ABSTRACT
Although tyrosine phosphorylation of extracellular proteins has been reported to occur extensively in vivo, no secreted protein tyrosine kinase has been identified. As a result, investigation of the potential role of extracellular tyrosine phosphorylation in physiological and pathological tissue regulation has not been possible. Here, we show that VLK, a putative protein kinase previously shown to be essential in embryonic development, is a secreted protein kinase, with preference for tyrosine, that phosphorylates a broad range of secreted and ER-resident substrate proteins. We find that VLK is rapidly and quantitatively secreted from platelets in response to stimuli and can tyrosine phosphorylate coreleased proteins utilizing endogenous as well as exogenous ATP sources. We propose that discovery of VLK activity provides an explanation for the extensive and conserved pattern of extracellular tyrosine phosphophorylation seen in vivo, and extends the importance of regulated tyrosine phosphorylation into the extracellular environment.
Subject(s)
Blood Platelets/enzymology , Embryo, Mammalian/enzymology , Protein Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Amino Acid Sequence , Animals , Embryonic Development , Glycosylation , Humans , Mice , Molecular Sequence Data , Phosphorylation , Protein Kinases/chemistry , Protein Kinases/genetics , Protein Processing, Post-Translational , Protein Structure, Tertiary , Protein-Tyrosine Kinases/chemistry , Secretory PathwayABSTRACT
Human immunodeficiency virus type 1 (HIV-1) does not replicate in primary cells of New World primates. To better understand this restriction, we expressed owl monkey (Aotus nancymaae) CD4 and CXCR4 in the owl monkey kidney cell line, OMK. An HIV-1 variant modified to evade the owl monkey restriction factor TRIM-cyp replicated efficiently in these cells but could not replicate in primary A. nancymaae CD4-positive T cells. To understand this difference, we examined APOBEC3G and tetherin orthologs from OMK cells and primary A. nancymaae cells. We observed that OMK cells expressed substantially lower levels of APOBEC3G than did A. nancymaae cells. A. nancymaae, but not marmoset (Callithrix jacchus), APOBEC3G was partially downregulated by HIV-1 vif and reduced but did not abolish HIV-1 replication when stably expressed in OMK cells. The functional difference between A. nancymaae and marmoset APOBEC3Gs mapped to residue 128, previously shown to distinguish African green monkey from human APOBEC3G. We also characterized tetherin orthologs from OMK and A. nancymaae cells. The A. nancymaae tetherin ortholog, but not OMK tetherin, prevented HIV-1 release. Alteration of threonine 181 of OMK tetherin rescued its function and its efficient N glycosylation. All alleles of Aotus lemurinus griseimembra examined, but none of A. nancymaae or Aotus vociferans, encoded this nonfunctional tetherin ortholog. Our data indicate that HIV-1 replication in owl monkeys is not restricted at entry but can be limited by APOBEC3G and tetherin. Further, A. lemurinus griseimembra does not restrict HIV-1 replication via tetherin, a property likely useful for the study of tetherin-restricted viruses.
Subject(s)
Antigens, CD/immunology , Aotidae/virology , HIV-1/growth & development , HIV-1/immunology , Membrane Glycoproteins/immunology , Amino Acid Sequence , Animals , Antigens, CD/genetics , Cell Line , Cells, Cultured , Cytidine Deaminase/genetics , Cytidine Deaminase/immunology , Gene Expression , Humans , Membrane Glycoproteins/genetics , Molecular Sequence Data , Receptors, CXCR4/biosynthesis , Receptors, CXCR4/genetics , Sequence Alignment , Sequence Analysis, DNA , T-Lymphocytes/virologyABSTRACT
Spike (S) proteins of coronaviruses, including the coronavirus that causes severe acute respiratory syndrome (SARS), associate with cellular receptors to mediate infection of their target cells. Here we identify a metallopeptidase, angiotensin-converting enzyme 2 (ACE2), isolated from SARS coronavirus (SARS-CoV)-permissive Vero E6 cells, that efficiently binds the S1 domain of the SARS-CoV S protein. We found that a soluble form of ACE2, but not of the related enzyme ACE1, blocked association of the S1 domain with Vero E6 cells. 293T cells transfected with ACE2, but not those transfected with human immunodeficiency virus-1 receptors, formed multinucleated syncytia with cells expressing S protein. Furthermore, SARS-CoV replicated efficiently on ACE2-transfected but not mock-transfected 293T cells. Finally, anti-ACE2 but not anti-ACE1 antibody blocked viral replication on Vero E6 cells. Together our data indicate that ACE2 is a functional receptor for SARS-CoV.
