scholarly journals Mutations in the Cytoplasmic Tail of Murine Leukemia Virus Envelope Protein Suppress Fusion Inhibition by R Peptide

2001 ◽  
Vol 75 (5) ◽  
pp. 2337-2344 ◽  
Author(s):  
Min Li ◽  
Chinglai Yang ◽  
Richard W. Compans
Keyword(s):  
Amino Acids ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Cytoplasmic Tail ◽  
Syncytium Formation ◽  
Inhibitory Effect ◽  
Fusion Activity ◽  
Virus Envelope ◽  
Env Protein ◽  
Murine Leukemia

ABSTRACT During viral maturation, the cytoplasmic tail of the murine leukemia virus (MuLV) envelope (Env) protein undergoes proteolytic cleavage by the viral protease to release the 16-amino-acid R peptide, and this cleavage event activates the Env protein's fusion activity. We introduced Gly and/or Ser residues at different positions upstream of the R peptide in the cytoplasmic tail of the Friend MuLV Env protein and investigated their effects on fusion activity. Expression in HeLa T4 cells of a mutant Env protein with a single Gly insertion after I619, five amino acids upstream from the R peptide, induced syncytium formation with overlaid XC cells. Env proteins containing single or double Gly-Ser insertions after F614, 10 amino acids upstream from the R peptide, induced syncytium formation, and mutant proteins with multiple Gly insertions induced various levels of syncytium formation between HeLa T4 and XC cells. Immunoprecipitation and surface biotinylation assays showed that most of the mutants had surface expression levels comparable to those of the wild-type or R peptide-truncated Env proteins. Fluorescence dye redistribution assays also showed no hemifusion in the Env proteins which did not induce fusion. Our results indicate that insertion mutations in the cytoplasmic tail of the MuLV Env protein can suppress the inhibitory effect of the R peptide on membrane fusion and that there are differences in the effects of insertions in two regions in the cytoplasmic tail upstream of the R peptide.

scholarly journals Cytoplasmic Tail of Moloney Murine Leukemia Virus Envelope Protein Influences the Conformation of the Extracellular Domain: Implications for Mechanism of Action of the R Peptide

2003 ◽  
Vol 77 (2) ◽  
pp. 1281-1291 ◽  
Author(s):  
Hector C. Aguilar ◽  
W. French Anderson ◽  
Paula M. Cannon
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Cytoplasmic Tail ◽  
Moloney Murine Leukemia Virus ◽  
Virus Envelope ◽  
Peptide Cleavage ◽  
Virus Envelope Protein ◽  
Env Protein ◽  
Membrane Spanning ◽  
Murine Leukemia

ABSTRACT The envelope (Env) protein of Moloney murine leukemia virus (MoMuLV) is a homotrimeric complex whose monomers consist of linked surface (SU) and transmembrane (TM) proteins cleaved from a precursor protein by a cellular protease. In addition, a significant fraction of virion-associated TM is further processed by the viral protease to remove the C-terminal 16 amino acids of the cytoplasmic domain, the R peptide. This cleavage greatly enhances the fusogenicity of the protein and is necessary for the formation of a fully functional Env protein complex. We have previously proposed that R peptide cleavage enhances fusogenicity by altering the conformation of the ectodomain of the protein (Y. Zhao et al., J. Virol. 72:5392-5398, 1998). Using a series of truncation and point mutants of MoMuLV Env, we now provide direct biochemical and immunological evidence that the cytoplasmic tail and the membrane-spanning region of Env can influence the overall structure of the ectodomain of the protein and alter the strength of the SU-TM interaction. The R-peptide-truncated form of the protein, in particular, exhibits a markedly different conformation than the full-length protein.

scholarly journals Palmitoylation of the Murine Leukemia Virus Envelope Protein Is Critical for Lipid Raft Association and Surface Expression

2002 ◽  
Vol 76 (23) ◽  
pp. 11845-11852 ◽  
Author(s):  
Min Li ◽  
Chinglai Yang ◽  
Suxiang Tong ◽  
Armin Weidmann ◽  
Richard W. Compans
Keyword(s):  
Lipid Rafts ◽  
Lipid Raft ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Viral Fusion ◽  
Deficient Mutant ◽  
Wild Type ◽  
Virus Envelope ◽  
Env Protein ◽  
Murine Leukemia

