Towards a mechanism of function of the viral ion channel Vpu from HIV-1

Vpu, an integral membrane protein encoded in HIV-1, is implicated in the release of new virus particles from infected cells, presumably mediated by ion channel activity of homo-oligomeric Vpu bundles. Reconstitution of both full length Vpu(1-81) and a short, the transmembrane (TM) domain comprising peptide Vpu(1-32) into bilayers under a constant electric field results in an asymmetric orientation of those channels. For both cases, channel activity with similar kinetics is observed. Channels can open and remain open within a broad series of conductance states even if a small or no electric potential is applied. The mean open time for Vpu peptide channels is voltage-independent. The rate of channel opening shows a biphasic voltage activation, implicating that the gating is influenced by the interaction of the dipole moments of the TM helices with an electric field.     

Chemical synthesis and single channel properties of tetrameric and pentameric TASPs (template-assembled synthetic proteins) derived from the transmembrane domain of HIV virus protein u (Vpu)

Vpu, an 81-residue membrane protein encoded by the genome of HIV-1, is involved in CD4 degradation and facilitates virion budding from infected cells. The latter activity requires an intact transmembrane (TM) domain; however, the mechanism remains unclear. Vpu forms ion channels, an activity linked to the TM domain and envisioned to arise by oligomerization. The precise number of Vpu monomers that structure the channel is not yet known. To address this issue, we have synthesized tetrameric and pentameric proteins consisting of a carrier template to which four or five peptides corresponding to the TM domain of Vpu are attached. Ketoxime-forming chemoselective ligation efficiently ligated four and five copies, respectively, of the linear transmembrane peptide that was solubilized by the addition of a cleavable polyethylene glycol-polyamide auxiliary to a template. Purified tetrameric and pentameric proteins, denoted as T(4)Vpu and T(5)Vpu, exhibit the predicted mass as determined by MS analysis and fold with a high helical content as evidenced by CD. Both T(4)Vpu and T(5)Vpu, after reconstitution in lipid bilayers, form discrete ion channels of distinct conductance and high propensity to be open. The most frequent openings have a single channel conductance of 42 +/- 5 pS for T(4)Vpu and 76 +/- 5 pS for T(5)Vpu in 0.5m KCl. These findings validate the notion that the channels formed by Vpu result from the self-assembly of monomers. We conclude that a five-helix bundle of the TM of Vpu may approximate the structural motif underlying the oligomeric state of the conductive channel.     

Towards a Mechanism of Function of the Viral Ion Channel Vpu from HIV-1

Abstract

Vpu, an integral membrane protein encoded in HIV-1, is implicated in the release of new virus particles from infected cells, presumably mediated by ion channel activity of homo- oligomeric Vpu bundles.

Reconstitution of both full length Vpu_1–81 and a short, the transmembrane (TM) domain comprising peptide Vpu_1-32 into bilayers under a constant electric field results in an asymmetric orientation of those channels. For both cases, channel activity with similar kinetics is observed. Channels can open and remain open within a broad series of conductance states even if a small or no electric potential is applied.

The mean open time for Vpu peptide channels is voltage-independent. The rate of channel opening shows a biphasic voltage activation, implicating that the gating is influenced by the interaction of the dipole moments of the TM helices with an electric field.     

Structural implications of mutations assessed by molecular dynamics: Vpu1–32 from HIV-1

Structural pore models are generated for Vpu_1–32WT from HIV-1 as well as for three mutants W23L, S24L and R31V. A computational methodology is employed which samples the whole conformational space of the pentameric assemblies of Vpu. The analysis of the related energy landscape reveals a small set of reasonable pore models, which are thoroughly investigated regarding their structural properties as well as their putative stability under native-like conditions. The models are also discussed in respect of earlier experimental findings about their channel activities. The study proposes functional pores reflecting the experimentally found conductance states of Vpu and its mutants.     

