Abstract
BackgroundThe 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.ResultsAll 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.ConclusionsFrom 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.
Highlights
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
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 Yand LL-based motifs were sequentially mutated along the Env CD
The results indicate that all of the Env CD mutants maintained at least wild type (WT) levels of surface expression, while introduction of the Y712C mutation into the CD resulted in an increase in glycoprotein cell surface expression, following immunostaining with all three antibodies
Summary
The gp 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. The envelope glycoprotein (Env) cytoplasmic domain (CD) is a key determinant in the replication of Human Immunodeficiency Virus type I (HIV-1) at two pivotal steps: (i) at the point of viral assembly, where Env must be incorporated into budding virions, and (ii) at the stage of viral entry into host target cells. The mature Env complex, which facilitates viral entry into host cells [15,16], is transported to and expressed on the cell surface, where either of two events may occur: Env is either incorporated into budding virions or it is rapidly internalized [10,11,12,13,17]
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