The Antimalarial Compound Atovaquone Inhibits Zika and Dengue Virus Infection by Blocking E Protein-Mediated Membrane Fusion.

Flaviviruses bear class II fusion proteins as their envelope (E) proteins. Here, we describe the development of an in vitro quantitative mosquito-cell-based membrane-fusion assay for the E protein using dual split proteins (DSPs). The assay does not involve the use of live viruses and allows the analysis of a membrane-fusion step independent of other events in the viral lifecycle, such as endocytosis. The progress of membrane fusion can be monitored continuously by measuring the activities of Renilla luciferase derived from the reassociation of DSPs during cell fusion. We optimized the assay to screen an FDA-approved drug library for a potential membrane fusion inhibitor using the E protein of Zika virus. Screening results identified atovaquone, which was previously described as an antimalarial agent. Atovaquone potently blocked the in vitro Zika virus infection of mammalian cells with an IC90 of 2.1 µM. Furthermore, four distinct serotypes of dengue virus were also inhibited by atovaquone with IC90 values of 1.6-2.5 µM, which is a range below the average blood concentration of atovaquone after its oral administration in humans. These findings make atovaquone a likely candidate drug to treat illnesses caused by Zika as well as dengue viruses. Additionally, the DSP assay is useful to study the mechanism of membrane fusion in Flaviviruses.

The Anticoagulant Nafamostat Potently Inhibits SARS-CoV-2 S Protein-Mediated Fusion in a Cell Fusion Assay System and Viral Infection In Vitro in a Cell-Type-Dependent Manner.

Although infection by SARS-CoV-2, the causative agent of coronavirus pneumonia disease (COVID-19), is spreading rapidly worldwide, no drug has been shown to be sufficiently effective for treating COVID-19. We previously found that nafamostat mesylate, an existing drug used for disseminated intravascular coagulation (DIC), effectively blocked Middle East respiratory syndrome coronavirus (MERS-CoV) S protein-mediated cell fusion by targeting transmembrane serine protease 2 (TMPRSS2), and inhibited MERS-CoV infection of human lung epithelium-derived Calu-3 cells. Here we established a quantitative fusion assay dependent on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S protein, angiotensin I converting enzyme 2 (ACE2) and TMPRSS2, and found that nafamostat mesylate potently inhibited the fusion while camostat mesylate was about 10-fold less active. Furthermore, nafamostat mesylate blocked SARS-CoV-2 infection of Calu-3 cells with an effective concentration (EC)50 around 10 nM, which is below its average blood concentration after intravenous administration through continuous infusion. On the other hand, a significantly higher dose (EC50 around 30 mM) was required for VeroE6/TMPRSS2 cells, where the TMPRSS2-independent but cathepsin-dependent endosomal infection pathway likely predominates. Together, our study shows that nafamostat mesylate potently inhibits SARS-CoV-2 S protein-mediated fusion in a cell fusion assay system and also inhibits SARS-CoV-2 infection in vitro in a cell-type-dependent manner. These findings, together with accumulated clinical data regarding nafamostat's safety, make it a likely candidate drug to treat COVID-19.

Cell-cell and virus-cell fusion assay-based analyses of alanine insertion mutants in the distal α9 portion of the JRFL gp41 subunit from HIV-1.

Membrane fusion is the first essential step in HIV-1 replication. The gp41 subunit of HIV-1 envelope protein (Env), a class I fusion protein, achieves membrane fusion by forming a structure called a six-helix bundle composed of N- and C-terminal heptad repeats. We have recently shown that the distal portion of the α9 helix in the C-terminal heptad repeat of X4-tropic HXB2 Env plays a critical role in the late-stage membrane fusion and viral infection. Here, we used R5-tropic JRFL Env and constructed six alanine insertion mutants, 641+A to 646+A, in the further distal portion of α9 where several glutamine residues are conserved (the number corresponds to the position of the inserted alanine in JRFL Env). 644+A showed the most severe defect in syncytia formation. Decreased fusion pore formation activity, revealed by a dual split protein assay, was observed in all mutants except 641+A. Sequence analysis and substitution of inserted 644A with Gln revealed that the glutamine residue at position 644 that forms complex hydrogen-bond networks with other polar residues on the surface of the six-helix bundle is critical for cell-cell fusion. We also developed a split NanoLuc® (Nluc) reporter-based assay specific to the virus-cell membrane fusion step to analyze several of the mutants. Interestingly syncytia-competent mutants failed to display Nluc activities. In addition to defective fusion activity, a reduction of Env incorporation into virions may further contribute to differences in cell-cell and virus-cell fusions.

