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• W2019469658 abstract "Article15 February 1997free access The membrane-proximal intracytoplasmic tyrosine residue of HIV-1 envelope glycoprotein is critical for basolateral targeting of viral budding in MDCK cells Robert Lodge Robert Lodge Département de Microbiologie et Immunologie Montréal (Québec), H3C 3J7 Canada Search for more papers by this author Jean-Philippe Lalonde Jean-Philippe Lalonde Département de Microbiologie et Immunologie Montréal (Québec), H3C 3J7 Canada Search for more papers by this author Guy Lemay Guy Lemay Département de Microbiologie et Immunologie Montréal (Québec), H3C 3J7 Canada Groupe de Recherche en Transport Membranaire, Université de Montréal, Montréal (Québec), H3C 3J7 Canada Search for more papers by this author Éric A. Cohen Corresponding Author Éric A. Cohen Département de Microbiologie et Immunologie Montréal (Québec), H3C 3J7 Canada Search for more papers by this author Robert Lodge Robert Lodge Département de Microbiologie et Immunologie Montréal (Québec), H3C 3J7 Canada Search for more papers by this author Jean-Philippe Lalonde Jean-Philippe Lalonde Département de Microbiologie et Immunologie Montréal (Québec), H3C 3J7 Canada Search for more papers by this author Guy Lemay Guy Lemay Département de Microbiologie et Immunologie Montréal (Québec), H3C 3J7 Canada Groupe de Recherche en Transport Membranaire, Université de Montréal, Montréal (Québec), H3C 3J7 Canada Search for more papers by this author Éric A. Cohen Corresponding Author Éric A. Cohen Département de Microbiologie et Immunologie Montréal (Québec), H3C 3J7 Canada Search for more papers by this author Author Information Robert Lodge1, Jean-Philippe Lalonde1, Guy Lemay1,2 and Éric A. Cohen 1 1Département de Microbiologie et Immunologie Montréal (Québec), H3C 3J7 Canada 2Groupe de Recherche en Transport Membranaire, Université de Montréal, Montréal (Québec), H3C 3J7 Canada The EMBO Journal (1997)16:695-705https://doi.org/10.1093/emboj/16.4.695 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Budding of retroviruses from polarized epithelial Madin–Darby canine kidney cells (MDCK) takes place specifically at the basolateral membrane surface. This sorting event is suspected to require a specific signal harbored by the viral envelope glycoprotein and it was previously shown that, as for most basolateral proteins, the intracytoplasmic domain plays a crucial role in this targeting phenomenon. It is well known that tyrosine-based motifs are a central element in basolateral targeting signals. In the present study, site-directed mutagenesis was used to generate conservative or non-conservative substitutions of each four intracytoplasmic tyrosines of the human immunodeficiency virus (HIV-1) envelope glycoprotein. This approach revealed that the membrane-proximal tyrosine is essential to ensure both the basolateral localization of envelope glycoprotein and the basolateral targeting of HIV-1 virions. Substitutions of the membrane-proximal tyrosine did not appear to affect incorporation of envelope glycoprotein into the virions, as assayed by virion infectivity and protein content, nor its capability to ensure its role in viral infection, as determined by viral multiplication kinetics. Altogether, these results indicate that the intracytoplasmic domain of the HIV-1 envelope glycoprotein harbors a unique, tyrosine-based, basolateral targeting signal. Such a tyrosine-based targeting signal may play a fundamental role in HIV transmission and pathogenesis. Introduction Maintaining the integrity of cellular architecture, through an asymmetrical distribution of surface and intracellular proteins, is essential to ensure that different cell types assume their role in tissue organization. Perhaps the best example of a specific subcellular distribution of plasma membrane proteins is observed at the surface of polarized epithelial cells (Simons and Fuller, 1985; Compans and Srinivas, 1991; Mellman et al., 1993; Matter and Mellman, 1994). These cells are best known as essential building blocks of the intestinal wall lining, kidney tubules and various mucosal surfaces. In epithelial cells, two plasma membrane domains are clearly differentiated and exhibit a distinct lipid and protein composition (Sargiacomo et al., 1989; van Meer, 1993). The apical domain, often abundant with microvilli, faces the lumen of the organ; its surface harbors proteases and glycolytic enzymes, and houses most of the proteins involved in specialized functions. The basolateral domain is found below the tight junctions that link cells together to form a tight epithelium; in mucosal surfaces, this membrane is thus oriented toward the underlying extracellular matrix, cells or blood flow. The basolateral membrane mostly harbors proteins associated with cellular adsorption and housekeeping functions common to all cells. The generation of different plasma membrane domains with the specific distribution of