ABSTRACT To investigate the association of the murine leukemia virus (MuLV) Env protein with lipid rafts, we compared wild-type and palmitoylation-deficient mutant Env proteins by using extraction with the mild detergent Triton X-100 (TX-100) followed by a sucrose gradient flotation assay. We found that the wild-type MuLV Env protein was resistant to ice-cold TX-100 treatment and floated to the top of the gradients. In contrast, we observed that the palmitoylation-deficient mutant Env protein was mostly soluble when extracted by ice-cold TX-100 and stayed at the bottom of the gradients. Both the wild-type and mutant Env proteins were found to be soluble when treated with methyl-β-cyclodextrin before extraction with ice-cold TX-100 or when treated with ice-cold octyl-β-glucoside instead of TX-100. These results indicate that the MuLV Env protein is associated with lipid rafts and that palmitoylation of the Env protein is critical for lipid raft association. Although the palmitoylation-deficient Env mutant was synthesized at a level similar to that of the wild-type Env, it was found to be expressed at reduced levels on the cell surface. We observed syncytium formation activity with both the wild-type and mutant Env proteins, indicating that palmitoylation or raft association is not required for MuLV viral fusion activity.

scholarly journals Characterizing the Murine Leukemia Virus Envelope Glycoprotein Membrane-Spanning Domain for Its Roles in Interface Alignment and Fusogenicity

2015 ◽  
Vol 89 (24) ◽  
pp. 12492-12500 ◽  
Author(s):  
Daniel J. Salamango ◽  
Marc C. Johnson
Keyword(s):  
Amino Acids ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Tail Domain ◽  
Virus Envelope ◽  
C Terminus ◽  
Viral Glycoproteins ◽  
N Terminus ◽  
Membrane Spanning ◽  
Murine Leukemia

ABSTRACTThe membrane-proximal region of murine leukemia virus envelope (Env) is a critical modulator of its functionality. We have previously shown that the insertion of one amino acid (+1 leucine) within the membrane-spanning domain (MSD) abolished protein functionality in infectivity assays. However, functionality could be restored to this +1 leucine mutant by either inserting two additional amino acids (+3 leucine) or by deleting the cytoplasmic tail domain (CTD) in the +1 leucine background. We inferred that the ectodomain and CTD have protein interfaces that have to be in alignment for Env to be functional. Here, we made single residue deletions to the Env mutant with the +1 leucine insertion to restore the interface alignment (gain of functionality) and therefore define the boundaries of the two interfaces. We identified the glycine-proline pairs near the N terminus (positions 147 and 148) and the C terminus (positions 159 and 160) of the MSD as being the boundaries of the two interfaces. Deletions between these pairs restored function, but deletions outside of them did not. In addition, the vast majority of the single residue deletions regained function if the CTD was deleted. The exceptions were four hydroxyl-containing amino acid residues (T139, T140, S143, and T144) that reside in the ectodomain interface and the proline at position 148, which were all indispensable for functionality. We hypothesize that the hydroxyl-containing residues at positions T139 and S143 could be a driving force for stabilizing the ectodomain interface through formation of a hydrogen-bonding network.IMPORTANCEThe membrane-proximal external region (MPER) and membrane-spanning domains (MSDs) of viral glycoproteins have been shown to be critical for regulating glycoprotein fusogenicity. However, the roles of these two domains are poorly understood. We report here that point deletions and insertions within the MPER or MSD result in functionally inactive proteins. However, when the C-terminal tail domain (CTD) is deleted, the majority of the proteins remain functional. The only residues that were found to be critical for function regardless of the CTD were four hydroxyl-containing amino acids located at the C terminus of the MPER (T139 and T140) and at the N terminus of the MSD (S143 and T144) and a proline near the beginning of the MSD (P148). We demonstrate that hydrogen-bonding at positions T139 and S143 is critical for protein function. Our findings provide novel insights into the role of the MPER in regulating fusogenic activity of viral glycoproteins.