Six-helix bundle completion in the distal C-terminal heptad repeat region of gp41 is required for efficient human immunodeficiency virus type 1 infection.

BACKGROUND: The native pre-fusion structure of gp120/gp41 complex of human immunodeficiency virus type 1 was recently revealed. In the model, the helices of gp41 (α6, α7, α8, and α9) form a four-helix collar underneath trimeric gp120. Gp41 is a class I fusion protein and mediates membrane fusion by forming a post-fusion structure called the six-helix bundle (6HB). The comparison of the pre- and post-fusion structures revealed the large conformational changes in gp41 during the antiparallel packing of the N- and C-terminal heptad repeats (NHRs and CHRs) in membrane fusion. Several mutagenesis studies of gp41 performed in the past were interpreted based on 6HB, the only available structure at that time. To obtain an insight about the current pre-fusion structural model and conformational changes during membrane fusion, alanine insertion mutagenesis of the NHR, CHR and connecting loop regions of HXB2 gp41 was performed. The effects of mutations on biosynthesis and membrane fusion were analyzed by immunoblotting and fusion assays, respectively. The extent of membrane fusion was evaluated by split luciferase-based pore formation and syncytia formation assays, respectively. RESULTS: Consistent with the current structural model, drastic negative effects of mutations on biosynthesis and membrane fusion were observed for NHR, loop, and proximal regions of CHR (up to amino acid position 643). The insertions in α9 after it leaves the four-helix collar were tolerable for biosynthesis. These CHR mutants showed varying effects on membrane fusion. Insertion at position 644 or 645 resulted in poor pore and syncytia formation. Efficient pore and syncytia formation almost similar to that of the wild type was observed for insertion at position 647, 648 or 649. However, recovery of virus infectivity was only observed for the insertions beyond position 648. CONCLUSIONS: The mutagenesis data for HXB2 gp41 is in agreement with the recent pre-fusion structure model. The virus infection data suggested that fusion pores sufficiently large enough for the release of the virus genome complex are formed after the completion of 6HB beyond position 648.

Identification of Nafamostat as a Potent Inhibitor of Middle East Respiratory Syndrome Coronavirus S Protein-Mediated Membrane Fusion Using the Split-Protein-Based Cell-Cell Fusion Assay.

Middle East respiratory syndrome (MERS) is an emerging infectious disease associated with a relatively high mortality rate of approximately 40%. MERS is caused by MERS coronavirus (MERS-CoV) infection, and no specific drugs or vaccines are currently available to prevent MERS-CoV infection. MERS-CoV is an enveloped virus, and its envelope protein (S protein) mediates membrane fusion at the plasma membrane or endosomal membrane. Multiple proteolysis by host proteases, such as furin, transmembrane protease serine 2 (TMPRSS2), and cathepsins, causes the S protein to become fusion competent. TMPRSS2, which is localized to the plasma membrane, is a serine protease responsible for the proteolysis of S in the post-receptor-binding stage. Here, we developed a cell-based fusion assay for S in a TMPRSS2-dependent manner using cell lines expressing Renilla luciferase (RL)-based split reporter proteins. S was stably expressed in the effector cells, and the corresponding receptor for S, CD26, was stably coexpressed with TMPRSS2 in the target cells. Membrane fusion between these effector and target cells was quantitatively measured by determining the RL activity. The assay was optimized for a 384-well format, and nafamostat, a serine protease inhibitor, was identified as a potent inhibitor of S-mediated membrane fusion in a screening of about 1,000 drugs approved for use by the U.S. Food and Drug Administration. Nafamostat also blocked MERS-CoV infection in vitro Our assay has the potential to facilitate the discovery of new inhibitors of membrane fusion of MERS-CoV as well as other viruses that rely on the activity of TMPRSS2.

