription attenuation mechanism. Tryptophan-activated TRAP binds to the nascent trp leader transcript by interacting with 11 (G/U)AG repeats, 6 of which are present in an antiterminator structure. TRAP binding to these repeats prevents formation of the antiterminator, thereby promoting formation of an overlapping intrinsic terminator. A third stem-loop structure that forms at the extreme 5' end of the trp leader transcript also plays a role in the transcription attenuation mechanism. The 5' stem-loop increases the affinity of TRAP for trp leader RNA. Results from RNA structure mapping experiments demonstrate that the 5' stem-loop consists of a 3-bp lower stem, a 5-by-2 asymmetric internal loop, a 6-bp upper stem, and a hexaloop at the apex of the structure. Footprinting results indicate that TRAP interacts with the 5' stem-loop and that this interaction differs depending on the number of downstream (G/U)AG repeats present in the transcript. Expression studies with trpE'-'lacZ translational fusions demonstrate that TRAP-5' stem-loop interaction is required for proper regulation of the trp operon. 3' RNA boundary experiments indicate that the 5' structure reduces the number of (G/U)AG repeats required for stable TRAP-trp leader RNA association. Thus, TRAP-5' stem-loop interaction may increase the likelihood that TRAP will bind to the (G/U)AG repeats in time to block antiterminator formation.

5' Untranslated Regions↗
PubMed2000

Probing the CD lumenal loop region of the D2 protein of photosystem II in Synechocystis sp. strain PCC 6803 by combinatorial mutagenesis.

The CD lumenal loop region of the photosystem II reaction center protein D2 contains residues involved in oxygen evolution. Since detailed structural information about this region is unavailable, an M13-based combinatorial mutagenesis approach was used to investigate structure-function relationships in this vital region of D2 in Synechocystis sp. strain PCC 6803. The CD loop coding region contains close to 100 nucleotides, and for effective mutagenesis, it was subdivided into four regions of seven to eight codons. A gain-of-function selection protocol was employed such that all mutants that were selected contained a functional D2 protein. In this way, conservation patterns of residues along with numbers and types of amino acid substitutions accommodated at each position for each set of mutants would indicate which residues in the CD loop may play important structural and functional roles. Results of this study have substantiated the importance of residues previously studied by site-directed mutagenesis such as Arg180 and His189 and have identified other previously unremarkable residues in the CD loop (such as Ser166, Phe169, and Ala170) that cannot be replaced by many other residues. In addition, the pliability of the CD loop was further tested using deletion and D1-D2 substitution constructs in M13. This showed that the length of the loop was important to its function, and in two cases, D2 could accommodate homologous sequences from D1, which forms a heterodimer with D2 in photosystem II, but not the other way around. This study of the CD loop in D2 provides valuable clues regarding the structural and functional requirements of the region.

Alanine↗
PubMed2002

Substrate specificity of the RND-type multidrug efflux pumps AcrB and AcrD of Escherichia coli is determined predominantly by two large periplasmic loops.

AcrAB-TolC is a constitutively expressed, tripartite efflux transporter complex that functions as the primary resistance mechanism to lipophilic drugs, dyes, detergents, and bile acids in Escherichia coli. TolC is an outer membrane channel, and AcrA is an elongated lipoprotein that is hypothesized to span the periplasm and coordinate efflux of such substrates by AcrB and TolC. AcrD is an efflux transporter of E. coli that provides resistance to aminoglycosides as well as to a limited range of amphiphilic agents, such as bile acids, novobiocin, and fusidic acid. AcrB and AcrD belong to the resistance nodulation division superfamily and share a similar topology, which includes a pair of large periplasmic loops containing more than 300 amino acid residues each. We used this knowledge to test several plasmid-encoded chimeric constructs of acrD and acrB for substrate specificity in a marR1 DeltaacrB DeltaacrD host. AcrD chimeras were constructed in which the large, periplasmic loops between transmembrane domains 1 and 2 and 7 and 8 were replaced with the corresponding loops of AcrB. Such constructs provided resistance to AcrB substrates at levels similar to native AcrB. Conversely, AcrB chimeras containing both loops of AcrD conferred resistance only to the typical substrates of AcrD. These results cannot be explained by simply assuming that AcrD, not hitherto known to interact with AcrA, acquired this ability by the introduction of the loop regions of AcrB, because (i) both AcrD and AcrA were found, in this study, to be required for the efflux of amphiphilic substrates, and (ii) chemical cross-linking in intact cells efficiently produced complexes between AcrD and AcrA. Since AcrD can already interact with AcrA, the alterations in substrate range accompanying the exchange of loop regions can only mean that substrate recognition (and presumably binding) is determined largely by the two periplasmic loops.

