ABSTRACT
There is evidence that a carboxyl-terminal valine residue is an anterograde transport signal for type I transmembrane proteins. Removal of the signal would either delay glycosylation in the Golgi complex of proteins destined to recycle to the endoplasmic reticulum or determine accumulation in the endoplasmic reticulum of newly synthesized proteins destined for the plasma membrane. We used the human CD8 alpha glycoprotein to investigate the role of the carboxyl-terminal valine in the exocytic pathway. Using immunofluorescence light microscopy, metabolic labeling, and cell fractionation, we demonstrate that removal of the carboxyl-terminal valine residue delays transport of CD8 alpha from the endoplasmic reticulum to the intermediate compartment. Removal of the residue did not affect the other steps of the exocytic pathway or the folding/dimerization and glycosylation processes. Therefore, it is likely that this signal plays a role in the transport of CD8 alpha from the endoplasmic reticulum to the intermediate compartment either before or during the formation of the transport vesicles that drive the exit the protein from the endoplasmic reticulum.
Subject(s)
CD8 Antigens/chemistry , CD8 Antigens/metabolism , Endoplasmic Reticulum/metabolism , Valine , Amino Acid Sequence , Amino Acid Substitution , Animals , Cats , Cattle , Cell Line , Cell Membrane/metabolism , Chickens , Cytosol/metabolism , Dimerization , Dogs , Exocytosis , Fluorescent Antibody Technique, Indirect , Humans , Mammals , Mice , Molecular Sequence Data , Mutagenesis, Site-Directed , Pongo pygmaeus , Protein Transport , Rats , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Deletion , Sequence Homology, Amino Acid , TransfectionABSTRACT
Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae of the order Nidovirales. The unsegmented, infectious genome of EAV is 12,704 nt in length [exclusive of the poly(A) tail] and contains eight overlapping genes that are expressed from a 3'-coterminal nested set of seven leader-containing mRNAs. To investigate the importance of the overlapping gene arrangement in the viral life-cycle and to facilitate the genetic manipulation of the viral genome, a series of mutant full-length cDNA clones was constructed in which either EAV open reading frames (ORFs) 4 and 5 or ORFs 5 and 6 or ORFs 4, 5, and 6 were separated by newly introduced AflII restriction endonuclease cleavage sites. RNA transcribed from each of these plasmids was infectious, demonstrating that the overlapping gene organization is not essential for EAV viability. Moreover, the recombinant viruses replicated with almost the same efficiency, i.e., reached nearly the same infectious titers as the wildtype virus, and stably maintained the mutations that were introduced. The AflII site engineered between ORFs 5 and 6 was subsequently used to generate a virus in which the ectodomain of the ORF 6-encoded M protein was extended with nine amino acids derived from the extreme N-terminus of the homologous protein of mouse hepatitis virus (MHV; family Coronaviridae, order Nidovirales). This nonapeptide contains a functional O-glycosylation signal as well as an epitope recognized by an MHV-specific monoclonal antibody, both of which were expressed by the recombinant virus. Although the hybrid virus had a clear growth disadvantage in comparison to the parental virus, three serial passages did not result in the loss of the foreign genetic material.
Subject(s)
DNA, Complementary/genetics , Epitopes/genetics , Equartevirus/genetics , Genes, Overlapping/genetics , Genes, Viral/genetics , Genetic Engineering , 5' Untranslated Regions/genetics , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Base Sequence , Cell Line , Cloning, Molecular , Coronavirus M Proteins , Deoxyribonucleases, Type II Site-Specific/metabolism , Epitopes/immunology , Equartevirus/physiology , Genome, Viral , Glycosylation , Molecular Sequence Data , Murine hepatitis virus/genetics , Murine hepatitis virus/immunology , Mutagenesis, Insertional/genetics , Open Reading Frames/genetics , RNA, Viral/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/metabolism , Viral Matrix Proteins/genetics , Viral Matrix Proteins/immunology , Viral Matrix Proteins/metabolism , Virus ReplicationABSTRACT
We obtained the cDNA sequence of the monkey homologue of the intermediate compartment protein ERGIC-53 by both cDNA library screening and RT-PCR amplification. The final sequence of 2422 nts of the monkey ERGIC-53 cDNA is 96.2% identical to the human ERGIC-53 cDNA and 87% and 67% identical to the rat and amphibian cDNA, respectively. The translated CV1 ERGIC-53 protein is 96.47% identical to the human ERGIC-53, 87% identical to the rat p58 and 66. 98% to the Xenopus laevis protein. Southern blot analysis of multiple genomic DNAs shows the presence of sequences similar to ERGIC-53 in different species. ERGIC-53 is expressed as a major transcript of about 5.5 kb in either monkey CV1 or in human CaCo2. A shorter transcript of 2.3 kb was detected in both cell lines and in mRNAs derived from human pancreas and placenta.