their proteins involves the sorting of these different proteins at the exit of the Golgi apparatus (Simons and Wandinger-Ness, 1990; Rodriguez-Boulan and Zurzolo, 1993). The exact nature of the hypothetical sorting signals still remains largely unknown. However, accumulating evidence points to the existence of distinct targeting signals on proteins destined to either the apical or basolateral membrane domain; in the absence of such signals, a roughly uniform distribution of the protein is generally observed at the cell surface (Wandinger-Ness et al., 1990). In the last few years, tyrosine-based signals, especially in Y-X-X-aliphatic/aromatic consensus motifs, have been found in the intracytoplasmic domain of basolateral membrane proteins and associated with targeted protein delivery (Hunziker et al., 1991; Matter et al., 1992; Dagermont et al., 1993; Thomas et al., 1993; Thomas and Roth, 1994; Höning and Hunziker, 1995; Ohno et al., 1995). Interestingly, these tyrosine-based signals have also been found to be used as endocytosis signals (Hopkins, 1992; Prill et al., 1993; Trowbridge et al., 1993; Pytowski et al., 1995). Viral envelope glycoproteins are synthesized and transported by the normal cellular machinery, and it has long been recognized that they are good representative models for the study of intracellular protein transport pathways (Rodriguez-Boulan and Sabatini, 1978; Rodriguez-Boulan, 1983; Rodriguez-Boulan et al., 1983; Tucker and Compans, 1993). Accordingly, different viruses have evolved mechanisms for the specific delivery of their envelope glycoproteins to one or the other membrane domains of polarized epithelial cells. Classical examples include the vesicular stomatitis virus G glycoprotein that harbors a critical intracytoplasmic tyrosine residue required for polarized basolateral targeting (Thomas et al., 1993; Thomas and Roth, 1994). Likewise, addition of a tyrosine residue to the intracytoplasmic domain of the normally apical hemagglutinin envelope glycoprotein of influenza virus redirects this protein to the basolateral surface (Brewer and Roth, 1991). The targeting of viral envelope glycoproteins has important consequences in viral maturation and release; although capsid proteins generally lack membrane domain targeting signals themselves, viral budding resulting in release of enveloped viruses specifically occurs through the membrane domain harboring an adequate envelope glycoprotein (Tucker and Compans, 1993). In the case of human immunodeficiency virus (HIV-1), targeted viral release was shown to occur mainly through the basolateral membrane surface (Owens and Compans, 1989; Fantini et al., 1991; Owens et al., 1991). A specific basolateral release has also been previously suggested for another retrovirus, namely murine leukemia virus (Roth et al., 1983; Kilpatrick et al., 1988). More recently, we showed that targeting of HIV budding requires the intracytoplasmic portion of the envelope glycoprotein transmembrane moiety, gp41. Large carboxy-terminal deletions of this intracytoplasmic region severely impair polarized release of the virus, whereas smaller deletions have little or no effect (Gabuzda et al., 1992b; Lodge et al., 1994). The actual incorporation of the glycoproteins into the budding virions was also found to be essential, since alterations of the matrix protein precluding their incorporation into the viral particles also prevented targeting of viral budding (Dorfman et al., 1994; Lodge et al., 1994). In the present study, we performed site-directed mutagenesis, individually substituting each of the intracytoplasmic tyrosine residues of the envelope glycoprotein of HIV-1. Non-conservative substitutions of the membrane-proximal tyrosine residue abolished basolateral targeting of both envelope glycoproteins and viral budding, but had no apparent effect on in vitro infectious potential of the resulting viruses. Such tyrosine-based basolateral signals may play a fundamental role in efficient urogenital transmission and pathogenesis of lentiviruses (Phillips and Bourinbaiar, 1992; Phillips et al., 1994), promoting viral dissemination in the host and access to various sensitive cell types. Results Rationale for mutagenesis Several viral and cellular membrane proteins have been shown to harbor tyrosine-based basolateral targeting determinants in their intracytoplasmic domains (Hunziker et al., 1991; Matter et al., 1992; Thomas et al., 1993; Thomas and Roth, 1994). The HIV-1 gp41 transmembrane glycoprotein harbors four tyrosine residues in its 150 amino acid-long intracytoplasmic region, as shown in Figure 1A. These four residues are highly conserved between HIV-1 isolates; of the four, residues 712 and 768 are found in a Y-X-X-L context. Interestingly, different retroviral envelope glycoproteins also harbor one or more intracytoplasmic tyrosine residues in a similar context; and this is despite the fact that other retroviruses possess much shorter intracytoplasmic domains compared with HIV-1 (Figure 1B). We thus undertook this site-directed mutagenesis study in order to clarify the possible occurrence of a tyrosine-based basolateral targeting signal in HIV-1 envelope glycoproteins. Figure 1.