doi: 10

doi: 10.1016/0022-2836(86)90395-5. (8). Rudiviruses infect members of the acidothermophilic crenarchaeal order of the and include four isolated viruses, SIRV1, SIRV2, Sulfolobus islandicus rod-shaped virus (SRV), and Acidianus rod-shaped virus (ARV) (9,C11), and a further two members, Sulfolobales Mexican rudivirus 1 (SMR1) and ARV2, assembled from metagenomes (12, 13). In this work, we provide evidence for the early formation of a replication focus in cells infected with SIRV2 where viral DNA synthesis Mouse monoclonal to CDC2 takes place and where viral single-stranded DNA (ssDNA) binding protein gp17 and host proteins host proliferating cell nuclear antigen (PCNA) and DNA polymerase I (Dpo1) are localized. In addition, it is shown that the host replicative Dpo1, rather than the other DNA polymerases (Dpo2 to Dpo4), is usually primarily responsible for viral DNA synthesis. RESULTS SIRV2 DNA replication is usually localized at a peripheral focus in infected cells. Recently, we showed that multiple copies of ssDNA intermediates are produced during SIRV2 replication (14). This allows us to determine the intracellular location of SIRV2 DNA synthesis by fluorescence microscopy using an anti-bromodeoxyuridine (BrdU) monoclonal antibody which exclusively targets BrdU in ssDNA but not in double-stranded DNA (dsDNA) (15). Since species do not carry a native thymidine kinase (gene was introduced into Sens1 which enabled incorporation of BrdU, a thymidine analog, into DNA in the transformant strain SsoTK. The expression of the gene in SsoTK cells was induced by arabinose in ACVY medium prior to contamination with SIRV2 (multiplicity of contamination [MOI] of 5) and MDV3100 addition of 1 1 mM BrdU to the culture. In the infected cells, the BrdU signal was clearly observed at 2 h postinfection (hpi), consistent with the presence of numerous ssDNA replication intermediates from the virus (Fig. 1A). In contrast, the uninfected SsoTK cells contained only dsDNA and DNA denaturation with HCl was required in order to detect BrdU-containing DNA (Fig. 1A). Notably, the BrdU signal in the infected cells was located at a single focus at the periphery of the cell (Fig. 1A). This strongly suggested that this SIRV2 replication was organized and confined to a single intracellular site. Open in a separate window FIG 1 Formation of a viral replication focus in SIRV2-infected cells. (A) Viral DNA synthesis occurs at discrete sites at the periphery of SIRV2-infected cells. Samples with (+) or without (?) SIRV2 contamination were subjected to immunostaining at 2 hpi with (+) or without (?) pretreatment with HCl. Images show the differential interference contrast (DIC) field, BrdU-Alexa Fluor 488 field (green), DAPI staining of DNA (blue), and merged images of the BrdU and DNA fields (Overlay). (B) Overlapping localizations of the BrdU focus and the gp17 viral protein in SIRV2-infected SsoTK cells. Images show the DIC field, the BrdU-Alexa Fluor 488 field (green), the viral gp17-Alexa Fluor 594 field (), and the DIC field merged with the BrdU and gp17 fluorescent signals (Overlay). (C) Time course of the distribution of gp17 in SIRV2-infected 5E6 cells (MOI, 5). Images show the DIC field, the viral gp17-Alexa Fluor 594 field (red), DAPI staining of DNA (blue), and the DIC field merged with the gp17 fluorescent signal (Overlay). MDV3100 White asterisks indicate cells without DNA. Bar, 1 m. To investigate whether viral replication proteins were recruited to the site of viral DNA synthesis, we MDV3100 localized viral protein gp17 in the infected SsoTK cells. This ssDNA-binding protein is considered to be essential for viral replication and is highly expressed throughout the SIRV2 replication cycle (16, 17). First, the SsoTK cells carrying the gene were infected with SIRV2 and supplemented with BrdU as described above. gp17 was then visualized by epifluorescence microscopy using a guinea pig anti-gp17 polyclonal antibody, and BrdU-containing ssDNA was visualized as described above. Physique 1B shows the result from the immunostaining of both gp17 (red) and BrdU-containing ssDNA (green). In a given cell, the gp17 protein was also located at a single peripheral focus that overlapped completely with the site of MDV3100 DNA synthesis, strongly suggesting that this focus constitutes a viral replication center. Next, the formation of the SIRV2 replication focus was examined throughout the viral life cycle, MDV3100 which lasts about 12 h (17, 18). To circumvent any deleterious effects caused by the incorporation of BrdU into the viral DNA, we excluded BrdU from the SIRV2-infected 5E6 cultures (MOI of 5) and monitored the formation of the viral replication focus by examining the distribution of gp17. The gp17 foci were detected.