Structure-function correlates of Vpu,a membrane protein of HIV-1

Vpu, a membrane protein from human immunodeficiency virus-1, folds into two distinct structural domains with different biological activities: a transmembrane (TM) helical domain involved in the budding of new virions from infected cells, and a cytoplasmic domain encompassing two amphipathic helices, which is implicated in CD4 degradation. The molecular mechanism by which Vpu facilitates virion budding is not clear. This activity of Vpu requires an intact TM helical domain. And it is known that oligomerization of the VPU TM domain results in the formation of sequence-specific, cation-selective channels. It has been shown that the channel activity of Vpu is confined to the TM domain, and that the cytoplasmic helices regulate the lifetime of the Vpu channel in the conductive state. Structure-function correlates based on the convergence of information about the channel activity of Vpu reconstituted in lipid bilayers and on its 3-D structure in membranes by a combination of solution and solid-state nuclear magnetic resonance spectroscopy may provide valuable insights to understand the role of Vpu in the pathogenesis of AIDS and for drug design aimed to block channel activity.     

Bundles consisting of extended transmembrane segments of Vpu from HIV-1: computer simulations and conductance measurements

Part of the genome of the human immunodeficiency virus type 1 (HIV-1) encodes for a short membrane protein Vpu, which has a length of 81 amino acids. It has two functional roles: (i) to downregulate CD4 and (ii) to support particle release. These roles are attributed to two distinct domains of the peptide, the cytoplasmic and transmembrane (TM) domains, respectively. It has been suggested that the enhanced particle release function is linked to the ion channel activity of Vpu, with a slight preference for cations over anions. To allow ion flux across the membrane Vpu would be required to assemble in homooligomers to form functional water-filled pores. In this study molecular dynamics simulations are used to address the role of particular amino acids in 4, 5, and 6 TM helix bundle structures. The helices (Vpu(6-33)) are extended to include hydrophilic residues such as Glu, Tyr, and Arg (EYR motif). Our simulations indicate that this motif destabilizes the bundles at their C-terminal ends. The arginines point into the pore to form a positive charged ring that could act as a putative selectivity filter. The helices of the bundles adopt slightly higher average tilt angles with decreasing number of helices. We also suggest that the helices are kinked. Conductance measurements on a peptide (Vpu(1-32)) reconstituted into lipid membranes show that the peptide forms ion channels with several conductance levels.     

Molecular Dynamics Simulations Reveal the HIV-1 Vpu Transmembrane Protein to Form Stable Pentamers

The human immunodeficiency virus type I (HIV-1) Vpu protein is 81 residues long and has two cytoplasmic and one transmembrane (TM) helical domains. The TM domain oligomerizes to form a monovalent cation selective ion channel and facilitates viral release from host cells. Exactly how many TM domains oligomerize to form the pore is still not understood, with experimental studies indicating the existence of a variety of oligomerization states. In this study, molecular dynamics (MD) simulations were performed to investigate the propensity of the Vpu TM domain to exist in tetrameric, pentameric, and hexameric forms. Starting with an idealized α-helical representation of the TM domain, a thorough search for the possible orientations of the monomer units within each oligomeric form was carried out using replica-exchange MD simulations in an implicit membrane environment. Extensive simulations in a fully hydrated lipid bilayer environment on representative structures obtained from the above approach showed the pentamer to be the most stable oligomeric state, with interhelical van der Waals interactions being critical for stability of the pentamer. Atomic details of the factors responsible for stable pentamer structures are presented. The structural features of the pentamer models are consistent with existing experimental information on the ion channel activity, existence of a kink around the Ile17, and the location of tetherin binding residues. Ser23 is proposed to play an important role in ion channel activity of Vpu and possibly in virus propagation.     

