scholarly journals A Tyrosine Motif in the Cytoplasmic Domain of Mason-Pfizer Monkey Virus Is Essential for the Incorporation of Glycoprotein into Virions

2003 ◽  
Vol 77 (9) ◽  
pp. 5192-5200 ◽  
Author(s):  
Chisu Song ◽  
Susan R. Dubay ◽  
Eric Hunter
Keyword(s):  
Amino Acid ◽  
Cytoplasmic Domain ◽  
Virus Infectivity ◽  
Fusion Activity ◽  
Tyrosine Residues ◽  
Glycoprotein Complex ◽  
Selective Incorporation ◽  
Uptake Studies ◽  
And Function ◽  
Immature Virus

ABSTRACT Mason-Pfizer monkey virus (M-PMV) encodes a transmembrane (TM) glycoprotein with a 38-amino-acid-long cytoplasmic domain. After the release of the immature virus, a viral protease-mediated cleavage occurs within the cytoplasmic domain, resulting in the loss of 17 amino acids from the carboxy terminus. This maturational cleavage occurs between a histidine at position 21 and a tyrosine at position 22 in the cytoplasmic domain of the TM protein. We have demonstrated previously that a truncated TM glycoprotein with a 21-amino-acid-long cytoplasmic tail showed enhanced fusogenicity but could not be incorporated into virions. These results suggest that postassembly cleavage of the cytoplasmic domain removes a necessary incorporation signal and activates fusion activity. To investigate the contribution of tyrosine residues to the function of the glycoprotein complex and virus replication, we have introduced amino acid substitutions into two tyrosine residues found in the cytoplasmic domain. The effects of these mutations on glycoprotein biosynthesis and function, as well as on virus infectivity, have been examined. Mutation of tyrosine 34 to alanine had little effect on glycoprotein function. In contrast, substitutions at tyrosine 22 modulated fusion activity in either a positive or negative manner, depending on the substituting amino acid. Moreover, any nonaromatic substitution at this position blocked glycoprotein incorporation into virions and abolished infectivity. These results demonstrate that M-PMV employs a tyrosine signal for the selective incorporation of glycoprotein into budding virions. Antibody uptake studies show that tyrosine 22 is part of an efficient internalization signal in the cytoplasmic domain of the M-PMV glycoprotein that can also be positively and negatively influenced by changes at this site.

scholarly journals Amino Acid Residues in the Cytoplasmic Domain of the Mason-Pfizer Monkey Virus Glycoprotein Critical for Its Incorporation into Virions

2005 ◽  
Vol 79 (18) ◽  
pp. 11559-11568 ◽  
Author(s):  
Chisu Song ◽  
Keith Micoli ◽  
Helena Bauerova ◽  
Iva Pichova ◽  
Eric Hunter
Keyword(s):  
Amino Acid ◽  
Transmembrane Protein ◽  
Critical Role ◽  
Cytoplasmic Domain ◽  
Virus Infectivity ◽  
Amino Acid Residues ◽  
Glycoprotein Complex ◽  
Uptake Studies ◽  
Domain Block ◽  
And Function

ABSTRACT Assembly of an infectious retrovirus requires the incorporation of the envelope glycoprotein complex during the process of particle budding. We have recently demonstrated that amino acid substitutions of a tyrosine residue in the cytoplasmic domain block glycoprotein incorporation into budding Mason-Pfizer monkey virus (M-PMV) particles and abrogate infectivity (C. Song, S. R. Dubay, and E. Hunter, J. Virol. 77:5192-5200, 2003). To investigate the contribution of other amino acids in the cytoplasmic domain to the process of glycoprotein incorporation, we introduced alanine-scanning mutations into this region of the transmembrane protein. The effects of the mutations on glycoprotein biosynthesis and function, as well as on virus infectivity, have been examined. Mutation of two cytoplasmic residues, valine 20 and histidine 21, inhibits viral protease-mediated cleavage of the cytoplasmic domain that is observed during virion maturation, but the mutant virions show only moderately reduced infectivity. We also demonstrate that the cytoplasmic domain of the M-PMV contains three amino acid residues that are absolutely essential for incorporation of glycoprotein into virions. In addition to the previously identified tyrosine at residue 22, an isoleucine at position 18 and a leucine at position 25 each mediate the process of incorporation and efficient release of virions. While isoleucine 18 may be involved in direct interactions with immature capsids, antibody uptake studies showed that leucine 25 and tyrosine 22 are part of an efficient internalization signal in the cytoplasmic domain of the M-PMV glycoprotein. These results demonstrate that the cytoplasmic domain of M-PMV Env, in part through its YXXL-mediated endocytosis and intracellular trafficking signals, plays a critical role in the incorporation of glycoprotein into virions.

scholarly journals Interaction between the Cytoplasmic Domain of ICAM-1 and Pr55Gag Leads to Acquisition of Host ICAM-1 by Human Immunodeficiency Virus Type 1