Subject(s)
Severe acute respiratory syndrome-related coronavirus/metabolism , Angiotensin-Converting Enzyme 2 , Animals , Antibodies/immunology , Antibodies/pharmacology , Carboxypeptidases/antagonists & inhibitors , Carboxypeptidases/genetics , Carboxypeptidases/immunology , Carboxypeptidases/metabolism , Cell Line , Chlorocebus aethiops , Giant Cells/cytology , Giant Cells/metabolism , Humans , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Molecular Weight , Peptidyl-Dipeptidase A/immunology , Peptidyl-Dipeptidase A/metabolism , Protein Binding , Protein Structure, Tertiary , Receptors, Virus/antagonists & inhibitors , Receptors, Virus/genetics , Receptors, Virus/immunology , Receptors, Virus/metabolism , Severe acute respiratory syndrome-related coronavirus/genetics , Severe acute respiratory syndrome-related coronavirus/growth & development , Solubility , Spike Glycoprotein, Coronavirus , Transfection , Vero Cells , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/genetics , Viral Envelope Proteins/metabolism , Virus Replication/drug effectsABSTRACT
In this report, the antiviral activity of 80R immunoglobulin G1 (IgG1), a human monoclonal antibody against severe acute respiratory syndrome coronavirus (SARS-CoV) spike (S) protein that acts as a viral entry inhibitor in vitro, was investigated in vivo in a mouse model. When 80R IgG1 was given prophylactically to mice at doses therapeutically achievable in humans, viral replication was reduced by more than 4 orders of magnitude to below assay limits. The essential core region of S protein required for 80R binding was identified as a conformationally sensitive fragment (residues 324 to 503) that overlaps the receptor ACE2-binding domain. Amino acids critical for 80R binding were identified. In addition, the effects of various 80R-binding domain amino acid substitutions which occur in SARS-like-CoV from civet cats, and which evolved during the 2002/2003 outbreak and in a 2003/2004 Guangdong index patient, were analyzed. The results demonstrated that the vast majority of SARS-CoVs are sensitive to 80R. We propose that by establishing the susceptibility and resistance profiles of newly emerging SARS-CoVs through early S1 genotyping of the core 180-amino-acid neutralizing epitope of 80R, an effective immunoprophylaxis strategy with 80R should be possible in an outbreak setting. Our study also cautions that for any prophylaxis strategy based on neutralizing antibody responses, whether by passive or active immunization, a genotyping monitor will be necessary for effective use.
Subject(s)
Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/immunology , Antibodies, Viral/administration & dosage , Antibodies, Viral/immunology , Membrane Glycoproteins/immunology , Severe Acute Respiratory Syndrome/prevention & control , Severe acute respiratory syndrome-related coronavirus/immunology , Viral Envelope Proteins/immunology , Viral Vaccines/administration & dosage , Viral Vaccines/immunology , Amino Acid Substitution , Animals , Disease Models, Animal , Epitope Mapping , Escherichia coli/metabolism , Female , Genotype , Humans , Immunization, Passive , Immunoglobulin G/administration & dosage , Immunoglobulin G/immunology , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/genetics , Mice , Mice, Inbred BALB C , Receptors, Virus/antagonists & inhibitors , Receptors, Virus/metabolism , Severe acute respiratory syndrome-related coronavirus/genetics , Severe acute respiratory syndrome-related coronavirus/isolation & purification , Severe Acute Respiratory Syndrome/virology , Spike Glycoprotein, Coronavirus , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/geneticsABSTRACT
Human angiotensin-converting enzyme 2 (ACE2) is a functional receptor for SARS coronavirus (SARS-CoV). Here we identify the SARS-CoV spike (S)-protein-binding site on ACE2. We also compare S proteins of SARS-CoV isolated during the 2002-2003 SARS outbreak and during the much less severe 2003-2004 outbreak, and from palm civets, a possible source of SARS-CoV found in humans. All three S proteins bound to and utilized palm-civet ACE2 efficiently, but the latter two S proteins utilized human ACE2 markedly less efficiently than did the S protein obtained during the earlier human outbreak. The lower affinity of these S proteins could be complemented by altering specific residues within the S-protein-binding site of human ACE2 to those of civet ACE2, or by altering S-protein residues 479 and 487 to residues conserved during the 2002-2003 outbreak. Collectively, these data describe molecular interactions important to the adaptation of SARS-CoV to human cells, and provide insight into the severity of the 2002-2003 SARS epidemic.
