Functional Analysis of the Disulfide-Bonded Loop/Chain Reversal Region of Human Immunodeficiency Virus Type 1 gp41 Reveals a Critical Role in gp120-gp41 Association

Human immunodeficiency virus type 1 (HIV-1) entry into cells is mediated by the surface-exposed envelope protein (SU) gp120, which binds to cellular CD4 and chemokine receptors, triggering the membrane fusion activity of the transmembrane (TM) protein gp41. The core of gp41 comprises an N-terminal triple-stranded coiled coil and an antiparallel C-terminal helical segment which is packed against the exterior of the coiled coil and is thought to correspond to a fusion-activated conformation. The available gp41 crystal structures lack the conserved disulfide-bonded loop region which, in human T-lymphotropic virus type 1 (HTLV-1) and murine leukemia virus TM proteins, mediates a chain reversal, connecting the antiparallel N- and C-terminal regions. Mutations in the HTLV-1 TM protein gp21 disulfide-bonded loop/chain reversal region adversely affected fusion activity without abolishing SU-TM association (A. L. Maerz, R. J. Center, B. E. Kemp, B. Kobe, and P. Poumbourios, J. Virol. 74:6614-6621, 2000). We now report that in contrast to our findings with HTLV-1, conservative substitutions in the HIV-1 gp41 disulfide-bonded loop/chain reversal region abolished association with gp120. While the mutations affecting gp120-gp41 association also affected cell-cell fusion activity, HIV-1 glycoprotein maturation appeared normal. The mutant glycoproteins were processed, expressed at the cell surface, and efficiently immunoprecipitated by conformation-dependent monoclonal antibodies. The gp120 association site includes aromatic and hydrophobic residues on either side of the gp41 disulfide-bonded loop and a basic residue within the loop. The HIV-1 gp41 disulfide-bonded loop/chain reversal region is a critical gp120 contact site; therefore, it is also likely to play a central role in fusion activation by linking CD4 plus chemokine receptor-induced conformational changes in gp120 to gp41 fusogenicity. These gp120 contact residues are present in diverse primate lentiviruses, suggesting conservation of function.     

A critical coiled coil motif in the small terminase, gp16, from bacteriophage T4: insights into DNA packaging initiation and assembly of packaging motor

Double-stranded DNA packaging in bacteriophages is driven by one of the most powerful force-generating molecular motors reported to date. The phage T4 motor is composed of the small terminase protein, gpl6 (18kDa), the large terminase protein, gp17 (70kDa), and the dodecameric portal protein gp20 (61kDa). gp16, which exists as an oligomer in solution, is involved in the recognition of the viral DNA substrate, the very first step in the DNA packaging pathway, and stimulates the ATPase and packaging activities associated with gp17. Sequence analyses using COILS2 revealed the presence of coiled coil motifs (CCMs) in gp16. Sixteen T4-family and numerous phage small terminases show CCMs in the corresponding region of the protein, suggesting a common structural and functional theme. Biochemical properties such as reversible thermal denaturation and analytical gel filtration data suggest that the central CCM-1 is critical for oligomerization of gp16. Mutations in CCM-1 that change the hydrophobicity of key residues, or pH 6.0, destabilized coiled coil interactions, resulting in a loss of gp16 oligomerization. The gp16 oligomers are in a dynamic equilibrium with lower M(r) intermediate species and monomer. Monomeric gp16 is unable to stimulate gp17-ATPase, an activity essential for DNA packaging, while conversion back into oligomeric form restored the activity. These data for the first time defined a CCM that is critical for structure and function of the small terminase. We postulate a packaging model in which the gp16 CCM is implicated in the regulation of packaging initiation and assembly of a supramolecular DNA packaging machine on the viral concatemer.     

Insertion of the membrane-proximal region of the neuronal SNARE coiled coil into the membrane

In the neuron, soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins assemble into an alpha-helical coiled coil that bridges the synaptic vesicle to the plasma membrane and drives membrane fusion, a required process for neurotransmitter release at the nerve terminal. How does coiled coil formation drive membrane fusion? To investigate the structural and energetic coupling between the coiled coil and membrane, the recombinant SNARE complex in the phospholipid bilayer was studied using fluorescence quenching and site-directed spin labeling EPR. Fluorescence analysis revealed that two native Trp residues at the membrane-proximal region of the coiled coil are inserted into the membrane, tightly coupling the coiled coil to the membrane. The EPR results indicate that the coiled coil penetrates into the membrane with an oblique angle, providing a favorable geometry for the basic residues to interact with negatively charged lipids. The result supports the proposition that core complex formation directly leads to the apposition of two membranes, which could facilitate lipid mixing. Trp residues and basic residues are abundant at the membrane-proximal region of transmembrane SNARE proteins, suggesting the generality of the proposed mechanism for the SNARE complex-membrane coupling.     