2004 ◽  
Vol 78 (21) ◽  
pp. 11916-11925 ◽  
Author(s):  
Yannick Beauséjour ◽  
Michel J. Tremblay
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Adhesion Molecule ◽  
Cytoplasmic Domain ◽  
Virus Infectivity ◽  
Immunodeficiency Virus ◽  
Selective Incorporation ◽  
Hiv 1

ABSTRACT We have examined the molecular basis for the selective incorporation of the adhesion molecule ICAM-1 within human immunodeficiency virus type 1 (HIV-1). The process of ICAM-1 incorporation was investigated by using different ICAM-1 constructs in combination with virus capture and immunoprecipitation studies, Western blot and confocal microscopy analyses, and infectivity assays. Experiments conducted with viruses bearing a truncated version of ICAM-1 revealed that the cytoplasmic domain of ICAM-1 governs insertion of this adhesion molecule into HIV-1. Further experiments suggested that there is an association between ICAM-1 and the virus-encoded Pr55Gag polyprotein. This study represents the first demonstration that structural Gag polyproteins play a key role in the uptake of a host-derived cell surface by the virus entity. Taken together, our results indicate that interactions between viral and cellular proteins are responsible for the selective uptake of host ICAM-1 by HIV-1. This observation describes a new strategy by which HIV-1 can modulate its replicative cycle, considering that insertion of ICAM-1 within nascent virions has been shown to increase virus infectivity.

scholarly journals The Pre-Transmembrane Domain of the Autographa californica Multicapsid Nucleopolyhedrovirus GP64 Protein Is Critical for Membrane Fusion and Virus Infectivity

2009 ◽  
Vol 83 (21) ◽  
pp. 10993-11004 ◽  
Author(s):  
Zhaofei Li ◽  
Gary W. Blissard
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Membrane Fusion ◽  
Transmembrane Domain ◽  
Aromatic Amino Acids ◽  
Autographa Californica ◽  
Fusion Pore ◽  
Virus Infectivity ◽  
Viral Fusion ◽  
Fusion Activity

ABSTRACT The envelope glycoprotein, GP64, of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) is a class III viral fusion protein that mediates pH-triggered membrane fusion during virus entry. Viral fusion glycoproteins from many viruses contain a short region in the ectodomain and near the transmembrane domain, referred to as the pre-transmembrane (PTM) domain. In some cases, the PTM domain is rich in aromatic amino acids and plays an important role in membrane fusion. Although the 23-amino-acid (aa) PTM domain of AcMNPV GP64 lacks aromatic amino acids, we asked whether this region might also play a significant role in membrane fusion. We generated alanine scanning and single and multiple amino acid substitutions in the GP64 PTM domain. We specifically focused on amino acid positions conserved between baculovirus GP64 and thogotovirus GP75 proteins, as well as hydrophobic and charged amino acids. For each PTM-modified construct, we examined trimerization, cell surface localization, and membrane fusion activity. Membrane merger and pore formation were also examined. We identified eight aa positions that are important for membrane fusion activity. Critical positions were not clustered in the linear sequence but were distributed throughout the PTM domain. While charged residues were not critical or essential, three hydrophobic amino acids (L465, L476, and L480) played an important role in membrane fusion activity and appear to be involved in formation of the fusion pore. We also asked whether selected GP64 constructs were capable of rescuing a gp64null AcMNPV virus. These studies suggested that several conserved residues (T463, G460, G462, and G474) were not required for membrane fusion but were important for budding and viral infectivity.

scholarly journals Activity of the Mason-Pfizer Monkey Virus Fusion Protein Is Modulated by Single Amino Acids in the Cytoplasmic Tail

2005 ◽  
Vol 79 (18) ◽  
pp. 11569-11579 ◽  
Author(s):  
Chisu Song ◽  
Keith Micoli ◽  
Eric Hunter
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Coiled Coil ◽  
Cytoplasmic Tail ◽  
Amino Acid Residues ◽  
Protein Fusion ◽  
Glycoprotein Complex ◽  
Transmembrane Glycoprotein ◽  
Immature Virus

ABSTRACT Mason-Pfizer monkey virus (M-PMV) encodes a transmembrane glycoprotein with a 38-amino-acid-long cytoplasmic tail. After the release of the immature virus, a viral protease-mediated cleavage of the cytoplasmic tail (CT) results in the loss of 17 amino acids from the carboxy terminus and renders the envelope protein fusion competent. To investigate the role of individual amino acid residues in the CT in fusion, a series of mutations was introduced, and the effects of these mutations on glycoprotein biosynthesis and fusion were examined. Most of the alanine-scanning mutations in the CT had little effect on fusion activity. However, four amino acid substitutions (threonine 4, lysine 7, glutamine 9, and isoleucine 10) resulted in substantially increased fusogenicity, while six (leucine 2, phenylalanine 5, isoleucine 13, lysine 16, proline 17, and glycine 31) resulted in much-reduced fusion. Interestingly, the bulk of these mutations are located upstream of the CT cleavage site in a region that has the potential to form a coiled-coil in the Env trimer. Substitutions at glutamine 9 and isoleucine 10 with alanine had the most dramatic positive effect and resulted in the formation of large syncytia. Taken together, these data demonstrate that individual residues within the cytoplasmic domain of M-PMV Env can modulate, in both a positive and negative manner, biological functions that are associated with the extracellular domains of the glycoprotein complex.

scholarly journals A critical cytoplasmic domain of the interleukin-5 (IL-5) receptor alpha chain and its function in IL-5-mediated growth signal transduction.