Dual Split Protein (DSP) Assay to Monitor Cell-Cell Membrane Fusion.

Fusion between viral and cellular membranes is the essential first step in infection of enveloped viruses. This step is mediated by viral envelope glycoproteins (Env) that recognize cellular receptors. The membrane fusion between the effector cells expressing viral Env and the target cells expressing its receptors can be monitored by several methods. We have recently developed a pair of chimeric reporter protein composed of split Renilla luciferase (RL) and split GFP. We named this reporter dual split protein (DSP), since it recovers both RL and GFP activities upon self reassociation. By using DSP, pore formation and content mixing between the effector and target cells can be monitored upon the recovery of RL and GFP activities after the membrane fusion. This quick assay provides quantitative as well as spatial information about membrane fusion mediated by viral Env.

Using a split luciferase assay (SLA) to measure the kinetics of cell-cell fusion mediated by herpes simplex virus glycoproteins.

Herpes simplex virus (HSV) entry and cell-cell fusion require the envelope proteins gD, gH/gL and gB. We propose that receptor-activated conformational changes to gD activate gH/gL, which then triggers gB (the fusogen) into an active form. To study this dynamic process, we have adapted a dual split protein assay originally developed to study the kinetics of human immunodeficiency virus (HIV) mediated fusion. This assay uses a chimera of split forms of renilla luciferase (RL) and green fluorescent protein (GFP). Effector cells are co-transfected with the glycoproteins and one of the split reporters. Receptor-bearing target cells are transfected with the second reporter. Co-culture results in fusion and restoration of RL, which can convert a membrane permeable substrate into a luminescent product, thereby enabling one to monitor initiation and extent of fusion in live cells in real time. Restoration of GFP can also be studied by fluorescence microscopy. Two sets of split reporters have been developed: the original one allows one to measure fusion kinetics over hours whereas the more recent version was designed to enhance the sensitivity of RL activity allowing one to monitor both initiation and rates of fusion in minutes. Here, we provide a detailed, step-by-step protocol for the optimization of the assay (which we call the SLA for split luciferase assay) using the HSV system. We also show several examples of the power of this assay to examine both the initiation and kinetics of cell-cell fusion by wild type forms of gD, gB, gH/gL of both serotypes of HSV as well as the effect of mutations and antibodies that alter the kinetics of fusion. The SLA can be applied to other viral systems that carry out membrane fusion.

Recent advance in the structural analysis of HIV-1 envelope protein.

Human immunodeficiency virus type I (HIV-1), a causative agent of AIDS, is affecting today more than 35 millions of people worldwide. The advance of anti-HIV chemotherapy has made AIDS a chronic non-fatal disease in resourceful countries. Long-awaited anti-HIV-1 vaccine is still not with us yet; however, great progress in structural analyses of the envelope protein of HIV-1 in recent years starts to shed light on rational intervention targeted at the envelope protein, as will be reviewed in this article.

The V4 and V5 Variable Loops of HIV-1 Envelope Glycoprotein Are Tolerant to Insertion of Green Fluorescent Protein and Are Useful Targets for Labeling.