Autographa californica multiple nucleopolyhedrovirus GP64 protein: roles of histidine residues in triggering membrane fusion and fusion pore expansion

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) GP64 protein mediates membrane fusion during entry. Fusion results from a low-pH-triggered conformational change in GP64 and subsequent interactions with the membrane bilayers. The low-pH sensor and trigger of the conformational change are not known, but histidine residues are implicated because the pK(a) of histidine is near the threshold for triggering fusion by GP64. We used alanine substitutions to examine the roles of all individual and selected clusters of GP64 histidine residues in triggering and mediating fusion by GP64. Three histidine residues (H152, H155, and H156), located in fusion loop 2, were identified as important for membrane fusion. These three histidine residues were important for efficient pore expansion but were not required for the pH-triggered conformational change. In contrast, a cluster of three histidine residues (H245, H304, and H430) located near the base of the central coiled coil was identified as a putative sensor for low pH. Three alanine substitutions in cluster H245/H304/H430 resulted in dramatically reduced membrane fusion and the apparent loss of the prefusion conformation at neutral pH. Thus, the H245/H304/H430 cluster of histidines may function or participate as a pH sensor by stabilizing the prefusion structure of GP64.     

De novo design of peptide immunogens that mimic the coiled coil region of human T-cell leukemia virus type-1 glycoprotein 21 transmembrane subunit for induction of native protein reactive neutralizing antibodies

Peptide vaccines able to induce high affinity and protective neutralizing antibodies must rely in part on the design of antigenic epitopes that mimic the three-dimensional structure of the corresponding region in the native protein. We describe the design, structural characterization, immunogenicity, and neutralizing potential of antibodies elicited by conformational peptides derived from the human T-cell leukemia virus type 1 (HTLV-1) gp21 envelope glycoprotein spanning residues 347-374. We used a novel template design and a unique synthetic approach to construct two peptides (WCCR2T and CCR2T) that would each assemble into a triple helical coiled coil conformation mimicking the gp21 crystal structure. The peptide B-cell epitopes were grafted onto the epsilon side chains of three lysyl residues on a template backbone construct consisting of the sequence acetyl-XGKGKGKGCONH2 (where X represents the tetanus toxoid promiscuous T cell epitope (TT) sequence 580-599). Leucine substitutions were introduced at the a and d positions of the CCR2T sequence to maximize helical character and stability as shown by circular dichroism and guanidinium hydrochloride studies. Serum from an HTLV-1-infected patient was able to recognize the selected epitopes by enzyme-linked immunosorbent assay (ELISA). Mice immunized with the wild-type sequence (WCCR2T) and the mutant sequence (CCR2T) elicited high antibody titers that were capable of recognizing the native protein as shown by flow cytometry and whole virus ELISA. Sera and purified antibodies from immunized mice were able to reduce the formation of syncytia induced by the envelope glycoprotein of HTLV-1, suggesting that antibodies directed against the coiled coil region of gp21 are capable of disrupting cell-cell fusion. Our results indicate that these peptides represent potential candidates for use in a peptide vaccine against HTLV-1.     

Structural characterization of the SARS-coronavirus spike S fusion protein core

The spike (S) glycoprotein of coronaviruses mediates viral entry into host cells. It is a type 1 viral fusion protein that characteristically contains two heptad repeat regions, denoted HR-N and HR-C, that form coiled-coil structures within the ectodomain of the protein. Previous studies have shown that the two heptad repeat regions can undergo a conformational change from their native state to a 6-helix bundle (trimer of dimers), which mediates fusion of viral and host cell membranes. Here we describe the biophysical analysis of the two predicted heptad repeat regions within the severe acute respiratory syndrome coronavirus S protein. Our results show that in isolation the HR-N region forms a stable alpha-helical coiled coil that associates in a tetrameric state. The HR-C region in isolation formed a weakly stable trimeric coiled coil. When mixed together, the two peptide regions (HR-N and HR-C) associated to form a very stable alpha-helical 6-stranded structure (trimer of heterodimers). Systematic peptide mapping showed that the site of interaction between the HR-N and HR-C regions is between residues 916-950 of HR-N and residues 1151-1185 of HR-C. Additionally, interchain disulfide bridge experiments showed that the relative orientation of the HR-N and HR-C helices in the complex was antiparallel. Overall, the structure of the hetero-stranded complex is consistent with the structures observed for other type 1 viral fusion proteins in their fusion-competent state.     

