TI - Results and discussion I5 is expressed as a post-replicative protein , does not appear to be PHOSphorylated , and is present in crescents , immature and mature virions . AB - The I5L gene encodes a protein of 78 aa , which is predicted to have two highly hydrophobic domains at the N- and C-termini and is likely to be an integral membrane protein (Fig 1A) . Indeed , VP13 was originally identified as a component of the membrane of mature virions [12] . The protein is highly conserved in diverse chordopoxviruses , as is evident in the alignment shown in Fig 1B . To enable a structural and functional characterization of I5 , we generated a number of recombinant viruses , for which the key genomic features are depicted in Fig 1C . These recombinant viruses will be discussed in more detail throughout the remainder of this report . First , we generated the vI5V5 virus , in which a V5 epitope tag has been inserted at the C-terminus of the endogenous I5 open reading frame ( see Fig 1C ) . Immunoblot analysis permitted the ready visualization of the I5 protein in cells that had been infected with vI5V5 for 8 h ( Fig 2A ) . However , inclusion of ara C (cytosine arabinoside) , which is an inhibitor of DNA replication and thus of intermediate and late gene expression , blocked the expression of I5 . Hence , I5 is expressed as a post-replicative gene . The G7 protein , which is a known late protein , served as an internal control for the immunoblot analysis [28,29] . The predicted amino acid sequence of I5 contains a number of highly conserved serine , threonine and tyrosine residues that could be modified by PHOSphorylation in vivo ( marked by black ovals in Fig 1B ; ser/thr present in >= 10 of 13 orthologs shown ) . Therefore , we also performed immunoprecipitation analysis on infected cells that had been metabolically labeled with either 35S-met or 32PPi (Fig 2B) . Although 35S-labeled I5 could be immunoprecipitated with the anti-V5 antibody , no 32P-labeled I5 was retrieved . In contrast , both 35S-labeled and 32P-labeled F18 , a known PHOSphoprotein that is expressed at post-replicative times and encapsidated in the virion core , were retrieved in immunoprecipitations performed with the anti-F18 serum [30-33] . Thus , it appears that the I5 protein is not PHOSphorylated in vivo . To monitor the intracellular localization of I5 , we used immunofluorescence microscopy to visualize the I5 protein in cells that had been infected with vI5V5 for 8 h , or with wt virus as a negative control . DAPI was included to visualize nuclei and viral replication factories . As shown in Fig 2C , the I5V5 protein showed a punctate distribution that overlapped the viral replication factories and extended throughout the cytoplasm . Limited background staining was seen in cells infected with wild-type virus . The punctate staining was consistent with localization of I5 to intracellular membranes , although I5 was not restricted to any sub-cellular compartment such as the ER , the Golgi , or the plasma membrane . Since the I5L gene encodes the protein identified as the VP13 component of purified virions , we utilized immunoelectron microscopy to determine whether I5 was associated with the classical intermediates in virion morphogenesis ( crescents and immature virions ) as well as with mature virions . As shown in Fig 2D , I5 was found to associate with crescents (A) , immature (A) and mature virions ( B-F ) .