(A) Conservation of tyrosine residues in the intracytoplasmic domain of various HIV or SIV Env glycoprotein isolates. Sequences surrounding the tyrosines are shown; gaps between the sequences are represented by dots. Predicted protein secondary structures are also included and amino acids are numbered from the first Met of gp160 (HXBc2). Single amino acid deletions are represented by a dash. (B) Positions of intracytoplasmic tyrosines in various retrovirus transmembrane (TM) glycoproteins. For comparative purposes, some known viral and cellular tyrosine-based basolateral signals are also included. Download figure Download PowerPoint Substitution mutagenesis of the HIV envelope intracytoplasmic tyrosines We used a technique of polymerase chain reaction (PCR) for the mutagenesis of an HIV-1 envelope gp160 plasmid expression vector as described in Materials and methods. Using this technique, each tyrosine was substituted for a serine; such a substitution maintains the presence of an hydroxyl side chain at this position, but eliminates the aromatic ring and should thus be considered as a non-conservative substitution. The plasmid expression vectors encoding wild-type or mutant envelope glycoproteins were then introduced into polarized monolayers of MDCK cells in order to complement an envelope-defective proviral DNA as described in Materials and methods. This trans-complementation assay, performed by lipofection of confluent cells forming a tight monolayer on semi-permeable filters, was described previously (Lodge et al., 1994). Released viral particles were then recovered in the upper chamber medium bathing the apical membrane or lower chamber medium bathing the basolateral membrane attached onto the semi-permeable filter. Recovered viral particles were then quantitated by a sensitive immunodetection method using an ELISA directed against the p24 viral capsid protein. Substitution of the membrane-proximal tyrosine (Y712S) completely abolished the polarized basolateral release. The ratio of apically to basolaterally released virions was similar to the levels obtained with envelope-negative viruses. In contrast, substitution of either of the three other carboxy-terminal tyrosines (Y768S, Y795S, Y802S) maintained viral release exclusively basolateral (Figure 2A). Figure 2.(A). Effect of tyrosine-to-serine mutations in the gp41 intracytoplasmic domain on polarized virus release. The env-negative HXBH10 proviral construct was co-transfected in equimolar amounts with plasmids encoding HIV wild-type (WT) or mutant (Y712S, Y768S, Y795S, Y802S) gp160 as described in the text. Apical and basolateral supernatants were harvested 48 h after lipofection, and p24 ELISA were performed as described in Materials and methods. (B) Effect of various substitutions (Y712A, Y712F) of the membrane-proximal gp41 intracytoplasmic tyrosine or a nearby proline (P714A). Viral release was detected as in (A). The mock results were obtained following introduction of env-negative proviral plasmid DNA alone and all results are presented as the average of separate values obtained from two separate lipofections plus or minus SD. Download figure Download PowerPoint Further analysis of the tyrosine-dependent basolateral viral budding signal To establish further the importance of tyrosine 712, we examined the effect of another non-conservative substitution to alanine (Y712A) as well as a conservative substitution for the aromatic amino acid, phenylalanine (Y712F). Furthermore, a proline residue found close to the tyrosine and well conserved among HIV viral isolates was changed for an alanine (P714A). This amino acid was chosen as a target for site-directed mutagenesis since it is known to promote the formation of β-turns and such a structure is suspected to play an important role as part of tyrosine-based targeting signals (Collawn et al., 1990; Hopkins, 1992; Prill et al., 1993; Trowbridge et al., 1993; Pytowski et al., 1995). When co-transfected in MDCK cells with the envelope-negative HIV proviral construct, the expression vector encoding the non-conservative Y712A substitution mutant did not rescue polarized budding of the virus. A significant amount of virus could also be detected in the apical-bathing medium of cells encoding the conservative Y712F mutant; however, viral release at the basolateral surface was still favored and it thus seems that this mutation does not completely eliminate the polarization signal (Figure 2B). Alanine substitution of the proline 714 residue abolished basolateral targeting and similar amounts of virus were released in both apical- and basolateral-bathing media. Taken together, these results indicate a critical role for an aromatic amino acid, preferably a tyrosine, in a membrane-proximal