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Mutagenesis of tyrosine and di-leucine motifs in the HIV-1 envelope cytoplasmic domain results in a loss of Env-mediated fusion and infectivity.

Bhakta SJ, Shang L, Prince JL, Claiborne DT, Hunter E - Retrovirology (2011)

Bottom Line: Sequential mutagenesis of the Y- and LL-motifs resulted in a generally progressive decrease in Env fusogenicity.However, additive mutation of dileucine and tyrosine motifs beyond the tyrosine at residue 768 resulted in the most dramatic effects on Env incorporation into virions, viral infectivity, and virus fusion with target cells.Because these mutant proteins are expressed at the cell surface, we conclude that tyrosine and di-leucine residues within the cytoplasmic domain of gp41 play critical roles in HIV-1 replication that are distinct from that of targeting the plasma membrane.

View Article: PubMed Central - HTML - PubMed

Affiliation: Emory Vaccine Center at the Yerkes National Primate Research Center and Department of Pathology and Laboratory Medicine, Emory University, Atlanta, Georgia 30329, USA.

ABSTRACT

Background: The gp41 component of the Human Immunodeficiency Virus (HIV) envelope glycoprotein (Env) contains a long cytoplasmic domain (CD) with multiple highly conserved tyrosine (Y) and dileucine (LL) motifs. Studies suggest that the motifs distal to major endocytosis motif (Y712HRL), located at residues 712-715 of Env, may contribute to Env functionality in the viral life cycle. In order to examine the biological contribution of these motifs in the biosynthesis, transport, and function of Env, we constructed two panels of mutants in which the conserved Y- and LL-motifs were sequentially substituted by alternative residues, either in the presence or absence of Y712. Additional mutants targeting individual motifs were then constructed.

Results: All mutant Envs, when expressed in the absence of other viral proteins, maintained at least WT levels of Env surface staining by multiple antibodies. The Y712 mutation (Y712C) contributed to at least a 4-fold increase in surface expression for all mutants containing this change. Sequential mutagenesis of the Y- and LL-motifs resulted in a generally progressive decrease in Env fusogenicity. However, additive mutation of dileucine and tyrosine motifs beyond the tyrosine at residue 768 resulted in the most dramatic effects on Env incorporation into virions, viral infectivity, and virus fusion with target cells.

Conclusions: From the studies reported here, we show that mutations of the Y- and LL-motifs, which effectively eliminate the amphipathic nature of the lytic peptide 2 (LLP2) domain or disrupt YW and LL motifs in a region spanning residues 795-803 (YWWNLLQYW), just C-terminal of LLP2, can dramatically interfere with biological functions of HIV-1 Env and abrogate virus replication. Because these mutant proteins are expressed at the cell surface, we conclude that tyrosine and di-leucine residues within the cytoplasmic domain of gp41 play critical roles in HIV-1 replication that are distinct from that of targeting the plasma membrane.

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Biosynthesis and processing of mutant glycoproteins. COS-1 cells transiently transfected with the Env expression vector pSRHS were metabolically labeled in a pulse, followed by a 4 h chase and immunoprecipitated with anti-HIV-1 patient sera. The locations of the Env precursor and the components of the mature Env complex are indicated at the left of the gel. The pulse-chase cell lysates of glycoproteins expressed from the pSRHS vector (A) are shown in the gel at the top, and the corresponding gel for the amount of gp120 shed into the supernatant (B), is shown in the gel at the bottom.
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Figure 2: Biosynthesis and processing of mutant glycoproteins. COS-1 cells transiently transfected with the Env expression vector pSRHS were metabolically labeled in a pulse, followed by a 4 h chase and immunoprecipitated with anti-HIV-1 patient sera. The locations of the Env precursor and the components of the mature Env complex are indicated at the left of the gel. The pulse-chase cell lysates of glycoproteins expressed from the pSRHS vector (A) are shown in the gel at the top, and the corresponding gel for the amount of gp120 shed into the supernatant (B), is shown in the gel at the bottom.

Mentions: In order to investigate the effects of this mutagenesis on the biosynthesis, processing, and stability of the glycoproteins, WT and mutant envelopes were expressed from the SV40-based pSRHS vector, which also expresses Rev and Tat. Env expression was under the control of the SV40 late promoter and polyadenylation signals were provided by the long terminal repeat (LTR) of the Mason-Pfizer monkey virus [19,21]. The WT and mutant glycoproteins were expressed in COS-1 cells, which have been shown to facilitate high expression of Env from pSRHS [19]. Two days after transfection, the Env proteins were metabolically labeled for 30 min with [35S] and further chased for 4 h in complete unlabeled media. Following lysis of the cells, the glycoproteins within the cell lysates and supernatants were immunoprecipitated with HIV-1 patient sera, resolved by SDS-PAGE, and visualized by autoradiography [19,21]. Sequential mutagenesis of the Y- and LL-based motifs in the CD mutants did not decrease the level of expression of gp160, or the processing of precursor to gp120 and gp41, indicating normal intracellular transport to the trans-Golgi network, as seen in a pulse-chase experiment in Figure 2A. Examination of the amount of gp120 shed into the supernatant also revealed that the mutagenesis of these motifs did not alter the stability of gp120, represented in Figure 2B. Similar results were seen in pulse-chase experiments conducted with the pSRHS-EB Env expression constructs (data not shown).