Peptide mimics of the M13 coat protein transmembrane segment. Retention of helix-helix interaction motifs

Sequence-specific noncovalent helix-helix interactions between transmembrane (TM) segments in proteins are investigated by incorporating selected TM sequences into synthetic peptides using the construct CKKK-TM-KKK. The peptides are of suitable hydrophobicity for spontaneous membrane insertion, whereas formation of an N-terminal S-S bond can bring pairs of TM helices into proximity and promote their parallel orientation. Using the propensity of the protein to undergo thermally induced alpha-helix --> beta-sheet transitions as a parameter for helix stability, we compared the wild type and mutant (V29A and V31A) bacteriophage M13 coat proteins with their corresponding TM peptide constructs (M13 residues 24-42). Our results demonstrated that the relevant helix-helix tertiary contacts found in the intact proteins persist in the peptide mimics. Molecular dynamics simulations support the tight "two in-two out" dimerization motif for V31A consistent with mutagenesis data. The overall results reinforce the notion of TM segments as autonomous folding domains and suggest that the generic peptide construct provides a viable reductionist system for membrane protein structural and computational analysis.     

Role of the membrane interface on the conformation of the caveolin scaffolding domain: a CD and NMR study

Circular dichroism (CD) and NMR spectroscopy were used to study the conformational properties of two synthetic peptides, D82-R101 and D82-I109, encompassing the caveolin scaffolding domain (D82-R101), in the presence of dodecylphosphocholine (DPC) micelles. Our data show that a stable helical conformation of the caveolin scaffolding domain in a membrane mimicking system is only obtained for the peptide including the L102-I109 hydrophobic stretch, a part of the caveolin intra-membrane domain. Through chemical shift variations, an ensemble of six residues of the D82-L109 peptide, mainly located in the V(94)TKYWFYR(101) motif were found to detect the presence of phosphatidylserine solubilized in DPC micelles. Our results constitute a first step for elucidating at a residue level the conformational properties of the central region of the caveolin-1 protein.     

Conformational changes induced by a single amino acid substitution in the trans-membrane domain of Vpu: implications for HIV-1 susceptibility to channel blocking drugs

The channel-forming trans-membrane domain of Vpu (Vpu TM) from HIV-1 is known to enhance virion release from the infected cells and is a potential target for ion-channel blockers. The substitution of alanine at position 18 by a histidine (A18H) has been shown to render HIV-1 infections susceptible to rimantadine, a channel blocker of M2 protein from the influenza virus. In order to describe the influence of the mutation on the structure and rimantadine susceptibility of Vpu, we determined the structure of A18H Vpu TM, and compared it to those of wild-type Vpu TM and M2 TM. Both isotropic and orientationally dependent NMR frequencies of the backbone amide resonance of His18 were perturbed by rimantadine, and those of Ile15 and Trp22 were also affected, suggesting that His18 is the key residue for rimantadine binding and that residues located on the same face of the TM helix are also involved. A18H Vpu TM has an ideal, straight alpha-helix spanning residues 6-27 with an average tilt angle of 41 degrees in C14 phospholipid bicelles, indicating that the tilt angle is increased by 11 degrees compared to that of wild-type Vpu TM. The longer helix formed by the A18H mutation has a larger tilt angle to compensate for the hydrophobic mismatch with the length of the phospholipids in the bilayer. These results demonstrate that the local change of the primary structure plays an important role in secondary and tertiary structures of Vpu TM in lipid bilayers and affects its ability to interact with channel blockers.     

Effect of nonpolar substitutions of the conserved Phe11 in the fusion peptide of HIV-1 gp41 on its function, structure, and organization in membranes.