position and also suggest a role for a local secondary structure, namely a putative β-turn, in the targeting signal. Plasma membrane distribution of the mutant envelope glycoprotein The membrane targeting of envelope glycoproteins, rather than the polarized budding of viruses, was examined to establish further the importance of the membrane-proximal intracytoplasmic tyrosine in basolateral targeting of Env. Since the lipofection procedure on confluent MDCK cells was not sufficiently efficient to detect envelope protein expression per se, an alternate method was devised using virions pseudotyped with the vesicular stomatitis virus G envelope glycoprotein (VSV-G), since VSV is able to infect MDCK cells. Viruses having incorporated a mosaic of HIV-Env and VSV-G glycoproteins, or representing a VSV-G-containing and HIV-Env-containing heterogeneous viral population, were generated in transfected COS cells as described in Materials and methods. Immunoprecipitation of labeled pseudotyped viral particles with both a rabbit anti-VSV serum and a HIV-positive human serum clearly show the presence of gp120 and VSV-G glycoproteins (Figure 3A, lane 4). VSV-G-mediated infection allowed the transduction of wild-type or mutant HIV viral genomes by infection of MDCK cells with the pseudotyped viruses, allowing HIV-Env expression in a large amount of cells. Radiolabeling and surface immunoprecipitation were then performed by applying a monoclonal anti-gp120 antibody to either the apical or basolateral membrane compartments of filter-grown cells. The results of this experiment, shown in Figure 3B, confirmed that the wild-type glycoprotein is restricted to the basolateral surface, where virus budding occurs (compare lanes 5 and 8). Similarly, the presence of the Y712S mutant glycoprotein at both membrane surfaces (lanes 6 and 9) correlates with equivalent viral release from both plasma membrane domains observed with this mutant. Immunoprecipitation of intracellular proteins with the same antibodies clearly shows that expression of wild-type Env and Y712S mutant glycoproteins was equivalent (lanes 2 and 3). Figure 3.(A) Incorporation of VSV-G glycoprotein in HIV virions. 106 COS cells were transfected with 10 μg of either proviral construct with or without 10 μg of SVCMV-VSV-G. Cells were labeled 40 h after transfection with 150 μCi/ml of [35S]methionine for 8 h. Cell-free supernatants were collected and ultracentrifuged, and viral pellets were then resuspended in RIPA lysis buffer. Viral proteins were immunoprecipitated with the HIV-positive human serum combined with the rabbit anti-VSV serum and loaded on a 10% SDS–PAGE gel as described in Materials and methods. Labeled proteins were then revealed by autoradiography. Detection of p24, VSV-G, gp120 and VSV-G trimeric forms is indicated by arrows. Positions of molecular weight markers are indicated. (B) MDCK cell surface expression of Env. Identical volumes of transfected COS cell-free supernatants containing the G-protein chimeric wild-type or mutant Env HIV viruses were added onto 106 MDCK cells grown in 100 mm diameter Petri dishes for 8 h. MDCK cells were then trypsinized and grown for 1–2 days on semi-permeable 1 μm pore diameter filter membranes. After an 8 h labeling with 150 μCi/ml of [35S]methionine, cells were washed with PBS and monoclonal anti-gp120 antibody was added to either their apical or basolateral surface. Cells were then washed, lysed with RIPA, and added to protein A–Sepharose beads. Intracellular proteins were obtained by immunoprecipitating the cell lysate supernatants of the surface protein immunoprecipitates. Immunocomplexes were separated by 10% SDS–PAGE and revealed by autoradiography as described in Materials and methods. The position of gp120 is indicated by an arrow and squares. Download figure Download PowerPoint Effects of amino acid substitutions on glycoprotein incorporation into the virions The intracytoplasmic tail has been shown to modulate various functions of retroviral glycoproteins. Viral envelope incorporation, glycoprotein cell surface expression, stability and infectious potential have all been shown to be altered by certain mutations in the intracytoplasmic domain of HIV gp41 (Dubay et al., 1992; Gabuzda et al., 1992b; Shimuzu et al., 1992; Ritter et al., 1993; Spies and Compans, 1994; Freed and Martin, 1995; LaBranche et al., 1995). To achieve clear conclusions concerning the mutant glycoproteins, it was therefore necessary to ensure that these molecules are incorporated into the budding viral particles at a level comparable to the wild-type protein. This is especially important since it was previously demonstrated that actual incorporation of the envelope glycoprotein into the budding virion is required for the polarized release of the virus (Lodge et al., 1994). In order to clarify this point for the different mutants, a trans-complementation infectivity assay was first performed using a reporter CAT gene (Helseth et al., 