The ectodomain of the viral receptor YueB forms a fiber that triggers ejection of bacteriophage SPP1 DNA

The irreversible binding of bacteriophages to their receptor(s) in the host cell surface triggers release of the naked genome from the virion followed by transit of viral DNA to the host cell cytoplasm. We have purified, for the first time, a receptor from a Gram-positive bacterium that is active to trigger viral DNA ejection in vitro. This extracellular region ("ectodomain") of the Bacillus subtilis protein YueB (YueB780) was a 7 S elongated dimer forming a 36.5-nm-long fiber. YueB780 bound to the tail tip of bacteriophage SPP1. Although a stable receptor-phage interaction occurred between 0 and 37 degrees C, complete blocking of phage DNA release or partial ejection events were observed at temperatures below 15 degrees C. We also showed that the receptor was exposed to the B. subtilis surface. YueB differed structurally from phage receptors from Gram-negative bacteria. Its properties revealed a fiber spanning the full length of the 30-nm-thick peptidoglycan layer. The fiber is predicted to be anchored in the cell membrane through transmembrane segments. These features, highly suitable for a virus receptor in Gram-positive bacteria, are very likely shared by a large number of phage receptors.     

New insights into pb5, the receptor binding protein of bacteriophage T5, and its interaction with its Escherichia coli receptor FhuA

The majority of bacterial viruses are bacteriophages bearing a tail that serves to recognise the bacterial surface and deliver the genome into the host cell. Infection is initiated by the irreversible interaction between the viral receptor binding protein (RBP) and a receptor at the surface of the bacterium. This interaction results ultimately in the phage DNA release in the host cytoplasm. Phage T5 infects Escherichia coli after binding of its RBP pb5 to the outer membrane ferrichrome transporter FhuA. Here, we have studied the complex formed by pb5 and FhuA by a variety of biophysical and biochemical techniques. We show that unlike RBPs of known structures, pb5 probably folds as a unique domain fulfilling both functions of binding to the host receptor and interaction with the rest of the phage. Pb5 likely binds to the domain occluding the β-barrel of FhuA as well as to external loops of the barrel. Furthermore, upon binding to FhuA, pb5 undergoes conformational changes, at the secondary and tertiary structure level that would be the key to the transmission of the signal through the tail to the capsid, triggering DNA release. This is the first structural information regarding the binding of a RBP to a proteic receptor.     

Synthesis of Bacteriophage and Host DNA in Toluene-Treated Cells Prepared from T4-Infected Escherichia coli: Role of Bacteriophage Gene D2a

We investigated the synthesis of DNA in toluene-treated cells prepared from Escherichia coli infected with bacteriophage T4. If the phage carry certain rII deletion mutations, those which extend into the nearby D2a region, the following results are obtained: (i) phage DNA synthesis occurs unless the phage carries certain DNA-negative mutations; and (ii) host DNA synthesis occurs even though the phage infection has already resulted in the cessation of host DNA synthesis in vivo. The latter result indicates that the phage-induced cessation of host DNA synthesis is not due to an irreversible inactivation of an essential component of the replication apparatus. If the phage are D2a(+), host DNA synthesis in toluene-treated infected cells is markedly reduced; phage DNA synthesis is probably also reduced somewhat. These D2a effects, considered along with our earlier work, suggest that a D2a-controlled nuclease, specific for cytosine-containing DNA, is active in toluene-treated cells.     