The mature human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) comprises the non-covalently associated gp120 and gp41 subunits generated from the gp160 precursor. Recent structural analyses have provided quaternary structural models for gp120/gp41 trimers, including the variable loops (V1-V5) of gp120. In these models, the V3 loop is located under V1/V2 at the apical center of the Env trimer, and the V4 and V5 loops project outward from the trimeric protomers. In addition, the V4 and V5 loops are predicted to have less movement upon receptor binding during membrane fusion events. We performed insertional mutagenesis using a GFP variant, GFPOPT, placed into the variable loops of HXB2 gp120. This allowed us to evaluate the current structural models and to simultaneously generate a GFP-tagged HIV-1 Env, which was useful for image analyses. All GFP-inserted mutants showed similar levels of whole-cell expression, although certain mutants, particularly V3 mutants, showed lower levels of cell surface expression. Functional evaluation of their fusogenicities in cell-cell and virus-like particle-cell fusion assays revealed that V3 was the most sensitive to the insertion and that the V1/V2 loops were less sensitive than V3. The V4 and V5 loops were the most tolerant to insertion, and certain tag proteins other than GFPOPT could also be inserted without functional consequences. Our results support the current structural models and provide a GFPOPT-tagged Env construct for imaging studies.

Co-expression of foreign proteins tethered to HIV-1 envelope glycoprotein on the cell surface by introducing an intervening second membrane-spanning domain.

The envelope glycoprotein (Env) of human immunodeficiency virus type I (HIV-1) mediates membrane fusion. To analyze the mechanism of HIV-1 Env-mediated membrane fusion, it is desirable to determine the expression level of Env on the cell surface. However, the quantification of Env by immunological staining is often hampered by the diversity of HIV-1 Env and limited availability of universal antibodies that recognize different Envs with equal efficiency. To overcome this problem, here we linked a tag protein called HaloTag at the C-terminus of HIV-1 Env. To relocate HaloTag to the cell surface, we introduced a second membrane-spanning domain (MSD) between Env and HaloTag. The MSD of transmembrane protease serine 11D, a type II transmembrane protein, successfully relocated HaloTag to the cell surface. The surface level of Env can be estimated indirectly by staining HaloTag with a specific membrane-impermeable fluorescent ligand. This tagging did not compromise the fusogenicity of Env drastically. Furthermore, fusogenicity of Env was preserved even after the labeling with the ligands. We have also found that an additional foreign peptide or protein such as C34 or neutralizing single-chain variable fragment (scFv) can be linked to the C-terminus of the HaloTag protein. Using these constructs, we were able to determine the required length of C34 and critical residues of neutralizing scFv for blocking membrane fusion, respectively.

Development of a rapid cell-fusion-based phenotypic HIV-1 tropism assay.

INTRODUCTION: A dual split reporter protein system (DSP), recombining Renilla luciferase (RL) and green fluorescent protein (GFP) split into two different constructs (DSP1-7 and DSP8-11), was adapted to create a novel rapid phenotypic tropism assay (PTA) for HIV-1 infection (DSP-Pheno). METHODS: DSP1-7 was stably expressed in the glioma-derived NP-2 cell lines, which expressed CD4/CXCR4 (N4X4) or CD4/CCR5 (N4R5), respectively. An expression vector with DSP8-11 (pRE11) was constructed. The HIV-1 envelope genes were subcloned in pRE11 (pRE11-env) and transfected into 293FT cells. Transfected 293FT cells were incubated with the indicator cell lines independently. In developing the assay, we selected the DSP1-7-positive clones that showed the highest GFP activity after complementation with DSP8-11. These cell lines, designated N4R5-DSP1-7, N4X4-DSP1-7 were used for subsequent assays. RESULTS: The env gene from the reference strains (BaL for R5 virus, NL4-3 for X4 virus, SF2 for dual tropic virus) subcloned in pRE11 and tested, was concordant with the expected co-receptor usage. Assay results were available in two ways (RL or GFP). The assay sensitivity by RL activity was comparable with those of the published phenotypic assays using pseudovirus. The shortest turnaround time was 5 days after obtaining the patient's plasma. All clinical samples gave positive RL signals on R5 indicator cells in the fusion assay. Median RLU value of the low CD4 group was significantly higher on X4 indicator cells and suggested the presence of more dual or X4 tropic viruses in this group of patients. Comparison of representative samples with Geno2Pheno [co-receptor] assay was concordant. CONCLUSIONS: A new cell-fusion-based, high-throughput PTA for HIV-1, which would be suitable for in-house studies, was developed. Equipped with two-way reporter system, RL and GFP, DSP-Pheno is a sensitive test with short turnaround time. Although maintenance of cell lines and laboratory equipment is necessary, it provides a safe assay system without infectious viruses. With further validation against other conventional analyses, DSP-Pheno may prove to be a useful laboratory tool. The assay may be useful especially for the research on non-B subtype HIV-1 whose co-receptor usage has not been studied much.