Anti-Bacterial Agents↗
PubMed1993

Inhibition of human immunodeficiency virus type 1 infection and syncytium formation in human cells by V3 loop synthetic peptides from gp120.

Because V3 loop-specific antibodies have been shown to inhibit human immunodeficiency virus type 1 (HIV-1) infection of human cells and because specific mutations in the V3 loop render the virus ineffective for infection and syncytium formation, we tested the anti-HIV effects of V3 loop peptides from different HIV-1 strains. We obtained evidence that V3 loop synthetic peptides of 8 to 15 amino acids at nanogram concentrations efficiently blocked HIV-1 IIIB infection of several human T-cell lines and of freshly prepared normal human T cells. More importantly, syncytium formation by three different primary clinical HIV isolates was inhibited by the V3 loop peptide from HIV-1 IIIB at a concentration of 1 micrograms/ml. Concentrations of V3 peptides up to 50 micrograms/ml were not toxic to any of the human cells studied. Additionally, V3 peptides incubated in normal human serum or plasma exhibited biological and physical stability for up to 24 h. Taken together, these results suggest that the V3 loop peptides have medical utility as therapeutic reagents to either prevent HIV-1 infection in humans or reduce the spread of virus infection in HIV-infected individuals. These findings are especially significant because a number of reports in the literature indicate that the V3 loop region in gp120 plays an important role in the initial stages of HIV-1 infection of cells.

Amino Acid Sequence↗
PubMed1994

Orientation-specific cis complementation by bulge- and loop-mutated human immunodeficiency virus type 1 TAR RNAs.

Tat activates human immunodeficiency type 1 gene expression by binding to TAR RNA. TAR comprises a partially base paired stem and hexanucleotide loop with a tripyrimidine bulge in the upper stem. In vitro, Tat binds to the bulge and upper stem, with no requirement for the loop. However, in vivo, loop sequences are critical for activation, implying that a loop binding cellular factor may be involved in the activation pathway. Given that activation appears to be a two-component system comprising a Tat-bulge interaction and a cellular factor-loop interaction, we considered that it might be possible to spatially separate the two components and retain activation. We have constructed a series of double TAR elements comprising various combinations of mutated TAR structures. Defective TARs with nucleotide substitutions in either the bulge or the loop complemented each other to give wild-type activation. However, the complementation was orientation specific, requiring the intact Tat binding site to reside on the 5'-proximal TAR. These data suggest that provided the wild-type orientation of the bulge and loop elements is retained, there is no requirement for them to coexist on the same TAR structure.

Bacterial Proteins↗
PubMed1995

Involvement of the V1/V2 variable loop structure in the exposure of human immunodeficiency virus type 1 gp120 epitopes induced by receptor binding.

The binding of human immunodeficiency virus type 1 (HIV-1) to the cellular receptor CD4 has been suggested to induce conformational changes in the viral envelope glycoproteins that promote virus entry. Conserved, discontinuous epitopes on the HIV-1 gp120 glycoprotein recognized by the 17b, 48d, and A32 antibodies are preferentially exposed upon the binding of soluble CD4 (sCD4). The binding of the 17b and 48d antibodies to the gp120 glycoprotein can also be enhanced by the binding of the A32 antibody. Here we constructed HIV-1 gp120 mutants in which the variable segments of the V1/V2 and V3 structures were deleted, individually or in combination, while the 17b, 48d, and A32 epitopes were retained. The effects of the variable loop deletions on the function of the HIV-1 envelope glycoproteins and on the exposure of epitopes induced by sCD4 or A32 binding to the monomeric gp120 glycoprotein were examined. The variable-loop-deleted envelope glycoproteins were able to mediate virus entry, albeit at lower efficiencies than those of the wild-type glycoproteins. Thus, the V1/V2 and V3 variable sequences contribute to the efficiency of HIV-1 entry but are not absolutely required for the process. Neither the V1/V2 nor V3 loops were necessary for the increase in exposure of the 17b/48d epitopes induced by binding of the A32 monoclonal antibody. By contrast, induction of the 17b, 48d, and A32 epitopes by sCD4 binding apparently involves a movement of the V1/V2 loops, which in the absence of CD4 partially mask these epitopes on the native gp120 monomer. The results obtained with a mutant glycoprotein containing a deletion of the V1 loop alone indicated that the contribution of the V2 loop to these phenomena was more significant than that of the V1 sequences. These results suggest that the V1/V2 loops, which have been previously implicated in CD4-modulated, postattachment steps in HIV-1 entry, contribute to CD4-induced gp120 conformational changes detected by the 17b, 48d, and A32 antibodies.

