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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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Infectivity and entry of Env cytoplasmic domain mutants. (A) Single round infectivity. Env-pseudotyped SG3ΔEnv viruses produced in 293T cells and p24-normalized, were used to infect TZM-bl indicator cells. After a 48 h incubation, the cell mixtures were lysed and luciferase activity was assayed. In this assay infection with virus pseudotyped with WT Env yielded 2.7 × 105 DLU and ∆Env virions an average of 1.65 × 104 DLU (B) Virus-cell fusion assay. Env CD mutants in NL4-3, pseudotyped with pCMV-BlaM-Vpr, were produced in 293T cells and subjected to gradient ultracentrifugation. Resuspended viral pellets were then normalized using p24 ELISA assays and used to infect TZM-bl indicator cells. The CCF2-AM fluorescent dye was loaded into the cells and incubated for 16 h at room temperature. The data represents results from three independent experiments conducted in triplicate. The error bars represent the standard deviation of the means. WT virus induced blue fluorescence in 5.48% of the 25,000 cells analyzed, a mock infected background of 0.46% blue cells was subtracted from all values.
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Figure 5: Infectivity and entry of Env cytoplasmic domain mutants. (A) Single round infectivity. Env-pseudotyped SG3ΔEnv viruses produced in 293T cells and p24-normalized, were used to infect TZM-bl indicator cells. After a 48 h incubation, the cell mixtures were lysed and luciferase activity was assayed. In this assay infection with virus pseudotyped with WT Env yielded 2.7 × 105 DLU and ∆Env virions an average of 1.65 × 104 DLU (B) Virus-cell fusion assay. Env CD mutants in NL4-3, pseudotyped with pCMV-BlaM-Vpr, were produced in 293T cells and subjected to gradient ultracentrifugation. Resuspended viral pellets were then normalized using p24 ELISA assays and used to infect TZM-bl indicator cells. The CCF2-AM fluorescent dye was loaded into the cells and incubated for 16 h at room temperature. The data represents results from three independent experiments conducted in triplicate. The error bars represent the standard deviation of the means. WT virus induced blue fluorescence in 5.48% of the 25,000 cells analyzed, a mock infected background of 0.46% blue cells was subtracted from all values.

Mentions: A luciferase-based single round virus entry assay was conducted, utilizing the same target cell fusion system as described above. Equivalent amounts of pseudotyped virus (normalized for p24), produced in COS-1 cells, were used to infect TZM-bl indicator cells. The cells were measured for luciferase activity at 48 h post-infection. The SG3Δenv virus was used as the background control. The results indicate that the sequential mutagenesis of the Env CD trafficking motifs resulted in much more pronounced defective phenotypes in the context of pseudotyped virus as shown in Figure 5A. In contrast to the cell-cell fusion results, where the maximum decrease observed for mutant E was 70%, infectivity of virus pseudotyped by this Env was reduced 99%. Even mutant B, in which just the Y768 motif and two adjacent dileucine motifs are mutated, exhibited only 16% the virus entry activity of WT Env. While the Y712C substitution in mutant Y had little effect on cell-cell fusion, the infectivity of viruses pseudotyped with this Env was 47% that of WT, and the remaining Y712C-containing mutants were reduced in virus entry by more than 94% compared to WT.


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)

Infectivity and entry of Env cytoplasmic domain mutants. (A) Single round infectivity. Env-pseudotyped SG3ΔEnv viruses produced in 293T cells and p24-normalized, were used to infect TZM-bl indicator cells. After a 48 h incubation, the cell mixtures were lysed and luciferase activity was assayed. In this assay infection with virus pseudotyped with WT Env yielded 2.7 × 105 DLU and ∆Env virions an average of 1.65 × 104 DLU (B) Virus-cell fusion assay. Env CD mutants in NL4-3, pseudotyped with pCMV-BlaM-Vpr, were produced in 293T cells and subjected to gradient ultracentrifugation. Resuspended viral pellets were then normalized using p24 ELISA assays and used to infect TZM-bl indicator cells. The CCF2-AM fluorescent dye was loaded into the cells and incubated for 16 h at room temperature. The data represents results from three independent experiments conducted in triplicate. The error bars represent the standard deviation of the means. WT virus induced blue fluorescence in 5.48% of the 25,000 cells analyzed, a mock infected background of 0.46% blue cells was subtracted from all values.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Infectivity and entry of Env cytoplasmic domain mutants. (A) Single round infectivity. Env-pseudotyped SG3ΔEnv viruses produced in 293T cells and p24-normalized, were used to infect TZM-bl indicator cells. After a 48 h incubation, the cell mixtures were lysed and luciferase activity was assayed. In this assay infection with virus pseudotyped with WT Env yielded 2.7 × 105 DLU and ∆Env virions an average of 1.65 × 104 DLU (B) Virus-cell fusion assay. Env CD mutants in NL4-3, pseudotyped with pCMV-BlaM-Vpr, were produced in 293T cells and subjected to gradient ultracentrifugation. Resuspended viral pellets were then normalized using p24 ELISA assays and used to infect TZM-bl indicator cells. The CCF2-AM fluorescent dye was loaded into the cells and incubated for 16 h at room temperature. The data represents results from three independent experiments conducted in triplicate. The error bars represent the standard deviation of the means. WT virus induced blue fluorescence in 5.48% of the 25,000 cells analyzed, a mock infected background of 0.46% blue cells was subtracted from all values.
Mentions: A luciferase-based single round virus entry assay was conducted, utilizing the same target cell fusion system as described above. Equivalent amounts of pseudotyped virus (normalized for p24), produced in COS-1 cells, were used to infect TZM-bl indicator cells. The cells were measured for luciferase activity at 48 h post-infection. The SG3Δenv virus was used as the background control. The results indicate that the sequential mutagenesis of the Env CD trafficking motifs resulted in much more pronounced defective phenotypes in the context of pseudotyped virus as shown in Figure 5A. In contrast to the cell-cell fusion results, where the maximum decrease observed for mutant E was 70%, infectivity of virus pseudotyped by this Env was reduced 99%. Even mutant B, in which just the Y768 motif and two adjacent dileucine motifs are mutated, exhibited only 16% the virus entry activity of WT Env. While the Y712C substitution in mutant Y had little effect on cell-cell fusion, the infectivity of viruses pseudotyped with this Env was 47% that of WT, and the remaining Y712C-containing mutants were reduced in virus entry by more than 94% compared to WT.

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