Dual split protein-based fusion assay reveals that mutations to herpes simplex virus (HSV) glycoprotein gB alter the kinetics of cell-cell fusion induced by HSV entry glycoproteins.

Herpes simplex virus (HSV) entry and cell-cell fusion require glycoproteins gD, gH/gL, and gB. We propose that receptor-activated changes to gD cause it to activate gH/gL, which then triggers gB into an active form. We employed a dual split-protein (DSP) assay to monitor the kinetics of HSV glycoprotein-induced cell-cell fusion. This assay measures content mixing between two cells, i.e., fusion, within the same cell population in real time (minutes to hours). Titration experiments suggest that both gD and gH/gL act in a catalytic fashion to trigger gB. In fact, fusion rates are governed by the amount of gB on the cell surface. We then used the DSP assay to focus on mutants in two functional regions (FRs) of gB, FR1 and FR3. FR1 contains the fusion loops (FL1 and FL2), and FR3 encompasses the crown at the trimer top. All FL mutants initiated fusion very slowly, if at all. However, the fusion rates caused by some FL2 mutants increased over time, so that total fusion by 8 h looked much like that of the WT. Two distinct kinetic patterns, "slow and fast," emerged for mutants in the crown of gB (FR3), again showing differences in initiation and ongoing fusion. Of note are the fusion kinetics of the gB syn mutant (LL871/872AA). Although this mutant was originally included as an ongoing high-rate-of-fusion control, its initiation of fusion is so rapid that it appears to be on a "hair trigger." Thus, the DSP assay affords a unique way to examine the dynamics of HSV glycoprotein-induced cell fusion.

[Joint collaboration in infectious diseases in China, the University of Tokyo].

Recent rapid developments in Asian and African countries bring an opportunity of cross-species transmission of pathogens through unprecedented contacts between people and wild animals. Furthermore, increase of global exchanges of people and products facilitates a rapid spread of infectious diseases worldwide. China has an enormous population with diverse ethnic groups within its wide territory; furthermore, it is experiencing very rapid urbanization. These conditions make China a potential epicenter of emerging infectious diseases. One good example is the SARS incidence in 2003. Therefore, it is essential to include China in a network of research groups of infectious diseases. Here we summarize the ongoing collaborations between the Institute of Medical Science, the University of Tokyo, and its Chinese counterparts.

Generation of a dual-functional split-reporter protein for monitoring membrane fusion using self-associating split GFP.

Split reporter proteins capable of self-association and reactivation have applications in biomedical research, but designing these proteins, especially the selection of appropriate split points, has been somewhat arbitrary. We describe a new methodology to facilitate generating split proteins using split GFP as a self-association module. We first inserted the entire GFP module at one of several candidate split points in the protein of interest, and chose clones that retained the GFP signal and high activity relative to the original protein. Once such chimeric clones were identified, a final pair of split proteins was generated by splitting the GFP-inserted chimera within the GFP domain. Applying this strategy to Renilla reniformis luciferase, we identified a new split point that gave 10 times more activity than the previous split point. The process of membrane fusion was monitored with high sensitivity using a new pair of split reporter proteins. We also successfully identified new split points for HaloTag protein and firefly luciferase, generating pairs of self-associating split proteins that recovered the functions of both GFP and the original protein. This simple method of screening will facilitate the designing of split proteins that are capable of self-association through the split GFP domains.

Dynamic appearance of antigenic epitopes effective for viral neutralization during membrane fusion initiated by interactions between HIV-1 envelope proteins and CD4/CXCR4.