A heptad repeat in herpes simplex virus 1 gH, located downstream of the alpha-helix with attributes of a fusion peptide, is critical for virus entry and fusion

Entry of herpes simplex virus 1 (HSV-1) into cells occurs by fusion with cell membranes; it requires gD as the receptor binding glycoprotein and the trigger of fusion, and the trio of the conserved glycoproteins gB, gH, and gL to execute fusion. Recently, we reported that the ectodomain of HSV-1 gH carries a hydrophobic alpha-helix (residues 377 to 397) with attributes of an internal fusion peptide (T. Gianni, P. L. Martelli, R. Casadio, and G. Campadelli-Fiume, J. Virol. 79:2931-2940, 2005). Downstream of this alpha-helix, a heptad repeat (HR) with a high propensity to form a coiled coil was predicted between residues 443 and 471 and was designated HR-1. The simultaneous substitution of two amino acids in HR-1 (E450G and L453A), predicted to abolish the coiled coil, abolished the ability of gH to complement the infectivity of a gH-null HSV mutant. When coexpressed with gB, gD, and gL, the mutant gH was unable to promote cell-cell fusion. These defects were not attributed to a defect in heterodimer formation with gL, the gH chaperone, or in trafficking to the plasma membrane. A 25-amino-acid synthetic peptide with the sequence of HR-1 (pep-gH(wt25)) inhibited HSV replication if present at the time of virus entry into the cell. A scrambled peptide had no effect. The effect was specific, as pep-gH(wt25) did not reduce HSV-2 and pseudorabies virus infection. The presence of a functional HR in the HSV-1 gH ectodomain strengthens the view that gH has attributes typical of a viral fusion glycoprotein.     

Structural basis for paramyxovirus-mediated membrane fusion

Paramyxoviruses are responsible for significant human mortality and disease worldwide, but the molecular mechanisms underlying their entry into host cells remain poorly understood. We have solved the crystal structure of a fragment of the simian parainfluenza virus 5 fusion protein (SV5 F), revealing a 96 A long coiled coil surrounded by three antiparallel helices. This structure places the fusion and transmembrane anchor of SV5 F in close proximity with a large intervening domain at the opposite end of the coiled coil. Six amino acids, potentially part of the fusion peptide, form a segment of the central coiled coil, suggesting that this structure extends into the membrane. Deletion mutants of SV5 F indicate that putative flexible tethers between the coiled coil and the viral membrane are dispensable for fusion. The lack of flexible tethers may couple a final conformational change in the F protein directly to the fusion of two bilayers.     

Identification and preliminary characterization of a mutant defective in the bacteriophage T4-induced unfolding of the Escherichia coli nucleoid.

The nucleoids of Escherichia coli S/6/5 cells are rapidly unfolded at about 3 min after infection with wild-type T4 bacteriophage or with nuclear disruption deficient, host DNA degradation-deficient multiple mutants of phage T4. Unfolding does not occur after infection with T4 phage ghosts. Experiments using chloramphenicol to inhibit protein synthesis indicate that the T4-induced unfolding of the E. coli chromosomes is dependent on the presence of one or more protein synthesized between 2 and 3 min after infection. A mutant of phage T4 has been isolated which fails to induce this early unfolding of the host nucleoids. This mutant has been termed "unfoldase deficient" (unf-) despite the fact that the function of the gene product defective in this strain is not yet known. Mapping experiments indicate that the unf- mutation is located near gene 63 between genes 31 and 63. The folded genomes of E. coli S/6/5 cells remain essentially intact (2,000-3,000S) at 5 min after infection with unfoldase-, nuclear disruption-, and host DNA degradation-deficient T4 phage. Nuclear disruption occurs normally after infection with unfoldase- and host DNA degradation-deficient but nuclear disruption-proficient (ndd+), T4 phage. The host chromosomes remain partially folded (1,200-1,800S) at 5 min after infection with the unfoldase single mutant unf39 x 5 or an unfoldase- and host DNA degradation-deficient, but nuclear disruption-proficient, T4 strain. The presence of the unfoldase mutation causes a slight delay in host DNA degradation in the presence of nuclear disruption but has no effect on the rate of host DNA degradation in the absence of nuclear disruption. Its presence in nuclear disruption- and host DNA degradation-deficient multiple mutants does not alter the shutoff to host DNA or protein synthesis.     

