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.

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 The Hypervariable Domain of the Murine Leukemia Virus Surface Protein Tolerates Large Insertions and Deletions, Enabling Development of a Retroviral Particle Display System

1999 ◽  
Vol 73 (3) ◽  
pp. 1802-1808 ◽  
Author(s):  
Samuel C. Kayman ◽  
Han Park ◽  
Maya Saxon ◽  
Abraham Pinter
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Single Chain ◽  
Virus Surface ◽  
Insertions And Deletions ◽  
N Terminus ◽  
Murine Leukemia

ABSTRACT The surface proteins (SU) of murine type-C retroviruses have a central hypervariable domain devoid of cysteine and rich in proline. This 41-amino-acid region of Friend ecotropic murine leukemia virus SU was shown to be highly tolerant of insertions and deletions. Viruses in which either the N-terminal 30 amino acids or the C-terminal 22 amino acids of this region were replaced by the 7-amino-acid sequence ASAVAGA were fully infectious. Insertions of this 7-amino-acid sequence at the N terminus, center, and the C terminus of the hypervariable domain had little effect on envelope protein (Env) function, while this insertion at a position 10 amino acids following the N terminus partially destabilized the association between the SU and transmembrane subunits of Env. Large, complex domains (either a 252-amino-acid single-chain antibody binding domain [scFv] or a 96-amino-acid V1/V2 domain of HIV-1 SU containing eight N-linked glycosylation sites and two disulfides) did not interfere with Env function when inserted in the center or C-terminal portions of the hypervariable domain. The scFv domain inserted into the C-terminal region of the hypervariable domain was shown to mediate binding of antigen to viral particles, demonstrating that it folded into the active conformation and was displayed on the surface of the virion. Both positive and negative enrichment of virions expressing the V1/V2 sequence were achieved by using a monoclonal antibody specific for a conformational epitope presented by the inserted sequence. These results indicated that the hypervariable domain of Friend ecotropic SU does not contain any specific sequence or structure that is essential for Env function and demonstrated that insertions into this domain can be used to extend particle display methodologies to complex protein domains that require expression in eukaryotic cells for glycosylation and proper folding.

scholarly journals Retrovirus Glycoprotein Functionality Requires Proper Alignment of the Ectodomain and the Membrane-Proximal Cytoplasmic Tail

2013 ◽  
Vol 87 (23) ◽  
pp. 12805-12813 ◽  
Author(s):  
Sanath Kumar Janaka ◽  
Devon A. Gregory ◽  
Marc C. Johnson
Keyword(s):  
Leukemia Virus ◽  
Coiled Coil ◽  
Cytoplasmic Tail ◽  
Wild Type ◽  
Virus Envelope ◽  
Viral Glycoproteins ◽  
Similar Phenotype ◽  
Membrane Spanning ◽  
Hiv 1 ◽  
Murine Leukemia

Nonnative viral glycoproteins, including Friend murine leukemia virus envelope (F-MLV Env) are actively recruited to HIV-1 assembly sites by an unknown mechanism. Because interactions with the lipid microenvironment at budding sites could contribute to recruitment, we examined the contribution of the hydrophobicity of the F-MLV Env membrane-spanning domain (MSD) to its incorporation into HIV-1 particles. A series of F-MLV Env mutants that added or deleted one, two, or three leucines in the MSD were constructed. All six mutants retained the ability to be incorporated into HIV-1 particles, but the −1L, −2L, −3L, +1L, and +2L mutants were not capable of producing infectious particles. Surprisingly, the +3L Env glycoprotein was able to produce infectious particles and was constitutively fusogenic. However, when the cytoplasmic tail domains (CTDs) in the Env constructs were deleted, all six of the MSD mutants were able to produce infectious particles. Further mutational analyses revealed that the first 10 amino acids of the CTD is a critical regulator of infectivity. A similar phenotype was observed in HIV-1 Env upon addition of leucines in the MSD, with +1 and +2 leucine mutations greatly reducing Env activity, but +3 leucine mutations behaving similar to the wild type. Unlike F-MLV Env (+1L and +2L), HIV-1 Env (+1L and +2L) infectivity was not restored by deletion of the CTD. We hypothesize that the CTD forms a coiled-coil that disrupts the protein's functionality if it is not in phase with the trimer interface of the ectodomain.