HIV-1 entry into cells is mediated by interactions between the envelope (Env) gp120 and gp41 proteins with CD4 and chemokine receptors via an intermediate called the viral fusion complex (vFC). Here, mAbs were used to find the dynamic changes in expression of antigenic epitopes during vFC formation. A CD4-specific mAb (R275) and anti-vFC mAbs, designated F12-1, F13-6 and F18-4 that recognize the epitopes only appeared by the co-culture of env-transfected 293FT and CD4-transfected 293 cells, were developed by immunizing ganp-gene transgenic mice with an vFC-like structure formed by the same co-culture. The epitopes recognized by the mAbs appeared at different time points during vFC formation: F18-4 appeared first, followed by F13-6, and finally F12-1. The anti-vFC mAbs had little effect on vFC formation or virus neutralization; however, interestingly F12-1 and F18-4 increased exposure of the OKT4-epitope on the domain 3 in the extracellular region of CD4. R275, which recognizes the epitope closely associated with the OKT4-determinant on the domain 3, showed the marked inhibition of vFC formation and viral neutralization activity. The Ab binding to the epitopes appeared during viral membrane fusion might reinforce the appearance of the target epitopes for effective neutralization activity.

Conserved arginine residue in the membrane-spanning domain of HIV-1 gp41 is required for efficient membrane fusion.

Despite the high mutation rate of HIV-1, the amino acid sequences of the membrane-spanning domain (MSD) of HIV-1 gp41 are well conserved. Arginine residues are rarely found in single membrane-spanning domains, yet an arginine residue, R(696) (the numbering is based on that of HXB2), is highly conserved in HIV-1 gp41. To examine the role of R(696), it was mutated to K, A, I, L, D, E, N, and Q. Most of these substitutions did not affect the expression, processing or surface distribution of the envelope protein (Env). However, a syncytia formation assay showed that the substitution of R(696) with amino acid residues other than K, a naturally observed mutation in the gp41 MSD, decreased fusion activity. Substitution with hydrophobic amino acid residues (A, I, and L) resulted in a modest decrease, while substitution with D or E, potentially negatively-charged residues, almost abolished the syncytia formation. All the fusion-defective mutants showed slower kinetics with the cell-based dual split protein (DSP) assay that scores the degree of membrane fusion based on pore formation between fusing cells. Interestingly, the D and E substitutions did show some fusion activity in the DSP assays, suggesting that proteins containing D or E substitutions retained some fusion pore-forming capability. However, nascent pores failed to develop, due probably to impaired activity in the pore enlargement process. Our data show the importance of this conserved arginine residue for efficient membrane fusion.

Monitoring viral-mediated membrane fusion using fluorescent reporter methods.

A simple and real-time cell-based assay of membrane fusion employing a pair of engineered novel reporter proteins is described. The reporter proteins are chimeras of split Renilla luciferase (RL) and split green fluorescent protein (GFP). This reporter allows us to perform both quantitative (RL mode) and visible (GFP mode) membrane fusion assays in live cells. The kinetic assay enabled by this method helps understand the mechanism of membrane fusion mediated by a viral envelope protein. This assay system is also suitable for the screening of potential inhibitors. The timing of inhibition by a particular inhibitor can be analyzed by time-dependent addition of the inhibitor. Although this unit demonstrates the application of the method for the analysis of HIV-1 envelope protein, the reporter can be applied to analyses of various other viral envelope proteins.

Interferon-gamma negatively regulates Th17-mediated immunopathology during mouse hepatitis virus infection.