Physiological and Genetic Aspects of Abortive Infection of a Shigella sonnei Strain by Coliphage T7

Phage T7 adsorbed to and lysed cells of Shigella sonnei D(2) 371-48, although the average burst size was only 0.1 phage per cell (abortive infection). No mechanism of host-controlled modification was involved. Upon infection, T7 rapidly degraded host deoxyribonucleic acid (DNA) to acid-soluble material. Phage-directed DNA synthesis was initiated normally, but after a few minutes the pool of phage DNA, including the parental DNA, was degraded. Addition of chloramphenicol, at the time of phage infection, prevented both the initiation of phage-directed DNA synthesis and the degradation of parental phage DNA. Addition of chloramphenicol 4.5 min after phage was added permitted the onset of phage-directed DNA synthesis but prevented breakdown of phage DNA. Mutants of T7 (ss(-) mutants) have been isolated which show normal growth in strain D(2) 371-48. Upon mixed infection of this strain with T7 wild type and an ss(-) mutant, infection was abortive; no complementation occurred. The DNA of the ss(-) mutants was degraded in mixed infection like that of the wild type. Revertant mutants which have lost their ability to grow on D(2) 371-48 were isolated from ss(-) mutants; they are, in essence, phenotypically like T7 wild type. Independently isolated revertants of ss(-) mutants did not produce ss(-) recombinants when they were crossed among themselves. When independently isolated ss(-) mutants were crossed with each other, wild-type recombinants were found; ss(-) mutants could then be mapped in a cluster compatible with the length of one cistron. We concluded that T7 codes for an active, chloramphenicol-sensitive function [ss(+) function (for suicide in Shigella)] which leads to the breakdown of phage DNA in the Shigella host.     

Phylogenetic occurrence of coiled coil proteins: Implications for tissue structure in metazoa via a coiled coil tissue matrix

We examined GenBank sequence files with a heptad repeat analysis program to assess the phylogenetic occurrence of coiled coil proteins, how heptad repeat domains are organized within them, and what structural/functional categories they comprise. Of 102,007 proteins analyzed, 5.95% (6,074) contained coiled coil domains; 1.26% (1,289) contained “extended” (> 75 amino acid) domains. While the frequency of proteins containing coiled coils was surprisingly constant among all biota, extended coiled coil proteins were fourfold more frequent in the animal kingdom and may reflect early events in the divergence of plants and animals. Structure/function categories of extended coils also revealed phylogenetic differences. In pathogens and parasites, many extended coiled coil proteins are external and bind host proteins. In animals, the majority of extended coiled coil proteins were identified as constituents of two protein categories: 1) myosins and motors; or 2) components of the nuclear matrix-intermediate filament scaffold. This scaffold, produced by sequential extraction of epithelial monolayers in situ, contains only 1–2% of the cell mass while accurately retaining morphological features of living epithelium and is greatly enriched in proteins with extensive, interrupted coiled coil forming domains. The increased occurrence of this type of protein in Metazoa compared with plants or protists leads us to hypothesize a tissue-wide matrix of coiled coil interactions underlying metazoan differentiated cell and tissue structure.     

Selection of a buried salt bridge by phage display

The α-helical coiled coil is a valuable folding motif for protein design and engineering. By means of phage display technology, we selected a capable binding partner for one strand of a coiled coil bearing a charged amino acid in a central hydrophobic core position. This procedure resulted in a novel coiled coil pair featuring an opposed Glu-Lys pair arranged staggered within the hydrophobic core of a coiled coil structure. Structural investigation of the selected coiled coil dimer by CD spectroscopy and MD simulations suggest that a buried salt bridge within the hydrophobic core enables the specific dimerization of two peptides.     

Isolation and characterization of a bacteriophage T5 mutant deficient in deoxynucleoside 5''-monophosphatase activity.

A bacteriophage T5 mutant has been isolated that is completely deficient in the induction of deoxynucleoside 5'-monophosphatase activity during infection of Escherichia coli F. The mutant bacteriophage has been shown to be deficient in the excretion of the final products of DNA degradation during infection of E. coli F, and about 30% of the host DNA's thymine residues were reinocorporated into phage DNA. During infection with this mutant, host DNA degradation to trichloroacetic acid-soluble products was normal, host DNA synthesis was shut off normally, and second-step transfer was not delayed. However, induction of early phage enzymes and production of DNA and phage were delayed by 5 to 15 min but eventually reached normal levels. The mutant's phenotype strongly suggests that the enzyme's role is to act at the final stage in the T5-induced system of host DNA degradation by hydrolyzing deoxynucleoside 5'-monophosphates to deoxynucleosides and free phosphate; failure to do this may delay expression of the second-step-transfer DNA.