Mitra Tanaji, Sosnovtsev Stanislav V, Green Kim Y
Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20892, USA.
J Virol. 2004 May;78(9):4931-5. doi: 10.1128/jvi.78.9.4931-4935.2004.
The genome of feline calicivirus (FCV) is an approximately 7.7-kb single-stranded positive-sense RNA molecule that is polyadenylated at its 3' end and covalently linked to a VPg protein (calculated mass, 12.6 kDa) at its 5' end. We performed a mutational analysis of the VPg protein in order to identify amino acids potentially involved in linkage to the genome and replication. The tyrosine residues at positions 12, 24, 76, and 104 were changed to alanines by mutagenesis of an infectious FCV cDNA clone. Viruses were recovered when Tyr-12, Tyr-76, or Tyr-104 of the VPg protein was changed to alanine, but virus was not recovered when Tyr-24 was changed to alanine. Growth properties of the recovered viruses were similar to those of the parental virus. We examined whether the amino acids serine, threonine, and phenylalanine could substitute for the tyrosine at position 24, but these mutations were lethal as well. A tyrosine at this relative position is conserved among all calicivirus VPg proteins examined thus far, suggesting that the VPg protein of caliciviruses, like those of picornaviruses and potyviruses, utilizes tyrosine in the formation of a covalent bond with RNA.
猫杯状病毒（FCV）的基因组是一个约7.7kb的单链正链RNA分子，其3'端进行了多聚腺苷酸化，5'端与一个VPg蛋白（计算分子量为12.6kDa）共价连接。我们对VPg蛋白进行了突变分析，以确定可能参与与基因组连接和复制的氨基酸。通过对感染性FCV cDNA克隆进行诱变，将第12、24、76和104位的酪氨酸残基替换为丙氨酸。当VPg蛋白的Tyr-12、Tyr-76或Tyr-104被替换为丙氨酸时，可回收病毒，但当Tyr-24被替换为丙氨酸时，未回收病毒。回收病毒的生长特性与亲本病毒相似。我们研究了丝氨酸、苏氨酸和苯丙氨酸是否可以替代第24位的酪氨酸，但这些突变也是致死性的。在迄今为止检测的所有杯状病毒VPg蛋白中，这个相对位置的酪氨酸是保守的，这表明杯状病毒的VPg蛋白与小RNA病毒和马铃薯Y病毒的VPg蛋白一样，在与RNA形成共价键时利用酪氨酸。