Subject(s)
Carboxypeptidases/metabolism , Membrane Glycoproteins/metabolism , Severe acute respiratory syndrome-related coronavirus/metabolism , Viral Envelope Proteins/metabolism , Amino Acid Sequence , Angiotensin-Converting Enzyme 2 , Animals , Binding Sites , Carboxypeptidases/chemistry , Carboxypeptidases/genetics , Catalytic Domain , Disease Outbreaks , Humans , Models, Molecular , Molecular Sequence Data , Peptidyl-Dipeptidase A , Protein Binding , Protein Structure, Tertiary , Rats , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Alignment , Severe Acute Respiratory Syndrome/epidemiology , Severe Acute Respiratory Syndrome/virology , Spike Glycoprotein, Coronavirus , Viverridae/virologyABSTRACT
Hepatitis delta virus (HDV) uses a host-encoded RNA-editing activity to express its two essential proteins from the same coding sequence. Adenosine deaminase that acts on RNA (ADAR)1 and ADAR2 are enzymes that catalyze such reactions, and each, when overexpressed, are capable of editing HDV RNA in vivo. However, the enzyme responsible for editing HDV RNA during replication has not been determined. Mammalian cells express two forms of ADAR1, a large form (ADAR1-L) that mainly localizes to the cytoplasm and a small form (ADAR1-S) that resides in the nucleus. Recently, we found that the specific activity of ADAR1-L within cells is much higher than that of ADAR1-S but only when the substrate can be edited in the cytoplasm. Here we observed that although both ADAR1-S and ADAR1-L were expressed throughout HDV replication, no ADAR2 could be observed at any time. Using expression vectors that individually overexpress either form of ADAR1, we found that ADAR1-S could stimulate editing during replication more efficiently. We next reduced ADAR1 levels during HDV replication. After transfection of an ADAR1-L-specific small interfering RNA (siRNA), we observed a significant loss of that protein and its associated cytoplasmic editing activity while the level of ADAR1-S remained unchanged. Transfection of this siRNA, however, did not reduce editing during HDV replication. In contrast, transfection of an siRNA that targets both forms of ADAR1 greatly reduced the expression of both proteins and potently inhibited editing during replication. We conclude that ADAR1-S edits HDV RNA during replication and that editing occurs in the nucleus.
Subject(s)
Adenosine Deaminase/genetics , Hepatitis Delta Virus/genetics , RNA Editing/genetics , RNA, Viral/genetics , Adenosine Deaminase/metabolism , Base Sequence , Cell Line , DNA Primers , DNA Replication , Genome, Viral , Hepatitis Delta Virus/physiology , Humans , Isoenzymes/genetics , Isoenzymes/metabolism , RNA, Small Interfering/genetics , RNA-Binding Proteins , Transcription, Genetic , Transfection , Virus Replication/geneticsABSTRACT
Mammalian cells express small and large forms of the RNA editing enzyme ADAR1, referred to as ADAR1-S and ADAR1-L, respectively. Here we observed that ADAR1-L was >70-fold more active than was ADAR1-S when assayed with a substrate that could be edited in either the nucleus or cytoplasm, and was also much more active when assayed with a substrate that was generated in the cytoplasm during viral replication. In contrast, when a substrate that could only be edited within the nucleus was assayed, the activity of ADAR1-S was found to be somewhat higher than that of ADAR1-L. We show here not only that editing could occur in the cytoplasm but also that the process was extremely efficient, occurred rapidly, and could occur in the absence of translation. Consistent with the observation that editing in the cytoplasm can be very efficient, deletion of the nuclear localization signal from ADAR2 resulted in a protein with 15-fold higher activity when tested with a substrate that contained an editing site in the mature message. In addition to its potential role in an antiviral response, we propose that ADAR1-L is the form primarily responsible for editing mRNAs in which the editing site is retained after processing.
Subject(s)
Adenosine Deaminase/chemistry , Adenosine Deaminase/metabolism , RNA Editing , Adenosine Deaminase/genetics , Base Sequence , Cell Line , Cell Nucleus/metabolism , Cytoplasm/metabolism , DNA-Directed RNA Polymerases/metabolism , Genes, Reporter , Humans , In Vitro Techniques , Kinetics , Mutagenesis, Site-Directed , Nuclear Localization Signals/chemistry , Nuclear Localization Signals/genetics , Nuclear Localization Signals/metabolism , Protein Structure, Tertiary , RNA/genetics , RNA/metabolism , RNA-Binding Proteins , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Deletion , Substrate Specificity , Viral ProteinsABSTRACT
The coronavirus spike (S) protein mediates infection of receptor-expressing host cells and is a critical target for antiviral neutralizing antibodies. Angiotensin-converting enzyme 2 (ACE2) is a functional receptor for the coronavirus (severe acute respiratory syndrome (SARS)-CoV) that causes SARS. Here we demonstrate that a 193-amino acid fragment of the S protein (residues 318-510) bound ACE2 more efficiently than did the full S1 domain (residues 12-672). Smaller S protein fragments, expressing residues 327-510 or 318-490, did not detectably bind ACE2. A point mutation at aspartic acid 454 abolished association of the full S1 domain and of the 193-residue fragment with ACE2. The 193-residue fragment blocked S protein-mediated infection with an IC(50) of less than 10 nm, whereas the IC(50) of the S1 domain was approximately 50 nm. These data identify an independently folded receptor-binding domain of the SARS-CoV S protein.