Fulminant hepatitis can cause acute liver failure and death in both humans and mice. However, the cellular and molecular mechanisms underlying the acute disease are still not well understood. Here, we examine the role of Th17 response in the development of the acute hepatitis following infection with mouse hepatitis virus (MHV). We show that IL-17 levels in serum are rapidly elevated and positively correlated to liver damage and death of the mice. In IFN-γR(-/-) mice, Th17 response is enhanced and the elevated IL-17 production contributes to severe liver damage as well as detrimental inflammation because neutralization of IL-17 effectively suppresses inflammation and protects mice from liver injury. We further show that IFN-γ facilitates antigen-induced apoptosis of Th17 cells and adoptive transferred IFN-γR(-/-), but not IFN-γR(+/+); CD4(+) T cells promotes an enhanced liver damage in wild-type mice. The results demonstrate an essential role of Th17 cells in MHV-induced immunopathology and the importance of IFN-γ in maintaining immune balance between Th1 and Th17 responses during acute viral infection.

Membrane topology analysis of HIV-1 envelope glycoprotein gp41.

BACKGROUND: The gp41 subunit of the HIV-1 envelope glycoprotein (Env) has been widely regarded as a type I transmembrane protein with a single membrane-spanning domain (MSD). An alternative topology model suggested multiple MSDs. The major discrepancy between the two models is that the cytoplasmic Kennedy sequence in the single MSD model is assigned as the extracellular loop accessible to neutralizing antibodies in the other model. We examined the membrane topology of the gp41 subunit in both prokaryotic and mammalian systems. We attached topological markers to the C-termini of serially truncated gp41. In the prokaryotic system, we utilized a green fluorescent protein (GFP) that is only active in the cytoplasm. The tag protein (HaloTag) and a membrane-impermeable ligand specific to HaloTag was used in the mammalian system. RESULTS: In the absence of membrane fusion, both the prokaryotic and mammalian systems (293FT cells) supported the single MSD model. In the presence of membrane fusion in mammalian cells (293CD4 cells), the data obtained seem to support the multiple MSD model. However, the region predicted to be a potential MSD is the highly hydrophilic Kennedy sequence and is least likely to become a MSD based on several algorithms. Further analysis revealed the induction of membrane permeability during membrane fusion, allowing the membrane-impermeable ligand and antibodies to cross the membrane. Therefore, we cannot completely rule out the possible artifacts. Addition of membrane fusion inhibitors or alterations of the MSD sequence decreased the induction of membrane permeability. CONCLUSIONS: It is likely that a single MSD model for HIV-1 gp41 holds true even in the presence of membrane fusion. The degree of the augmentation of membrane permeability we observed was dependent on the membrane fusion and sequence of the MSD.

The membrane-spanning domain of gp41 plays a critical role in intracellular trafficking of the HIV envelope protein.

BACKGROUND: The sequences of membrane-spanning domains (MSDs) on the gp41 subunit are highly conserved among many isolates of HIV-1. The GXXXG motif, a potential helix-helix interaction motif, and an arginine residue (rare in hydrophobic MSDs) are especially well conserved. These two conserved elements are expected to locate on the opposite sides of the MSD, if the MSD takes a α-helical secondary structure. A scanning alanine-insertion mutagenesis was performed to elucidate the structure-function relationship of gp41 MSD. RESULTS: A circular dichroism analysis of a synthetic gp41 MSD peptide determined that the secondary structure of the gp41 MSD was α-helical. We then performed a scanning alanine-insertion mutagenesis of the entire gp41 MSD, progressively shifting the relative positions of MSD segments around the helix axis. Altering the position of Gly694, the last residue of the GXXXG motif, relative to Arg696 (the number indicates the position of the amino acid residues in HXB2 Env) around the axis resulted in defective fusion. These mutants showed impaired processing of the gp160 precursor into gp120 and gp41. Furthermore, these Env mutants manifested inefficient intracellular transport in the endoplasmic reticulum and Golgi regions. Indeed, a transplantation of the gp41 MSD portion into the transmembrane domain of another membrane protein, Tac, altered its intracellular distribution. Our data suggest that the intact MSD α-helix is critical in the intracellular trafficking of HIV-1 Env. CONCLUSIONS: The relative position between the highly conserved GXXXG motif and an arginine residue around the gp41 MSD α-helix is critical for intracellular trafficking of HIV-1 Env. The gp41 MSD region not only modulates membrane fusion but also controls biosynthesis of HIV-1 Env.