The fusion domain of the HIV-1 envelope glycoprotein (gp120-gp41) is a conserved hydrophobic region located at the N-terminus of the transmembrane subunit (gp41). A prominent feature of this domain is a conserved five-residue "FLGFL" sequence at positions 8-12. Mutation of the highly conserved Phe(11) to Val (F11V), presumed not to significantly affect the hydrophobicity and the structure of this region, has been shown to decrease the level of syncytium formation and virus infectivity. Here we show that the substitution of Gly for Phe(11) (F11G) reduces cell-cell fusion activity by 80-90%. To determine the effect of these mutations on the properties of the fusion peptide, a 33-residue peptide (WT) identical to the extended fusion domain and its F11V and F11G mutants were synthesized, fluorescently labeled, and studied with respect to their function, structure, and organization in phospholipid membranes. The WT peptide alone induced fusion of both zwitterionic (PC/Chol) and negatively charged (PS/PC/Chol and POPG) vesicles, in contrast to a 23-mer fusion peptide lacking the C-terminal domain which has been shown to be inactive with PC vesicles but able to induce fusion of POPG vesicles which had been preaggragated with Ca(2+) or Mg(2+). The F11V peptide preserved 50% activity, and the F11G peptide was virtually inactive. ATR-FTIR spectroscopy indicated similar secondary structure of the peptides in multibilayers that was independent of membrane composition. Furthermore, all the peptides increased the extent of lipid disorder to a similar extent, but the kinetics of amide II H to D exchange was in the following order: F11G > F11V > WT. Fluorescence studies in the presence of membranes, as well as SDS-PAGE, revealed that the WT and F11V peptides self-associate to similar levels while F11G exhibited a decreased level of self-association. The data suggest that the FLGFL motif contributes to the functional organization of the HIV-1 fusion peptide and that the C-terminal domain following the fusion peptide contributes to the membrane fusion process.     

Influenza A virus M2 ion channel protein: a structure-function analysis.

A structure-function analysis of the influenza A virus M2 ion channel protein was performed. The M2 protein of human influenza virus A/Udorn/72 and mutants containing changes on one face of the putative alpha helix of the M2 transmembrane (TM) domain, several of which lead to amantadine resistance when found in virus, were expressed in oocytes of Xenopus laevis. The membrane currents of oocytes expressing mutant M2 ion channels were measured at both normal and low pH, and the amantadine-resistant mutant containing the change of alanine at residue 30 to threonine was found to have a significantly attenuated low pH activation response. The specific activity of the channel current of the amantadine-resistant mutants was investigated by measuring the membrane current of individual oocytes followed by quantification of the amount of M2 protein expressed in these single oocytes by immunoblotting analysis. The data indicate that changing residues on this face of the putative alpha helix of the M2 TM domain alters properties of the M2 ion channel. Some of the M2 proteins containing changes in the TM domain were found to be modified by addition of an N-linked carbohydrate chain at an asparagine residue that is membrane proximal and which is not modified in the wild-type M2 protein. These N-linked carbohydrate chains were further modified by addition of polylactosaminoglycan. A glycosylated M2 mutant protein (M2 + V, A30T) exhibited an ion channel activity with a voltage-activated, time-dependent kinetic component. Prevention of carbohydrate addition did not affect the altered channel activity. The ability of the M2 protein to tolerate deletions in the TM domain was examined by expressing three mutants (del29-31, del28-31, and del27-31) containing deletions of three, four, and five residues in the TM domain. No ion channel activity was detected from expression of M2 del29-31 and del27-31, whereas expression of M2 del28-31 resulted in an ion channel activity that was activated by hyperpolarization (and not low pH) and was resistant to amantadine block. Examination of the oligomeric form of M2 del28-31 indicated that the oligomer is different from wild-type M2, and the data were consistent with M2 del28-31 forming a pentamer.     

Structure and dynamics of the second and third transmembrane domains of human glycine receptor

A 61-residue polypeptide resembling the second and third transmembrane domains (TM23) of the alpha-1 subunit of human glycine receptor and its truncated form, both with the wild-type loop linking the two TM domains (the "23" loop), were studied using high-resolution NMR. Well-defined domain structures can be identified for the TM2, 23 loop, and TM3 regions. Contrary to the popular model of a long and straight alpha-helical structure for the pore-lining TM2 domain for the Cys-loop receptor family, the last three residues of the TM2 domain and the first eight residues of the 23 loop (S16-S26) seem to be intrinsically nonhelical and highly flexible even in trifluoroethanol, a solvent known to promote and stabilize alpha-helical structures. The six remaining residues of the 23 loop and most of the TM3 domain exhibit helical structures with a kinked pi-helix (or a pi-turn) from W34 to C38 and a kink angle of 159 +/- 3 degrees . The tertiary fold of TM3 relative to TM2 is defined by several unambiguously identified long-range NOE cross-peaks within the loop region and between TM2 and TM3 domains. The 20 lowest-energy structures show a left-handed tilt of TM3 relative to TM2 with a tilting angle of 44 +/- 2 degrees between TM2 (V1-Q14) and TM3 (L39-E48) helix axes. This left-handed TM2-TM3 arrangement ensures a neatly packed right-handed quaternary structure of five subunits to form an ion-conducting pore. This is the first time that two TM domains of the glycine receptor linked by the important 23 loop have ever been analyzed at atomistic resolution. Many structural characteristics of the receptor can be inferred from the structural and dynamical features identified in this study.     