Amino Acid Sequence↗
PubMed1997

Neutralizing antibodies against the V3 loop of human immunodeficiency virus type 1 gp120 block the CD4-dependent and -independent binding of virus to cells.

The CD4 molecule is an essential receptor for human immunodeficiency virus type 1 (HIV-1) through high-affinity interactions with the viral external envelope glycoprotein gp120. Previously, neutralizing monoclonal antibodies (MAbs) specific to the third hypervariable domain of gp120 (the V3 loop) have been thought to block HIV infection without affecting the binding of HIV particles to CD4-expressing human cells. However, here we demonstrate that this conclusion was not correct and was due to the use of soluble gp120 instead of HIV particles. Indeed, neutralizing anti-V3 loop MAbs inhibited completely the binding and entry of HIV particles into CD4+ human cells. In contrast, the binding of virus was only partially inhibited by neutralizing anti-CD4 MAbs against the gp120 binding site in CD4, which, like the anti-V3 loop MAbs, completely inhibited HIV entry and infection. Nonneutralizing control MAbs against either the V3 loop or the N or C terminus of gp120 had no significant effect on HIV binding and entry. HIV-1 particles were also found to bind human and murine cells expressing or not expressing the human CD4 molecule. Interestingly, the binding of HIV to CD4+ murine cells was inhibited by both anti-V3 and anti-CD4 MAbs, whereas the binding to human and murine CD4- cells was affected only by anti-V3 loop MAbs. The effect of anti-V3 loop neutralizing MAbs on the HIV binding to cells appears not to be the direct consequence of gp120 shedding from HIV particles or of a decreased affinity of CD4 or gp120 for binding to its surface counterpart. Taken together, our results suggest the existence of CD4-dependent and -independent binding events involved in the attachment of HIV particles to cells; in both of these events, the V3 loop plays a critical role. As murine cells lack the specific cofactor CXCR4 for HIV-1 entry, other cell surface molecules besides CD4 might be implicated in stable binding of HIV particles to cells.

Animals↗
PubMed1999

Analysis of the critical domain in the V3 loop of human immunodeficiency virus type 1 gp120 involved in CCR5 utilization.

Human immunodeficiency virus type 1 (HIV-1) infection of CD4(+) lymphocytes and macrophages involves interaction of the surface subunit of the envelope protein (gp120) with coreceptors. Isolates have been found with specific tropism for macrophages and/or T-cell lines, through the utilization of chemokine receptor CCR5 (R5) or CXCR4 (X4). The third hypervariable loop (V3 loop) of gp120 is the major determinant of tropism. Using chimeric envelopes between HXB2 (X4) and ADA (R5), we found that the C-terminal half of the V3 loop was sufficient to confer on HXB2 the ability to infect CCR5-expressing cells. A sequence motif was identified at positions 289 to 292 allowing 30% of wild-type levels of infection, whereas full activity was achieved with the conversion of Lys to Glu at position 287 in addition to the above motif. Moreover, V3 loops from either SF2 (X4R5) or SF162 (R5) also allowed infection of CCR5-expressing cells, supporting the importance of V3 loops in influencing CCR5 utilization. The effects of amino acid changes at position 287 on the level of infection via CCR5 showed that negatively charged residues (Glu and Asp) were optimal for efficient interaction whereas only bulky hydrophobic residues drastically reduced infection. In addition, sequences at the N terminus of the V3 loop independently modulated the level of infection via CCR5. This study also examined the susceptibility of chimeric envelopes to neutralization by anticoreceptor antibodies and suggested the presence of differential interaction between the chimeric envelopes and CCR5. These findings highlight the critical residues in the V3 loop that mediate HIV-1 infection.