1994 ◽  
Vol 14 (11) ◽  
pp. 7404-7413 ◽  
Author(s):  
S Takaki ◽  
H Kanazawa ◽  
M Shiiba ◽  
K Takatsu
Keyword(s):  
Signal Transduction ◽  
Protein Tyrosine Kinase ◽  
Cytoplasmic Domain ◽  
Beta Chain ◽  
Cytoplasmic Domains ◽  
Interleukin 5 ◽  
Alpha Chain ◽  
Gm Csf ◽  
Response Expression ◽  
And Function

Interleukin-5 (IL-5) regulates the production and function of B cells, eosinophils, and basophils. The IL-5 receptor (IL-5R) consists of two distinct membrane proteins, alpha and beta. The alpha chain (IL-5R alpha) is specific to IL-5. The beta chain is the common beta chain (beta c) of receptors for IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF). The cytoplasmic domains of both alpha and beta chains are essential for signal transduction. In this study, we generated cDNAs of IL-5R alpha having various mutations in their cytoplasmic domains and examined the function of these mutants by expressing them in IL-3-dependent FDC-P1 cells. The membrane-proximal proline-rich sequence of the cytoplasmic domain of IL-5R alpha, which is conserved among the alpha chains of IL-5R, IL-3R, and GM-CSF receptor (GM-CSFR), was found to be essential for the IL-5-induced proliferative response, expression of nuclear proto-oncogenes such as c-jun, c-fos, and c-myc, and tyrosine phosphorylation of cellular proteins including JAK2 protein-tyrosine kinase. In addition, analysis using chimeric receptors which consist of the extracellular domain of IL-5R alpha and the cytoplasmic domain of beta c suggested that dimerization of the cytoplasmic domain of beta c may be an important step in activating the IL-5R complex and transducing intracellular growth signals.

scholarly journals Acquisition of Cell–Cell Fusion Activity by Amino Acid Substitutions in Spike Protein Determines the Infectivity of a Coronavirus in Cultured Cells

PLoS ONE ◽  
2009 ◽  
Vol 4 (7) ◽  
pp. e6130 ◽  
Author(s):  
Yoshiyuki Yamada ◽  
Xiao Bo Liu ◽  
Shou Guo Fang ◽  
Felicia P. L. Tay ◽  
Ding Xiang Liu
Keyword(s):  
Amino Acid ◽  
Cell Fusion ◽  
Cultured Cells ◽  
Spike Protein ◽  
Amino Acid Substitutions ◽  
Fusion Activity ◽  
Cell Cell

scholarly journals Pichia pastoris and the Recombinant Human Heterodimeric Amino Acid Transporter 4F2hc-LAT1: From Clone Selection to Pure Protein

2021 ◽  
Vol 4 (3) ◽  
pp. 51
Author(s):  
Satish Kantipudi ◽  
Daniel Harder ◽  
Sara Bonetti ◽  
Dimitrios Fotiadis ◽  
Jean-Marc Jeckelmann
Keyword(s):  
Amino Acid ◽  
Pichia Pastoris ◽  
Protein Complexes ◽  
Amino Acid Transporter ◽  
Amino Acid Transporters ◽  
Expression Host ◽  
Amino Acids And Derivatives ◽  
Heterodimeric Complex ◽  
Acid Transporter ◽  
And Function

Heterodimeric amino acid transporters (HATs) are protein complexes composed of two subunits, a heavy and a light subunit belonging to the solute carrier (SLC) families SLC3 and SLC7. HATs transport amino acids and derivatives thereof across the plasma membrane. The human HAT 4F2hc-LAT1 is composed of the type-II membrane N-glycoprotein 4F2hc (SLC3A2) and the L-type amino acid transporter LAT1 (SLC7A5). 4F2hc-LAT1 is medically relevant, and its dysfunction and overexpression are associated with autism and tumor progression. Here, we provide a general applicable protocol on how to screen for the best membrane transport protein-expressing clone in terms of protein amount and function using Pichia pastoris as expression host. Furthermore, we describe an overexpression and purification procedure for the production of the HAT 4F2hc-LAT1. The isolated heterodimeric complex is pure, correctly assembled, stable, binds the substrate L-leucine, and is thus properly folded. Therefore, this Pichia pastoris-derived recombinant human 4F2hc-LAT1 sample can be used for downstream biochemical and biophysical characterizations.

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