Characterization of R peptide of murine leukemia virus envelope glycoproteins in syncytium formation and entry

2007 ◽  
Vol 152 (12) ◽  
pp. 2169-2182 ◽  
Author(s):  
Y. Kubo ◽  
C. Tominaga ◽  
H. Yoshii ◽  
H. Kamiyama ◽  
C. Mitani ◽  
...  
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Syncytium Formation ◽  
Envelope Glycoproteins ◽  
Virus Envelope ◽  
Murine Leukemia

scholarly journals Functional Characterization of the N Termini of Murine Leukemia Virus Envelope Proteins

2001 ◽  
Vol 75 (9) ◽  
pp. 4357-4366 ◽  
Author(s):  
Chi-Wei Lu ◽  
Monica J. Roth
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Functional Characterization ◽  
Virus Titer ◽  
Envelope Proteins ◽  
Virus Envelope ◽  
C Terminus ◽  
N Terminus ◽  
Env Protein ◽  
Murine Leukemia

ABSTRACT The function of the N terminus of the murine leukemia virus (MuLV) surface (SU) protein was examined. A series of five chimeric envelope proteins (Env) were generated in which the N terminus of amphotropic 4070A was replaced by equivalent sequences from ecotropic Moloney MuLV (M-MuLV). Viral titers of these chimeras indicate that exchange with homologous sequences could be tolerated, up to V17eco/T15ampho (crossover III). Constructs encoding the first 28 amino acids (aa) of ecotropic M-MuLV resulted in Env expression and binding to the receptor; however, the virus titer was reduced 5- to 45-fold, indicating a postbinding block. Additional exchange beyond the first 28 aa of ecotropic MuLV Env resulted in defective protein expression. These N-terminal chimeras were also introduced into the AE4 chimeric Env backbone containing the amphotropic receptor binding domain joined at the hinge region to the ecotropic SU C terminus. In this backbone, introduction of the first 17 aa of the ecotropic Env protein significantly increased the titer compared to that of its parental chimera AE4, implying a functional coordination between the N terminus of SU and the C terminus of the SU and/or transmembrane proteins. These data functionally dissect the N-terminal sequence of the MuLV Env protein and identify differential effects on receptor-mediated entry.

scholarly journals Murine Leukemia Virus R Peptide Inhibits Influenza Virus Hemagglutinin-Induced Membrane Fusion

2006 ◽  
Vol 80 (12) ◽  
pp. 6106-6114 ◽  
Author(s):  
Min Li ◽  
Zhu-Nan Li ◽  
Qizhi Yao ◽  
Chinglai Yang ◽  
David A. Steinhauer ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Membrane Fusion ◽  
Leukemia Virus ◽  
Cytoplasmic Tail ◽  
Inhibitory Effect ◽  
Fusion Pore ◽  
Fusion Activity ◽  
Wild Type ◽  
Env Protein ◽  
Ha Protein

ABSTRACT The cytoplasmic tail of the murine leukemia virus (MuLV) envelope (Env) protein is known to play an important role in regulating viral fusion activity. Upon removal of the C-terminal 16 amino acids, designated as the R peptide, the fusion activity of the Env protein is activated. To extend our understanding of the inhibitory effect of the R peptide and investigate the specificity of inhibition, we constructed chimeric influenza virus-MuLV hemagglutinin (HA) genes. The influenza virus HA protein is the best-studied membrane fusion model, and we investigated the fusion activities of the chimeric HA proteins. We compared constructs in which the coding sequence for the cytoplasmic tail of the influenza virus HA protein was replaced by that of the wild-type or mutant MuLV Env protein or in which the cytoplasmic tail sequence of the MuLV Env protein was added to the HA cytoplasmic domain. Enzyme-linked immunosorbent assays and Western blot analysis showed that all chimeric HA proteins were effectively expressed on the cell surface and cleaved by trypsin. In BHK21 cells, the wild-type HA protein had a significant ability after trypsin cleavage to induce syncytium formation at pH 5.1; however, neither the chimeric HA protein with the full-length cytoplasmic tail of MuLV Env nor the full-length HA protein followed by the R peptide showed any syncytium formation. When the R peptide was truncated or mutated, the fusion activity was partially recovered in the chimeric HA proteins. A low-pH conformational-change assay showed that similar conformational changes occurred for the wild-type and chimeric HA proteins. All chimeric HA proteins were capable of promoting hemifusion and small fusion pore formation, as shown by a dye redistribution assay. These results indicate that the R peptide of the MuLV Env protein has a sequence-dependent inhibitory effect on influenza virus HA protein-induced membrane fusion and that the inhibitory effect occurs at a late stage in fusion pore enlargement.

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