Identification of amino acids in the human tetherin transmembrane domain responsible for HIV-1 Vpu interaction and susceptibility

Tetherin, also known as BST-2/CD317/HM1.24, is an antiviral cellular protein that inhibits the release of HIV-1 particles from infected cells. HIV-1 viral protein U (Vpu) is a specific antagonist of human tetherin that might contribute to the high virulence of HIV-1. In this study, we show that three amino acid residues (I34, L37, and L41) in the transmembrane (TM) domain of human tetherin are critical for the interaction with Vpu by using a live cell-based assay. We also found that the conservation of an additional amino acid at position 45 and two residues downstream of position 22, which are absent from monkey tetherins, are required for the antagonism by Vpu. Moreover, computer-assisted structural modeling and mutagenesis studies suggest that an alignment of these four amino acid residues (I34, L37, L41, and T45) on the same helical face in the TM domain is crucial for the Vpu-mediated antagonism of human tetherin. These results contribute to the molecular understanding of human tetherin-specific antagonism by HIV-1 Vpu.     

Revisited and large-scale synthesis and purification of the mutated and wild type neu/erbB-2 membrane-spanning segment.

Solid-phase syntheses of the hydrophobic peptides Neu(TM35) ((1)EQRASPVTFIIATVVGVLLFLILVVVVGILIKRRR(35)) and Neu*(TM35) ((1)EQRASPVTFIIATVEGVLLFLILVVVVGILIKRRR(35)), corresponding to the native and mutated (V15E) transmembrane domain of the neu/erbB-2 tyrosine kinase receptor, respectively, were accomplished using Fmoc chemistry. The use of a new resin and cleavage and purification conditions led to large increases in yields and peptide purity. Two (15)N-labelled versions of both wild type and mutated peptides were also synthesized. Approximately 20-40 mg of peptide was obtained using a small-scale synthesis, whereas ca 100 mg of pure peptide was collected on a medium scale. Peptide purity, as monitored by HPLC and mass spectrometry, ranged from 95 to 98% for the six peptides synthesized. Secondary structure as determined by UV circular dichroism (CD) in trifluoroethanol (TFE) showed ca 74% alpha-helical content for the native peptide and ca 63% for that bearing the mutation. Secondary structure of Neu(TM35) was retained in DMPC (dimyristoylphosphatidylcholine)/DCPC (dicaproylphosphatidylcholine) membrane bicelles, and evidences for dimers/oligomers in the lipid bilayer were found.     

Hetero-assembly between all-L- and all-D-amino acid transmembrane domains: forces involved and implication for inactivation of membrane proteins

Protein-protein interactions within the membrane, partially or fully mediated by transmembrane (TM) domains, are involved in many vital cellular processes. Since the unique feature of the membrane environment enables protein-protein assembly that otherwise is not energetically favored in solution, the structural restrictions involved in the assembly of soluble proteins are not necessarily valid for the assembly of TM domains. Here we used the N-terminal TM domain (Tar-1) of the Escherichia coli aspartate receptor as a model system for examining the stereospecificity of TM-TM interactions in vitro and in vivo in isolated systems, and in the context of the full receptor. For this propose, we synthesized Tar-1 all-l and all-d amino acid TM peptides, a mutant TM peptide and an unrelated TM peptide. The data revealed: (i) Tar-1 all-d specifically associated with Tar-1 all-l within a model lipid membrane, as determined by using fluorescence energy transfer experiments; (ii) Tar-1 all-l and all-d, but not the control peptides, demonstrated a dose-dependant dominant negative effect on the Tar-1 TM homodimerization in the bacterial ToxR assembly system, suggesting a wild-type-like interaction; and most interestingly, (iii) both Tar-1 all-l and all-d showed a remarkable ability to inhibit the chemotaxis response of the full-length receptor, in vivo. Peptide binding to the bacteria was confirmed through confocal imaging, and Western blotting confirmed that ToxR Tar-1 chimera protein levels are not affected by the presence of the exogenous peptides. These findings present the first evidence that an all-d TM domain peptide acts in vivo similarly to its parental all-l peptide and suggest that the dimerization of the TM domains is mainly mediated by side-chain interactions, rather than geometrically fitted conformations. In addition, the study provides a new approach for modifying the function of membrane proteins by proteolysis-free peptides.     