1990). The envelope-negative proviral construct was modified to replace the non-essential nef gene by the CAT gene. This proviral construct was co-introduced with an expression plasmid for wild-type or either of the different mutant glycoproteins; MDCK cells grown in Petri dishes being used in these experiments. Identical amounts of recovered virus, as determined by RT (viral reverse transcriptase) measurement, was then used to infect susceptible CD4+ Jurkat lymphocytes and the resulting CAT activity was measured in the cell lysates, as described in Materials and methods. All HIV-1 glycoproteins harboring substitution mutations did confer the infectious potential to the envelope-negative CAT provirus (Figure 4). The capacity of the different envelope proteins to promote viral-mediated transfer of CAT activity is thus indicative of normal envelope incorporation into the virion and the capability to interact with the viral receptor to initiate infection. These results were also observed when viruses harboring mutant Y712S glycoprotein were recovered from apical or basolateral supernatants of cells grown on semi-permeable membranes. Viruses from both apical and basolateral supernatants displayed similar ability to mediate CAT transfer, indicating that the Env glycoproteins incorporated into these viruses were functional (Figure 5A). This, again, clearly demonstrates that transport of the mutant glycoproteins themselves was no longer polarized and that virions can acquire the mutant Y712S envelope glycoprotein from either apical or basolateral cell surfaces. Furthermore, we directly confirmed that wild-type amounts of viral glycoproteins were present on virions released from both membrane surfaces in MDCK cells transfected with the Y712S mutant as determined by Western blot analysis (Figure 5B) and radioimmunoprecipitation of labeled viral particles (data not shown). Probing of the same nitrocellulose filter with the human HIV-positive serum detected similar amounts of major viral capsid protein p24 in the apical and basolateral supernatants of cells expressing the Y712S mutant, demonstrating that virions were recovered from both surfaces, while only basolateral virion-associated proteins were detected in the presence of wild-type envelope glycoprotein (data not shown). Figure 4.Effect of intracytoplasmic tyrosine or proline substitutions on HIV Env glycoprotein incorporation and virus infectivity. The modified env-negative HIV proviral construct encompassing a reporter CAT gene was co-transfected in MDCK cells with an equimolar amount of wild-type or mutant env-expressing plasmids as described in Materials and methods. Viruses were harvested from transfected MDCK cell supernatants and used to infect Jurkat cells. CAT activity was measured 48 h after infection as described in Materials and methods, and analyzed by TLC followed by autoradiography. Positions of acetylated products and non-acetylated chloramphenicol substrate are indicated. Results shown represent the mean of two independent assays. Download figure Download PowerPoint Figure 5.(A) Infectivity of apical and basolateral viruses. The modified env-negative HIV proviral construct encompassing a reporter CAT gene was co-transfected in MDCK cells seeded on semi-permeable membranes, with an equimolar amount of wild-type or mutant env-expressing plasmids as described in Materials and methods. The apical or basolateral supernatants of three pooled transfected MDCK cell monolayers were used to infect Jurkat cells. CAT activity was measured 48 h after infection as described in Materials and methods, and analyzed by TLC followed by autoradiography. Positions of acetylated products and non-acetylated chloramphenicol substrate are indicated. (B) Western blot analysis was performed on viral pellets obtained by ultracentrifugation of either apical or basolateral cell-free pooled supernatants of three MDCK cell monolayers. The cells were transfected and viruses treated as in the viral polarized budding assay described in Materials and methods. Virus pellets were resuspended in RIPA and viral proteins separated on 10% SDS–PAGE, transferred onto nitrocellulose and incubated with the monoclonal antibody against gp120. Bound antibodies were then detected with horseradish peroxidase-linked anti-mouse immunoglobulin and revealed by using an enhanced chemiluminescence detection system, as described in Materials and methods. The position of gp120 is indicated by an arrow. Download figure Download PowerPoint Replication kinetics of mutant HIV viruses In order to establish further that the mutant HIV envelope glycoproteins possess normal functional properties, appropriate mutations were introduced into an infectious proviral clone. The different proviral DNA constructs were then transfected into Jurkat CD4+ T cells and the kinetics of viral multiplication were followed through the measurement of reverse transcriptase activity in the supernatant (Figure 6B). Virus production