Subject(s)
Carboxypeptidases/chemistry , Membrane Glycoproteins/chemistry , Severe acute respiratory syndrome-related coronavirus/metabolism , Viral Envelope Proteins/chemistry , Alanine/chemistry , Angiotensin-Converting Enzyme 2 , Carboxypeptidases/metabolism , Cell Line , Cysteine/chemistry , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Flow Cytometry , Glutamic Acid/chemistry , Humans , Inhibitory Concentration 50 , Mutation , Peptidyl-Dipeptidase A , Point Mutation , Protein Binding , Protein Structure, Tertiary , Spike Glycoprotein, Coronavirus , TransfectionABSTRACT
Infection of receptor-bearing cells by coronaviruses is mediated by their spike (S) proteins. The coronavirus (SARS-CoV) that causes severe acute respiratory syndrome (SARS) infects cells expressing the receptor angiotensin-converting enzyme 2 (ACE2). Here we show that codon optimization of the SARS-CoV S-protein gene substantially enhanced S-protein expression. We also found that two retroviruses, simian immunodeficiency virus (SIV) and murine leukemia virus, both expressing green fluorescent protein and pseudotyped with SARS-CoV S protein or S-protein variants, efficiently infected HEK293T cells stably expressing ACE2. Infection mediated by an S-protein variant whose cytoplasmic domain had been truncated and altered to include a fragment of the cytoplasmic tail of the human immunodeficiency virus type 1 envelope glycoprotein was, in both cases, substantially more efficient than that mediated by wild-type S protein. Using S-protein-pseudotyped SIV, we found that the enzymatic activity of ACE2 made no contribution to S-protein-mediated infection. Finally, we show that a soluble and catalytically inactive form of ACE2 potently blocked infection by S-protein-pseudotyped retrovirus and by SARS-CoV. These results permit studies of SARS-CoV entry inhibitors without the use of live virus and suggest a candidate therapy for SARS.
Subject(s)
Carboxypeptidases/metabolism , Leukemia Virus, Murine/physiology , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Severe acute respiratory syndrome-related coronavirus/genetics , Simian Immunodeficiency Virus/physiology , Viral Envelope Proteins/genetics , Viral Envelope Proteins/metabolism , Amino Acid Sequence , Angiotensin-Converting Enzyme 2 , Animals , Carboxypeptidases/genetics , Cell Line , HIV-1/genetics , Humans , Leukemia Virus, Murine/genetics , Leukemia Virus, Murine/metabolism , Molecular Sequence Data , Peptidyl-Dipeptidase A , Receptors, Virus/metabolism , Simian Immunodeficiency Virus/genetics , Simian Immunodeficiency Virus/metabolism , Spike Glycoprotein, Coronavirus , Virion/chemistry , Virion/metabolism , Virus ReplicationABSTRACT
Effective prophylaxis and antiviral therapies are urgently needed in the event of reemergence of the highly contagious and often fatal severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) infection. We have identified eight recombinant human single-chain variable region fragments (scFvs) against the S1 domain of spike (S) protein of the SARS-CoV from two nonimmune human antibody libraries. One scFv 80R efficiently neutralized SARS-CoV and inhibited syncytia formation between cells expressing the S protein and those expressing the SARS-CoV receptor angiotensin-converting enzyme 2 (ACE2). Mapping of the 80R epitope showed it is located within the N-terminal 261-672 amino acids of S protein and is not glycosylation-dependent. 80R scFv competed with soluble ACE2 for association with the S1 domain and bound S1 with high affinity (equilibrium dissociation constant, Kd=32.3 nM). A human IgG1 form of 80R bound S1 with a 20-fold higher affinity of 1.59 nM comparable to that of ACE2 (Kd=1.70 nM), and neutralized virus 20-fold more efficiently than the 80R scFv. These data suggest that the 80R human monoclonal antibody may be a useful viral entry inhibitor for the emergency prophylaxis and treatment of SARS, and that the ACE2-binding site of S1 could be an attractive target for subunit vaccine and drug development.