Mutagenesis of tyrosine and di-leucine motifs in the HIV-1 envelope cytoplasmic domain results in a loss of Env-mediated fusion and infectivity.

Bhakta SJ, Shang L, Prince JL, Claiborne DT, Hunter E - Retrovirology (2011)

Biosynthesis and processing of mutant glycoproteins. COS-1 cells transiently transfected with the Env expression vector pSRHS were metabolically labeled in a pulse, followed by a 4 h chase and immunoprecipitated with anti-HIV-1 patient sera. The locations of the Env precursor and the components of the mature Env complex are indicated at the left of the gel. The pulse-chase cell lysates of glycoproteins expressed from the pSRHS vector (A) are shown in the gel at the top, and the corresponding gel for the amount of gp120 shed into the supernatant (B), is shown in the gel at the bottom.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3117779&req=5

Figure 2: Biosynthesis and processing of mutant glycoproteins. COS-1 cells transiently transfected with the Env expression vector pSRHS were metabolically labeled in a pulse, followed by a 4 h chase and immunoprecipitated with anti-HIV-1 patient sera. The locations of the Env precursor and the components of the mature Env complex are indicated at the left of the gel. The pulse-chase cell lysates of glycoproteins expressed from the pSRHS vector (A) are shown in the gel at the top, and the corresponding gel for the amount of gp120 shed into the supernatant (B), is shown in the gel at the bottom.
Mentions: In order to investigate the effects of this mutagenesis on the biosynthesis, processing, and stability of the glycoproteins, WT and mutant envelopes were expressed from the SV40-based pSRHS vector, which also expresses Rev and Tat. Env expression was under the control of the SV40 late promoter and polyadenylation signals were provided by the long terminal repeat (LTR) of the Mason-Pfizer monkey virus [19,21]. The WT and mutant glycoproteins were expressed in COS-1 cells, which have been shown to facilitate high expression of Env from pSRHS [19]. Two days after transfection, the Env proteins were metabolically labeled for 30 min with [35S] and further chased for 4 h in complete unlabeled media. Following lysis of the cells, the glycoproteins within the cell lysates and supernatants were immunoprecipitated with HIV-1 patient sera, resolved by SDS-PAGE, and visualized by autoradiography [19,21]. Sequential mutagenesis of the Y- and LL-based motifs in the CD mutants did not decrease the level of expression of gp160, or the processing of precursor to gp120 and gp41, indicating normal intracellular transport to the trans-Golgi network, as seen in a pulse-chase experiment in Figure 2A. Examination of the amount of gp120 shed into the supernatant also revealed that the mutagenesis of these motifs did not alter the stability of gp120, represented in Figure 2B. Similar results were seen in pulse-chase experiments conducted with the pSRHS-EB Env expression constructs (data not shown).

Bottom Line: Sequential mutagenesis of the Y- and LL-motifs resulted in a generally progressive decrease in Env fusogenicity.However, additive mutation of dileucine and tyrosine motifs beyond the tyrosine at residue 768 resulted in the most dramatic effects on Env incorporation into virions, viral infectivity, and virus fusion with target cells.Because these mutant proteins are expressed at the cell surface, we conclude that tyrosine and di-leucine residues within the cytoplasmic domain of gp41 play critical roles in HIV-1 replication that are distinct from that of targeting the plasma membrane.

View Article: PubMed Central - HTML - PubMed

Affiliation: Emory Vaccine Center at the Yerkes National Primate Research Center and Department of Pathology and Laboratory Medicine, Emory University, Atlanta, Georgia 30329, USA.

ABSTRACT

Background: The gp41 component of the Human Immunodeficiency Virus (HIV) envelope glycoprotein (Env) contains a long cytoplasmic domain (CD) with multiple highly conserved tyrosine (Y) and dileucine (LL) motifs. Studies suggest that the motifs distal to major endocytosis motif (Y712HRL), located at residues 712-715 of Env, may contribute to Env functionality in the viral life cycle. In order to examine the biological contribution of these motifs in the biosynthesis, transport, and function of Env, we constructed two panels of mutants in which the conserved Y- and LL-motifs were sequentially substituted by alternative residues, either in the presence or absence of Y712. Additional mutants targeting individual motifs were then constructed.

Results: All mutant Envs, when expressed in the absence of other viral proteins, maintained at least WT levels of Env surface staining by multiple antibodies. The Y712 mutation (Y712C) contributed to at least a 4-fold increase in surface expression for all mutants containing this change. Sequential mutagenesis of the Y- and LL-motifs resulted in a generally progressive decrease in Env fusogenicity. However, additive mutation of dileucine and tyrosine motifs beyond the tyrosine at residue 768 resulted in the most dramatic effects on Env incorporation into virions, viral infectivity, and virus fusion with target cells.

Conclusions: From the studies reported here, we show that mutations of the Y- and LL-motifs, which effectively eliminate the amphipathic nature of the lytic peptide 2 (LLP2) domain or disrupt YW and LL motifs in a region spanning residues 795-803 (YWWNLLQYW), just C-terminal of LLP2, can dramatically interfere with biological functions of HIV-1 Env and abrogate virus replication. Because these mutant proteins are expressed at the cell surface, we conclude that tyrosine and di-leucine residues within the cytoplasmic domain of gp41 play critical roles in HIV-1 replication that are distinct from that of targeting the plasma membrane.

Show MeSH
Related in: MedlinePlus