Expression, purification, and activities of full-length and truncated versions of the integral membrane protein Vpu from HIV-1

Vpu is an 81-residue accessory protein of HIV-1. Because it is a membrane protein, it presents substantial technical challenges for the characterization of its structure and function, which are of considerable interest because the protein enhances the release of new virus particles from cells infected with HIV-1 and induces the intracellular degradation of the CD4 receptor protein. The Vpu-mediated enhancement of the virus release rate from HIV-1-infected cells is correlated with the expression of an ion channel activity associated with the transmembrane hydrophobic helical domain. Vpu-induced CD4 degradation and, to a lesser extent, enhancement of particle release are both dependent on the phosphorylation of two highly conserved serine residues in the cytoplasmic domain of Vpu. To define the minimal folding units of Vpu and to identify their activities, we prepared three truncated forms of Vpu and compared their structural and functional properties to those of full-length Vpu (residues 2-81). Vpu(2-37) encompasses the N-terminal transmembrane alpha-helix; Vpu(2-51) spans the N-terminal transmembrane helix and the first cytoplasmic alpha-helix; Vpu(28-81) includes the entire cytoplasmic domain containing the two C-terminal amphipathic alpha-helices without the transmembrane helix. Uniformly isotopically labeled samples of the polypeptides derived from Vpu were prepared by expression of fusion proteins in E. coli and were studied in the model membrane environments of lipid micelles by solution NMR spectroscopy and oriented lipid bilayers by solid-state NMR spectroscopy. The assignment of backbone resonances enabled the secondary structure of the constructs corresponding to the transmembrane and the cytoplasmic domains of Vpu to be defined in micelle samples by solution NMR spectroscopy. Solid-state NMR spectra of the polypeptides in oriented lipid bilayers demonstrated that the topology of the domains is retained in the truncated polypeptides. The biological activities of the constructs of Vpu were evaluated. The ion channel activity is confined to the transmembrane alpha-helix. The C-terminal alpha-helices modulate or promote the oligomerization of Vpu in the membrane and stabilize the conductive state of the channel, in addition to their involvement in CD4 degradation.     

Impact of histidine residues on the transmembrane helices of viroporins

Abstract

The role of histidine in channel-forming transmembrane (TM) helices was investigated by comparing the TM helices from Virus protein ‘u' (Vpu) and the M2 proton channel. Both proteins are members of the viroporin family of small membrane proteins that exhibit ion channel activity, and have a single TM helix that is capable of forming oligomers. The TM helices from both proteins have a conserved tryptophan towards the C-terminus. Previously, alanine 18 of Vpu was mutated to histidine in order to artificially introduce the same HXXXW motif that is central to the proton channel activity of M2. Interestingly, the mutated Vpu TM resulted in an increase in helix tilt angle of 11° in lipid bilayers compared to the wild-type Vpu TM. Here, we find the reverse, when histidine 37 of the HXXXW motif in M2 was mutated to alanine, it decreased the helix tilt by 10° from that of wild-type M2. The tilt change is independent of both the helix length and the presence of tryptophan. In addition, compared to wild-type M2, the H37A mutant displayed lowered sensitivity to proton concentration. We also found that the solvent accessibility of histidine-containing M2 is greater than without histidine. This suggests that the TM helix may increase the solvent exposure by changing its tilt angle in order to accommodate a polar/charged residue within the hydrophobic membrane region. The comparative results of M2, Vpu and their mutants demonstrated the significance of histidine in a transmembrane helix and the remarkable plasticity of the function and structure of ion channels stemming from changes at a single amino acid site.     