was maximum at day 12 for the positive wild-type provirus control HXBc2. Similarly, all proviruses containing a substitution at tyrosine 712 (Y712S, Y712A, Y712F) or tyrosine 768 (Y768S, used as a control) exhibited equivalent levels of reverse transcriptase activity at day 12. These results indicate that there is no apparent loss of function in the in vitro replicative capacity of these proviruses containing substitutions in the tyrosine-based basolateral targeting signal. Furthermore, as determined by radioimmunoprecipitation of transfected Jurkat lymphocytes just before the peak of viral replication (day 12), expression levels of mutant viral glycoproteins, as well as their incorporation into the virion, were shown to be similar to the wild-type glycoprotein. This further establishes that defects in polarized release are not due to a deficient processing or incorporation of the mutant glycoproteins (Figure 6A). Figure 6.(A) Detection of glycoproteins in the Jurkat-transfected cells and released virions. Jurkat cells were transfected with wild-type or various mutant provirus constructs as described in Materials and methods. Cells were subjected to metabolic labeling 12 days post-transfection with 150 μCi/ml of [35S]methionine for 5 h. Cell-free supernatants were harvested and cleared by ultracentrifugation; cells and viral pellets were then resuspended in RIPA lysis buffer. Viral proteins were immunoprecipitated with the HIV-positive human serum and resolved on a 10% SDS–PAGE gel. Labeled proteins were then revealed by autoradiography. Molecular weight markers are indicated. Virion-associated proteins are indicated by arrows. (B) Replication kinetics of mutant viruses. Aliquots of transfected Jurkat cell supernatants were recovered and analyzed for reverse transcriptase activity at different times post-transfection. Results are presented as trichloroacetic acid-precipitable values per 106 cells. Download figure Download PowerPoint Discussion The identification of targeting signals resulting in polarized basolateral budding of HIV-1 is clearly an essential prerequisite for a better understanding of its interactions with epithelial cells. The intracytoplasmic domain of several basolateral membrane proteins has been shown to encompass the molecular determinants involved in their polarized transport (Hunziker et al., 1991; Matter et al., 1992; Geffen et al., 1993). More recently, tyrosine-based basolateral targeting signals have been identified in the cellular polymeric immunoglobulin and LDL receptors as well as in the intracytoplasmic tail of the VSV-G (Figure 1B) (Casanova et al., 1991; Aroeti et al., 1993; Thomas et al., 1993; Thomas and Roth, 1994). The significance of tyrosine-based signals in polarized basolateral transport was further supported when it was reported that a single substitution incorporating a tyrosine in the intracytoplasmic domain of influenza virus hemagglutinin, a naturally apical glycoprotein, can redirect its transport to the basolateral domain in MDCK cells (Brewer and Roth, 1991). Since then, several basolateral tyrosine-based signals in viral or cellular proteins have been identified, although not all known basolateral determinants are associated with tyrosines (Hunziker and Fumey, 1994). The comprehensive study of basolateral and apical targeting signals on additional proteins is thus still required in order to get a better overall understanding of the phenomenon. In the present study, we pursued the identification of the signal responsible for basolateral budding of lentiviruses, and more specifically HIV-1. Having demonstrated that the intracytoplasmic domain of HIV gp41 mediates the basolateral budding of the virus (Lodge et al., 1994), we thus substituted the four tyrosines of the intracytoplasmic tail of gp41. Using this approach, we clearly identified the membrane-proximal tyrosine as the sole tyrosine involved in the signal required for specific basolateral budding of viral particles. Radioimmunoprecipitation analysis confirmed the presence of the mutant glycoprotein in roughly equal amounts on both cell surfaces, in contrast to the wild-type protein. This again supports the notion that the targeting signal responsible for HIV polarized budding resides in the envelope glycoprotein. These findings also clearly show that, in the presence of envelope glycoprotein, viral budding is restricted to this membrane surface due to Env–Gag protein interactions. Similarly to other published reports of tyrosine-based signals (Hopkins, 1992), conservative substitution for an aromatic phenylalanine residue had a somewhat less pronounced effect on basolateral targeting of viral buddi" @default.
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• W2019469658 title "The membrane-proximal intracytoplasmic tyrosine residue of HIV-1 envelope glycoprotein is critical for basolateral targeting of viral budding in MDCK cells" @default.
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