Site-Directed Mutagenesis of HIV-1 vpu Gene Demonstrates Two Clusters of Replication-Defective Mutants with Distinct Ability to Down-Modulate Cell Surface CD4 and Tetherin

Apoptotic cells expose phosphatidylserine (PtdSer) on their surface as an “eat me” signal. Mammalian Xk-related (Xkr) protein 8, which is predicted to contain six transmembrane regions, and its Caenorhabditis elegans homolog CED-8 promote apoptotic PtdSer exposure. The mouse and human Xkr families consist of eight and nine members, respectively. Here, we found that mouse Xkr family members, with the exception of Xkr2, are localized to the plasma membrane. When Xkr8-deficient cells, which do not expose PtdSer during apoptosis, were transformed by Xkr family members, the transformants expressing Xkr4, Xkr8, or Xkr9 responded to apoptotic stimuli by exposing cell surface PtdSer and were efficiently engulfed by macrophages. Like Xkr8, Xkr4 and Xkr9 were found to possess a caspase recognition site in the C-terminal region and to require its direct cleavage by caspases for their function. Site-directed mutagenesis of the amino acid residues conserved among CED-8, Xkr4, Xkr8, and Xkr9 identified several essential residues in the second transmembrane and second cytoplasmic regions. Real time PCR analysis indicated that unlike Xkr8, which is ubiquitously expressed, Xkr4 and Xkr9 expression is tissue-specific.

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Probing the RNA Binding Surface of the HIV-1 Nucleocapsid Protein by Site-Directed Mutagenesis

Biochemistry ◽

10.1021/bi400125z ◽

2013 ◽

Vol 52 (19) ◽

pp. 3358-3368 ◽

Cited By ~ 5

Author(s):

Wei Ouyang ◽

Stephen Okaine ◽

Mark P. McPike ◽

Yong Lin ◽

Philip N. Borer

Keyword(s):

Nucleocapsid Protein ◽

Rna Binding ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Binding Surface ◽

Hiv 1

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Site-directed mutagenesis of the conserved Asp-443 and Asp-498 carboxy-terminal residues of HIV-1 reverse transcriptase

Nucleic Acids Research ◽

10.1093/nar/18.18.5359 ◽

1990 ◽

Vol 18 (18) ◽

pp. 5359-5363 ◽

Cited By ~ 38

Author(s):

Valerie Mizrahi ◽

Martine T. Usdin ◽

Alexis Harington ◽

Lindsay R. Dudding

Keyword(s):

Reverse Transcriptase ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Carboxy Terminal ◽

Hiv 1

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Identification of the glycosaminoglycan-binding site on the glycoprotein Erns of bovine viral diarrhoea virus by site-directed mutagenesis

Journal of General Virology ◽

10.1099/0022-1317-83-9-2153 ◽

2002 ◽

Vol 83 (9) ◽

pp. 2153-2159 ◽

Cited By ~ 23

Author(s):

Munir Iqbal ◽

John W. McCauley

Keyword(s):

Cell Surface ◽

Binding Site ◽

Bovine Viral Diarrhoea Virus ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Bovine Viral Diarrhoea ◽

Surface Binding ◽

C Terminus ◽

Diarrhoea Virus ◽

Lysine Residues

Bovine viral diarrhoea virus (BVDV) envelope glycoprotein Erns interacts with highly sulphated heparin-like glycosaminoglycans (GAGs) located on the cell surface as an early step in virus infection of cells. Site-directed mutagenesis of recombinant Erns was undertaken and analysis of mutants by heparin-affinity chromatography and cell surface binding showed that a cluster of basic amino acids (480KKLENKSK487) near the C terminus of Erns was essential for binding. Mutants with amino acid substitutions of lysine residues 481 and 485 in Erns reduced the binding of Erns to immobilized heparin and cellular GAGs but retained ribonuclease activity. In contrast to normal Erns, Erns that was unable to bind to cells also failed to inhibit BVDV infection of cells when the cells were pre-incubated with Erns. It is proposed that the cluster of basic residues (480KKLENKSK487) localized at the C-terminal end of Erns constitutes a GAG-binding site.

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