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 Repression of the Chromatin-Tethering Domain of Murine Leukemia Virus p12

2016 ◽  
Vol 90 (24) ◽  
pp. 11197-11207 ◽  
Author(s):  
Jonathon D. Brzezinski ◽  
Apexa Modi ◽  
Mengdan Liu ◽  
Monica J. Roth
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Foamy Virus ◽  
Viral Assembly ◽  
Chromatin Binding ◽  
Wild Type ◽  
Late Domain ◽  
C Terminus ◽  
N Terminus ◽  
Murine Leukemia

ABSTRACTMurine leukemia virus (MLV) p12, encoded within Gag, binds the viral preintegration complex (PIC) to the mitotic chromatin. This acts to anchor the viral PIC in the nucleus as the nuclear envelope re-forms postmitosis. Mutations within the p12 C terminus (p12 PM13 to PM15) block early stages in viral replication. Within the p12 PM13 region (p1260PSPMA65), our studies indicated that chromatin tethering was not detected when the wild-type (WT) p12 protein (M63) was expressed as a green fluorescent protein (GFP) fusion; however, constructs bearing p12-I63 were tethered. N-terminal truncations of the activated p12-I63-GFP indicated that tethering increased further upon deletion of p1225DLLTEDPPPY34, which includes the late domain required for viral assembly. The p12 PM15 sequence (p1270RREPP74) is critical for wild-type viral viability; however, virions bearing the PM15 mutation (p1270AAAAA74) with a second M63I mutant were viable, with a titer 18-fold lower than that of the WT. The p12 M63I mutation amplified chromatin tethering and compensated for the loss of chromatin binding of p12 PM15. Rescue of the p12-M63-PM15 nonviable mutant with prototype foamy virus (PFV) and Kaposi's sarcoma herpesvirus (KSHV) tethering sequences confirmed the function of p1270–74in chromatin binding. Minimally, full-strength tethering was seen with only p1261SPIASRLRGRR71fused to GFP. These results indicate that the p12 C terminus alone is sufficient for chromatin binding and that the presence of the p1225DLLTEDPPPY34motif in the N terminus suppresses the ability to tether.IMPORTANCEThis study defines a regulatory mechanism controlling the differential roles of the MLV p12 protein in early and late replication. During viral assembly and egress, the late domain within the p12 N terminus functions to bind host vesicle release factors. During viral entry, the C terminus of p12 is required for tethering to host mitotic chromosomes. Our studies indicate that the p12 domain including the PPPY late sequence temporally represses the p12 chromatin tethering motif. Maximal p12 tethering was identified with only an 11-amino-acid minimal chromatin tethering motif encoded at p1261–71. Within this region, the p12-M63I substitution switches p12 into a tethering-competent state, partially rescuing the p12-PM15 tethering mutant. A model for how this conformational change regulates early versus late functions is presented.

scholarly journals The Role of the Membrane-spanning Domain Sequence in Glycoprotein-mediated Membrane Fusion

1999 ◽  
Vol 10 (9) ◽  
pp. 2803-2815 ◽  
Author(s):  
Gwen M. Taylor ◽  
David Avram Sanders
Keyword(s):  
Membrane Fusion ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Envelope Proteins ◽  
Viral Fusion ◽  
Virus Envelope ◽  
Membrane Spanning ◽  
Membrane Spanning Domains ◽  
Murine Leukemia

The role of glycoprotein membrane-spanning domains in the process of membrane fusion is poorly understood. It has been demonstrated that replacing all or part of the membrane-spanning domain of a viral fusion protein with sequences that encode signals for glycosylphosphatidylinositol linkage attachment abrogates membrane fusion activity. It has been suggested, however, that the actual amino acid sequence of the membrane-spanning domain is not critical for the activity of viral fusion proteins. We have examined the function of Moloney murine leukemia virus envelope proteins with substitutions in the membrane-spanning domain. Envelope proteins bearing substitutions for proline 617 are processed and incorporated into virus particles normally and bind to the viral receptor. However, they possess greatly reduced or undetectable capacities for the promotion of membrane fusion and infectious virus particle formation. Our results imply a direct role for the residues in the membrane-spanning domain of the murine leukemia virus envelope protein in membrane fusion and its regulation. They also support the thesis that membrane-spanning domains possess a sequence-dependent function in other protein-mediated membrane fusion events.

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 Sequential activation of the three protomers in the Moloney murine leukemia virus Env

2017 ◽  
Vol 114 (10) ◽  
pp. 2723-2728 ◽  
Author(s):  
Mathilda Sjöberg ◽  
Robin Löving ◽  
Birgitta Lindqvist ◽  
Henrik Garoff
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Virus Envelope ◽  
Sequential Activation ◽  
Virus Envelope Protein ◽  
Membrane Fusion Proteins ◽  
Viral Membrane Fusion ◽  
Murine Leukemia

Viral membrane fusion proteins of class I are trimers in which the protomeric unit is a complex of a surface subunit (SU) and a fusion active transmembrane subunit (TM). Here we have studied how the protomeric units of Moloney murine leukemia virus envelope protein (Env) are activated in relation to each other, sequentially or simultaneously. We followed the isomerization of the SU-TM disulfide and subsequent SU release from Env with biochemical methods and found that this early activation step occurred sequentially in the three protomers, generating two asymmetric oligomer intermediates according to the scheme (SU-TM)3→ (SU-TM)2TM → (SU-TM)TM2→ TM3. This was the case both when activation was triggered in vitro by depleting stabilizing Ca2+from solubilized Env and when viral Env was receptor triggered on rat XC cells. In the latter case, the activation reaction was too fast for direct observation of the intermediates, but they could be caught by alkylation of the isomerization active thiol.

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