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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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HIV-1 Env cytoplasmic domain Y- and LL-motif mutagenesis strategy. The key amino acids of the Y- and LL-motifs targeted for mutagenesis are listed under the corresponding location in the sequence of NL4-3 envelope cytoplasmic domain. WT residues are indicated by regular-faced type, and the residues in bold-faced type represent the mutations for each mutant. The glycoproteins are separated by those containing the WT Y712 motif and by those containing the Y712C mutation.
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Figure 1: HIV-1 Env cytoplasmic domain Y- and LL-motif mutagenesis strategy. The key amino acids of the Y- and LL-motifs targeted for mutagenesis are listed under the corresponding location in the sequence of NL4-3 envelope cytoplasmic domain. WT residues are indicated by regular-faced type, and the residues in bold-faced type represent the mutations for each mutant. The glycoproteins are separated by those containing the WT Y712 motif and by those containing the Y712C mutation.

Mentions: The unusually long CD of gp41 contains multiple Y- and LL-motifs. In order to define the functional role played by these motifs in the HIV-1 life cycle, a progressive mutagenesis strategy was employed in which the Y- and LL-based motifs were sequentially mutated along the Env CD. Several of these motifs overlap the Rev open reading frame, necessitating substitutions that maintain Rev function. The mutants were classified based on their location in the NL4-3 Env and are shown in Figure 1. Site-directed mutagenesis was employed to introduce the trafficking motif mutations into the env gene. A complex overlapping PCR strategy was then utilized to create progressive mutants in the CD. Introduction of the L765H/Y768S mutations into the env sequence generated mutant A. The subsequent addition of L771S/LLLI774SHSN to mutant A results in mutant B, the addition of LL784HQ to mutant B results in mutant C, the additional changes of Y795S/LL799HQ/Y802S to mutant C produce mutant D, and LL814AA/LL855AA was combined with mutant D to create mutant E. Introduction of the Y712C mutation to WT and the Env mutants A, B, C, D, and E resulted in the generation of the Y, YA, YB, YC, YD, and YE mutants. The role of individual motifs was then probed by an additional set of mutations (Figure 1). All Env CD mutants were cloned into the Env expression vectors pSRHS and pSRHS-EB, as well as the proviral vector pNL4.3.


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)

HIV-1 Env cytoplasmic domain Y- and LL-motif mutagenesis strategy. The key amino acids of the Y- and LL-motifs targeted for mutagenesis are listed under the corresponding location in the sequence of NL4-3 envelope cytoplasmic domain. WT residues are indicated by regular-faced type, and the residues in bold-faced type represent the mutations for each mutant. The glycoproteins are separated by those containing the WT Y712 motif and by those containing the Y712C mutation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: HIV-1 Env cytoplasmic domain Y- and LL-motif mutagenesis strategy. The key amino acids of the Y- and LL-motifs targeted for mutagenesis are listed under the corresponding location in the sequence of NL4-3 envelope cytoplasmic domain. WT residues are indicated by regular-faced type, and the residues in bold-faced type represent the mutations for each mutant. The glycoproteins are separated by those containing the WT Y712 motif and by those containing the Y712C mutation.
Mentions: The unusually long CD of gp41 contains multiple Y- and LL-motifs. In order to define the functional role played by these motifs in the HIV-1 life cycle, a progressive mutagenesis strategy was employed in which the Y- and LL-based motifs were sequentially mutated along the Env CD. Several of these motifs overlap the Rev open reading frame, necessitating substitutions that maintain Rev function. The mutants were classified based on their location in the NL4-3 Env and are shown in Figure 1. Site-directed mutagenesis was employed to introduce the trafficking motif mutations into the env gene. A complex overlapping PCR strategy was then utilized to create progressive mutants in the CD. Introduction of the L765H/Y768S mutations into the env sequence generated mutant A. The subsequent addition of L771S/LLLI774SHSN to mutant A results in mutant B, the addition of LL784HQ to mutant B results in mutant C, the additional changes of Y795S/LL799HQ/Y802S to mutant C produce mutant D, and LL814AA/LL855AA was combined with mutant D to create mutant E. Introduction of the Y712C mutation to WT and the Env mutants A, B, C, D, and E resulted in the generation of the Y, YA, YB, YC, YD, and YE mutants. The role of individual motifs was then probed by an additional set of mutations (Figure 1). All Env CD mutants were cloned into the Env expression vectors pSRHS and pSRHS-EB, as well as the proviral vector pNL4.3.

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