Role of the myristoylation site in expressing exogenous functional proteins in coxsackieviral vector.
We generated a cardiotropic replication-competent chimeric coxsackievirus B3 (CVB3) to express alcohol dehydrogenase (ADH). Although exogenously expressed ADH was found by Western blot analysis, its enzyme function was repressed. To define the factor that inhibits the enzymatic function of ADH, we introduced a site-directed mutation at the second amino acid (MGAQEF···) of the CVB3 VP0 capsid protein, effectively changing glycine to alanine. This glycine is known to be a myristoylation site during viral capsid protein maturation in infected cells. In contrast to the unmodified virus, ADH expression and enzymatic function were readily detectable in the mutated rCVB3-ADH (G2A) virus. While expression of ADH required mutation of the CVB3 VP0 myristoylation site for proper function, another chimeric virus that expresses green fluorescent protein (rCVB3-GFP (G or A)) worked independently of the myristoylation site. Indeed, infected HeLa cells displayed GFP under a fluorescent microscope. These results indicate that the myristoylation site in the VP0 capsid protein inhibited the expression of enzymatically active ADH but not GFP. VP0 myristoylation is dispensable for chimeric CVB3 virus replication.
Department of Medicine, Sungkyunkwan University School of Medicine, Cardiac and Vascular Center, Samsung Medical Center, Seoul, Korea.
Byung-Kwan
BK
Lim
Soo-hyeon
SH
Yun
Eun-Seon
ES
Ju
Chae-Ok
CO
Gil
Duk-Kyung
DK
Kim
Eun-Seok
ES
Jeon
D000409:Alanine / Q000737:chemistry / Q000378:metabolism; D000426:Alcohol Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D036022:Capsid Proteins / Q000235:genetics* / Q000378:metabolism; D002522:Chlorocebus aethiops; D004330:Drosophila; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D029822:Enterovirus B, Human / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D005998:Glycine / Q000737:chemistry / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D019814:Myristic Acid / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication
D036022:Capsid Proteins; D029721:Drosophila Proteins; D011993:Recombinant Fusion Proteins; D019814:Myristic Acid; D049452:Green Fluorescent Proteins; D000426:Alcohol Dehydrogenase; D000409:Alanine; D005998:Glycine