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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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Replication in CEM (A) and H9 (B) cells. Viruses were generated by transfecting 293T cells with proviral DNAs. T cell lines were infected with an M.O.I. of 0.05. The supernatants were collected at day 2, 4, 6, 8, 10, and 12, then subjected to a reverse transcriptase assay to quantitate the amount of virions released at each time point.
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Figure 8: Replication in CEM (A) and H9 (B) cells. Viruses were generated by transfecting 293T cells with proviral DNAs. T cell lines were infected with an M.O.I. of 0.05. The supernatants were collected at day 2, 4, 6, 8, 10, and 12, then subjected to a reverse transcriptase assay to quantitate the amount of virions released at each time point.

Mentions: In order to examine the influence of the Env CD mutants on virus replication, we measured the replication kinetics of these mutants over a period of 12 days in both the H9 and CEM T cell lines by a reverse transcriptase assay. NL4-3 proviral constructs were transfected into 293T cells, supernatant virus was titered on TZM-bl cells, then CEM or H9 cells were infected with an equal MOI (0.05). As shown in Figure 8A, in CEM cells, single-motif mutants, S4 (LL784HQ) and S8 (LL814AA) showed similar replication capacities in H9 cells, with an initial replication rate comparable to WT. Mutants Y and A, as might be predicted from single round infections, demonstrated delayed replication kinetics, with peak RT values equivalent to, but 2 days after, WT. The additional mutation of the hydrophobic core of LLP2 in mutant B completely abrogated viral replication, with RT values dropping progressively over the course of the experiment, indicative of the infection of cells by the initial inoculum but then loss of RT production because the virus is unable to assemble infectious virus in T cells. The fact that mutant S3 (LLLI774SHSN) exhibits a significant but incomplete replication defect in CEM cells suggests that combining these mutations with mutant A, as in mutant B, is highly detrimental to the virus. Three mutants S5 (YW795SL), S6 (LL799HQ), and S7 (YW802SL) demonstrated a 6-8 day-delay before virus replication accelerated - and for S5 and S6 the peak of virus remained approximately 10-fold below that of 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)

Replication in CEM (A) and H9 (B) cells. Viruses were generated by transfecting 293T cells with proviral DNAs. T cell lines were infected with an M.O.I. of 0.05. The supernatants were collected at day 2, 4, 6, 8, 10, and 12, then subjected to a reverse transcriptase assay to quantitate the amount of virions released at each time point.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: Replication in CEM (A) and H9 (B) cells. Viruses were generated by transfecting 293T cells with proviral DNAs. T cell lines were infected with an M.O.I. of 0.05. The supernatants were collected at day 2, 4, 6, 8, 10, and 12, then subjected to a reverse transcriptase assay to quantitate the amount of virions released at each time point.
Mentions: In order to examine the influence of the Env CD mutants on virus replication, we measured the replication kinetics of these mutants over a period of 12 days in both the H9 and CEM T cell lines by a reverse transcriptase assay. NL4-3 proviral constructs were transfected into 293T cells, supernatant virus was titered on TZM-bl cells, then CEM or H9 cells were infected with an equal MOI (0.05). As shown in Figure 8A, in CEM cells, single-motif mutants, S4 (LL784HQ) and S8 (LL814AA) showed similar replication capacities in H9 cells, with an initial replication rate comparable to WT. Mutants Y and A, as might be predicted from single round infections, demonstrated delayed replication kinetics, with peak RT values equivalent to, but 2 days after, WT. The additional mutation of the hydrophobic core of LLP2 in mutant B completely abrogated viral replication, with RT values dropping progressively over the course of the experiment, indicative of the infection of cells by the initial inoculum but then loss of RT production because the virus is unable to assemble infectious virus in T cells. The fact that mutant S3 (LLLI774SHSN) exhibits a significant but incomplete replication defect in CEM cells suggests that combining these mutations with mutant A, as in mutant B, is highly detrimental to the virus. Three mutants S5 (YW795SL), S6 (LL799HQ), and S7 (YW802SL) demonstrated a 6-8 day-delay before virus replication accelerated - and for S5 and S6 the peak of virus remained approximately 10-fold below that of 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