Structural analysis of a 64-kDa major structural protein of human cytomegalovirus (Towne): identification of a phosphorylation site and comparison to pp65 of HCMV (AD169).
The major 64-kDa structural protein of human cytomegalovirus (pp64) was isolated from a guanidinium chloride extract of the virions and dense bodies of HCMV (Towne) by reverse-phase HPLC. Purified pp64 was reduced and alkylated followed by digestion with trypsin. The molecular mass of each of the tryptic peptides was determined by fast atom bombardment/mass spectrometry and compared with the predicted molecular mass of the fragments deduced from the corresponding DNA-derived peptide sequence of pp65 from HCMV (AD169). Microsequence analysis was employed to confirm selected peptides. Results of protein sequence analysis of pp64 from HCMV (Towne) are in complete agreement with the DNA-derived protein sequence of pp65 predicted for HCMV (AD169) with the following exceptions and modifications. The protein isolated from HCMV (Towne) was found to contain an Ala at position 448 instead of Ser448 reported for the protein from HCMV (AD169). We also identified Ser472 as a site of phosphorylation in pp64 from HCMV (Towne). Finally, on the basis of the sequence of HCMV (AD169) DNA fragment encoding the matrix protein and on S1 nuclease protection analysis, it has been predicted that one version of the matrix protein (possibly the lower matrix protein, Mr 65K) is encoded by an mRNA that is formed through splicing of a short intron. However, we have obtained peptides that contain sequences spanning through the splice-junction region, suggesting that in HCMV (Towne), the matrix protein is encoded by an unspliced message.
Pande
H
H
Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, California.
Lee
T D
TD
Churchill
M A
MA
Zaia
J A
JA
D000595:Amino Acid Sequence; D003587:Cytomegalovirus / Q000032:analysis*; D004279:DNA, Viral / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000096:biosynthesis / Q000235:genetics*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000096:biosynthesis; D014763:Viral Matrix Proteins / Q000096:biosynthesis / Q000235:genetics*; D015678:Viral Structural Proteins / Q000096:biosynthesis / Q000235:genetics*
D004279:DNA, Viral; D010750:Phosphoproteins; D012333:RNA, Messenger; D014763:Viral Matrix Proteins; D015678:Viral Structural Proteins; C065418:cytomegalovirus matrix protein 65kDa; C065417:major structural protein, cytomegalovirus 64kDa