(F) Electron micrograph of viral particle. gene expression was terminated in late protein-expressing cells. Finally, isolation of fluorescence-positive cells from nonresponders increased dynamic ranges of downstream experiments 10-fold. Thus, this study demonstrates a tool to examine heterogenic responses of KSHV reactivation for a deeper understanding of KSHV replication. IMPORTANCE Sensitivity and resolution of molecular analysis are often compromised by the use of techniques that measure the ensemble average of large cell populations. Having a Lapaquistat acetate research tool to nondestructively identify the KSHV replication stage in an infected cell would not only allow us to effectively isolate cells of interest from cell populations but also enable more precise sample selection for advanced single-cell analysis. We prepared a recombinant KSHV that can report on its replication stage in host cells by differential fluorescence emission. Consistent with previous host gene expression studies, our experiments reveal the highly heterogenic nature of KSHV replication/gene expression at individual cell levels. The utilization of a newly developed reporter-KSHV and initial characterization of KSHV replication in single cells are presented. infection. The indicated DNA copy numbers of virions were infected into iSLK cells, and the GFP-positive cell population was quantified with FACS 48?h after infection. (F) Electron micrograph of viral particle. KSHV viral particles in reactivated cells (left and Lapaquistat acetate middle) and purified from tissue culture supernatant (right) were visualized by electron microscopy. Rainbow-KSHV replication. We next examined viral particle production in culture supernatant. The results showed a clear increase of KSHV virions in culture media after stimulation. Rainbow-KSHV produces amounts of virions in supernatant comparable to those of rKSHV2.19 and about one-fourth of that produced by BAC16 wild-type virus (Fig. 2C). infection of the iSLK cells released mCardinal-ORF52, a tegument protein, and therefore increased the number of mCardinal-positive cells. Numbers of GFP-positive cells were also increased in a dose-dependent manner, and the infection ratio was comparable with that of the BAC16 wild-type virus (Fig. 2D and ?andE).E). Under electron microscopy, we observed Lapaquistat acetate densely stained Lapaquistat acetate virus-like particles in both reactivated cells and purified supernatant (Fig. 2F). Together, these results confirmed that fluorescence protein tags did not significantly interfere with viral DNA replication or virion production. Dynamic expression of fluorescence-tagged protein. We next reactivated the cells for different time points, and we fixed and observed fluorescence signals values of late genes; this likely reflects the smaller proportion of late gene-expressing cells in the cell population. Interestingly, a cell fraction (mCard+/mBFP?) showed very little late gene expression despite the presence of mCardinal protein, indicating late gene expression occurs very transiently and happens spontaneously with viral DNA replication. The largest amount of late gene expression was indeed seen in the double-positive cell fraction (mCard+/mBFP2+). With fractionation, we also observed clear downregulation of most of the viral genes in the mCardinal+/mBFP? cell fraction. The inclusion of proteasome or lysosomal inhibitors did not recover mBFP2 expression in the mCardinal-positive fraction, suggesting that downregulation of mBFP2 was primarily at transcriptional levels (Fig. S7). Downregulation of early genes in mCardinal-positive cells was further confirmed by RNA-fluorescent hybridization (FISH) with PAN RNA probes. Strong PAN RNA signals (red) were observed mostly in the mBFP-ORF6-positive cells (blue). Consistent with quantitative PCR (qPCR) results, mCardinal-positive cells (yellow) showed much lower intensities of PAN RNA signals (Fig. 6C and Fig. S8). Of note, the RNA-FISH signal of PAN RNA was extremely strong, so the exposure time for the red channel was set to 1/100?s. With this setting, we avoided having overlapping signals from mCherry-LANA, which requires a minimum of 0.5?s of exposure with the same setting. Although differences of cellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression among three groups (blue, Rabbit Polyclonal to Sumo1 brown, and purple; mBFP- and/or mCardinal-positive fractions) were statistically significant (infection. Based on our detection and measurement of mCardinal fluorescence, we think only a small fraction of cells produce viral particles, and of these, an even smaller subpopulation are generating infectious particles in the culture supernatant. It is interesting to determine.