Amino Acid Sequence↗
PubMed1999

Role of naturally occurring basic amino acid substitutions in the human immunodeficiency virus type 1 subtype E envelope V3 loop on viral coreceptor usage and cell tropism.

To assess the role of naturally occurring basic amino acid substitutions in the V3 loop of human immunodeficiency virus type 1 (HIV-1) subtype E on viral coreceptor usage and cell tropism, we have constructed a panel of chimeric viruses with mutant V3 loops of HIV-1 subtype E in the genetic background of HIV-1LAI. The arginine substitutions naturally occurring at positions 8, 11, and 18 of the V3 loop in an HIV-1 subtype E X4 strain were systematically introduced into that of an R5 strain to generate a series of V3 loop mutant chimera. These chimeric viruses were employed in virus infectivity assays using HOS-CD4 cells expressing either CCR5 or CXCR4, peripheral blood mononuclear cells, T-cell lines, or macrophages. The arginine substitution at position 11 of the V3 loop uniformly caused the loss of infectivity in HOS-CD4-CCR5 cells, indicating that position 11 is critical for utilization of CCR5. CXCR4 usage was conferred by a minimum of two arginine substitutions, regardless of combination, whereas arginine substitutions at position 8 and 11 were required for T-cell line tropism. Nonetheless, macrophage tropism was not conferred by the V3 loop of subtype E R5 strain per se. We found that the specific combinations of amino acid changes in HIV-1 subtype E env V3 loop are critical for determining viral coreceptor usage and cell tropism. However, the ability to infect HOS-CD4 cells through either CXCR4 or CCR5 is not necessarily correlated with T-cell or macrophage tropism, suggesting that cellular tropism is not dictated solely by viral coreceptor utilization.

Amino Acid Sequence↗
PubMed2001

N-linked glycosylation sites adjacent to and within the V1/V2 and the V3 loops of dualtropic human immunodeficiency virus type 1 isolate DH12 gp120 affect coreceptor usage and cellular tropism.

The envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) is extensively glycosylated, containing approximately 23 asparagine (N)-linked glycosylation sites on its gp120 subunit. In this study, specific glycosylation sites on gp120 of a dualtropic primary HIV-1 isolate, DH12, were eliminated by site-directed mutagenesis and the properties of the resulting mutant envelopes were evaluated using a recombinant vaccinia virus-based cell-to-cell fusion assay alone or in the context of viral infections. Of the glycosylation sites that were evaluated, those proximal to the V1/V2 loops (N135, N141, N156, N160) and the V3 loops (N301) of gp120 were functionally critical. The glycosylation site mutations near the V1/V2 loop compromised the use of CCR5 and CXCR4 equally. In contrast, a mutation within the V3 loop preferentially inhibited the usage of CCR5; although this mutant protein completely lost its CCR5-dependent fusion activity, it retained 50% of the wild-type fusion activity with CXCR4. The replication of a virus containing this mutation was severely compromised in peripheral blood mononuclear cells, MT-4 cells, and primary monocyte-derived macrophages. A revertant virus, which acquired second site changes in the V3 loop that resulted in an increase in net positive charge, was isolated. The revertant virus fully recovered the usage of CXCR4 but not of CCR5, thereby altering the tropism of the parental virus from dualtropic to T-tropic. These results suggest that carbohydrate moieties near the V1/V2 and the V3 loops play critical roles in maintaining proper conformation of the variable loops for optimal interaction with receptors. Our results, combined with those of previously reported studies, further demonstrate that the function of individual glycans may be virus isolate dependent.

Base Sequence↗
PubMed2004

A conserved histidine in the ij loop of the Semliki Forest virus E1 protein plays an important role in membrane fusion.