The two biological activities of human immunodeficiency virus type 1 Vpu protein involve two separable structural domains.

The human immunodeficiency virus type 1 (HIV-1) Vpu protein is an integral membrane phosphoprotein that induces CD4 degradation in the endoplasmic reticulum and enhances virus release from the cell surface. CD4 degradation is specific, requires phosphorylation of Vpu, and involves the interaction between Vpu and the CD4 cytoplasmic domain. In contrast, regulation of virus release is less specific and not restricted to HIV-1 and may be mechanistically-distinct from CD4 degradation. We show here that a mutant of Vpu, Vpu35, lacking most of its cytoplasmic domain has residual biological activity for virus release but is unable to induce CD4 degradation. This finding suggests that the N terminus of Vpu encoding the transmembrane (TM) anchor represents an active domain important for the regulation of virus release but not CD4 degradation. To better define the functions of Vpu's TM anchor and cytoplasmic domain, we designed a mutant, VpuRD, containing a scrambled TM sequence with a conserved amino acid composition and alpha-helical structure. The resulting protein was integrated normally into membranes, was able to form homo-oligomers, and exhibited expression levels, protein stability, and subcellular localization similar to those of wild-type Vpu. Moreover, VpuRD was capable of binding to CD4 and to induce CD4 degradation with wild-type efficiency, confirming proper membrane topology and indicating that the alteration of the Vpu TM domain did not interfere with this function of Vpu. However, VpuRD was unable to enhance the release of virus particles from infected or transfected cells, and virus encoding VpuRD had replication characteristics in T cells indistinguishable from those of a Vpu-deficient HIV-1 isolate. Mutation of the phosphorylation sites in VpuRD resulted in a protein which was unable to perform either function of Vpu. The results of our experiments suggest that the two biological activities of Vpu operate via two distinct molecular mechanisms and involve two different structural domains of the Vpu protein.     

Structural changes in a hydrophobic domain of the prion protein induced by hydration and by ala-->Val and pro-->Leu substitutions

X-ray diffraction was used to study the structure of assemblies formed by synthetic peptide fragments of the prion protein (PrP) that include the hydrophobic domain implicated in the Gerstmann-Str?ussler-Scheinker (GSS) mutation (P102L). The effects of hydration on polypeptide assembly and of Ala-->Val substitutions in the hydrophobic domain were characterized. Synthetic peptides included: (i) Syrian hamster (SHa) hydrophobic core, SHa106-122 (KTNMKHMAGAAAAGAVV); (ii) SHa104-122(3A-V), with A-->V mutations at 113, 115 and 118 (KPKTNMKHMVGVAAVGAVV); (iii) mouse (Mo) wild-type sequence of the N-terminal hydrophobic domain, Mo89-143WT; and (iv) the same mouse sequence with leucine substitution for proline at residue number 101, Mo89-143(P101L). Samples of SHa106-122 that formed assemblies while drying under ambient conditions showed X-ray patterns indicative of 33 A thick slab-like structures having extensive H-bonding and intersheet stacking. By contrast, lyophilized peptide that was equilibrated against 100 % relative humidity showed assemblies with only a few layers of beta-sheets. The Ala-->Val substitutions in SHa104-122 and Mo89-143(P101L) resulted in the formation of 40 A wide, cross-beta fibrils. Observation of similar size beta-sheet fibrils formed by peptides SHa104-122(3A-V) and the longer Mo89-143(P101L) supports the notion that the hydrophobic sequence forms a template or core that promotes the beta-folding of the longer peptide. The substitution of amino acids in the mutants, e.g. 3A-->V and P101L, enhances the folding of the peptide into compact structural units, significantly enhancing the formation of the extensive beta-sheet fibrils.