Intracellular localization and DNA-binding activity of a class of viral early phosphoproteins in human fibroblasts infected with human cytomegalovirus (Towne strain)

Abstract

Indirect immunofluorescence (IF) with monoclonal antibody M23 prepared against the nuclei of human embryo lung (HEL) cells infected with human cytomegalovirus (HCMV) Towne strain showed that the M23 antigen reactive with the M23 antibody was localized within distinct foci throughout the nucleus of infected HEL cells shortly after infection, even at 2 h post-infection (p.i.). The foci increased in size by 24 h p.i. and then the IF patterns changed to show the nuclear inclusion body-like structures at 72 h p.i. Treatment with phosphono-acetic acid, a HCMV DNA replication inhibitor, resulted in a nuclear pattern similar to that observed shortly after infection. The double-labelled IF test revealed that the HCMV UL44 antigen essential for viral DNA replication colocalized with the M23 antigen in the same intranuclear structure shortly after infection whereas neither viral antigen appeared to colocalize in most cells later after infection. The M23 antibody immunoprecipitated four proteins. 34K, 43K, 50K and 84K, in infected cells. To examine whether these proteins correspond to four early phosphoproteins encoded by the HCMV strain AD169 genome, the Towne strain DNA sequence corresponding to that encoding both the 34K and 43K proteins of strain AD169 was determined and transiently expressed in COS-7, Vero and HEL cells. These proteins were detected by the M23 antibody within the foci of these nuclei as found in the nuclei of productively infected cells shortly after infection. In addition, the 34K, 43K and 50K proteins at least were shown to be DNA-binding proteins by double- and single-stranded DNA-cellulose column chromatography. The relationship of these proteins to the status of viral DNA replication is discussed.