The enveloped alphavirus Semliki Forest virus (SFV) infects cells via a low pH-triggered membrane fusion reaction mediated by the E1 protein. E1 is a class II fusion protein that contains the hydrophobic fusion peptide loop and converts to a stable homotrimer during the fusion reaction. Intriguingly, the fusion loop is closely associated with a loop connecting the i and j beta-strands. This ij loop plays a role in the cholesterol dependence of membrane fusion and is specifically susceptible to proteolysis in the protease-resistant E1 homotrimer. The SFV ij loop contains a histidine residue at position 230. Sequence comparisons revealed that an analogous histidine is completely conserved in all alphavirus and flavivirus fusion proteins. An E1 H230A mutant was constructed using the SFV infectious clone. Although cells infected with H230A RNA produced virus particles, these virions were completely noninfectious and were blocked in both cell-cell fusion and lipid mixing assays. The H230A virions efficiently bound to cell surface receptors and responded to low pH by undergoing acid-dependent conformational changes including dissociation of the E1/E2 dimer, exposure of the fusion loop, association with target liposomes, exposure of acid-conformation-specific epitopes, and formation of the stable E1 homotrimer. Studies with a soluble fragment of E1 showed that the mutant protein was defective in lipid-dependent conformational changes. Our results indicate that the E1 ij loop and the conserved H230 residue play a critical role in alphavirus-membrane fusion and suggest the presence of a previously undescribed late intermediate in the fusion reaction.

Amino Acid Sequence↗
PubMed2005

Stem-loop IV in the 5' untranslated region is a cis-acting element in bovine coronavirus defective interfering RNA replication.

The 210-nucleotide (nt) 5' untranslated region (UTR) in the positive-strand bovine coronavirus (BCoV) genome is predicted to contain four higher-order structures identified as stem-loops I to IV, which may function as cis-acting elements in genomic RNA replication. Here, we describe evidence that stem-loop IV, a bulged stem-loop mapping at nt 186 through 215, (i) is phylogenetically conserved among group 2 coronaviruses and may have a homolog in groups 1 and 3, (ii) exists as a higher-order structure on the basis of enzyme probing, (iii) is required as a higher-order element for replication of a BCoV defective interfering (DI) RNA in the positive but not the negative strand, and (iv) as a higher-order structure in wild-type (wt) and mutant molecules that replicate, specifically binds six cellular proteins in the molecular mass range of 25 to 58 kDa as determined by electrophoretic mobility shift and UV cross-linking assays; binding to viral proteins was not detected. Interestingly, the predicted stem-loop IV homolog in the severe acute respiratory syndrome (SARS) coronavirus appears to be group 1-like in that it is in part duplicated with a group 1-like conserved loop sequence and is not group 2-like, as would be expected by the SARS coronavirus group 2-like 3' UTR structure. These results together indicate that stem-loop IV in the BCoV 5' UTR is a cis-acting element for DI RNA replication and that it might function through interactions with cellular proteins. It is postulated that stem-loop IV functions similarly in the virus genome.

5' Untranslated Regions↗
PubMed2006

A small loop in the capsid protein of Moloney murine leukemia virus controls assembly of spherical cores.

We report the identification of a novel domain in the Gag protein of Moloney murine leukemia virus (MoLV) that is important for the formation of spherical cores. Analysis of 18 insertional mutations in the N-terminal domain of the capsid protein (CA) identified 3 that were severely defective for viral assembly and release. Transmission electron microscopy of cells producing these mutants showed assembly of Gag proteins in large, flat or dome-shaped patches at the plasma membrane. Spherical cores were not formed, and viral particles were not released. This late assembly/release block was partially rescued by wild-type virus. All three mutations localized to the small loop between alpha-helices 4 and 5 of CA, analogous to the cyclophilin A-binding loop of human immunodeficiency virus type 1 CA. In the X-ray structure of the hexameric form of MLV CA, this loop is located at the periphery of the hexamer. The phenotypes of mutations in this loop suggest that formation of a planar lattice of Gag is unhindered by mutations in the loop. However, the lack of progression of these planar structures to spherical ones suggests that mutations in this loop may prevent formation of pentamers or of stable pentamer-hexamer interactions, which are essential for the formation of a closed, spherical core. This region in CA, focused to a few residues of a small loop, may offer a novel therapeutic target for retroviral diseases.

Animals↗