A sensitive luminescence syncytium induction assay (LuSIA) based on a reporter plasmid containing a mutation in the glucocorticoid response element in the long terminal repeat U3 region of bovine leukemia virus

Bovine leukemia virus (BLV), which belongs to family Retroviridae, is an oncogenic member of genus Deltaretrovirus that causes enzootic bovine leukosis (EBL), the most common neoplastic disease of cattle [1]. BLV infects cattle worldwide and causes serious problems for the cattle industry. For example, BLV infection decreases milk production and cow longevity without onset of leukosis [2].

BLV infectivity is typically measured using the syncytium induction assay (SIA) [3, 4]. Recently, we developed a new method for assessing BLV infectivity, the luminescence syncytium induction assay (LuSIA) [5], which uses CC81-BLU3G as the reporter cell line. CC81-BLU3G cells are stably transfected with a pBLU3-EGFP reporter plasmid harboring the BLV-LTR U3 region as the promoter and enhanced green fluorescent protein (EGFP) as the reporter gene. When these CC81-BLU3G cells are infected with BLV, they form large multinuclear syncytia that express EGFP. Thus, LuSIA facilitates detection and quantitative analysis of BLV infectivity.

The BLV long terminal repeat (LTR) consists of three regions: U3, R, and U5. The U3 region contains three Tax-responsive elements (TxREs) that are recognized by the BLV protein Tax, the main regulator of viral replication [6‐8]. In particular, the binding of Tax to TxRE-2 is predominantly responsible for BLV replication [6]. Moreover, binding of Tax to BLV TxREs is mediated by the cAMP response element–binding protein (CREB) [9]. By contrast, the BLV-LTR contains multiple binding sites for several translation factors: A binding site for the interferon responding factor is present in the U5 region, and two AP-4 sites, a glucocorticoid response element (GRE), and a PU.1/Spi-B–binding site are present in the U3 region. These binding sites regulate BLV transcription, either dependent on or independently of BLV-Tax expression [7, 10‐14]. However, their effects on viral replication (i.e., up- or down-regulation) differ among target cell lines [11‐13]. In addition, BLV transcriptional activity is affected by acetylation and methylation of these binding sites [15, 16]. The GRE-mutated BLV-LTR promoter decreases BLV replication activity in the absence of Tax expression [11, 12] and is not affected by acetylation [14], implying that this promoter could decrease the background of BLV-LTR–derived transcription.

Here, we constructed reporter plasmids in which the EGFP reporter gene was expressed under control of the GRE-mutated LTR-U3 promoter (pBLU3_GREM-EGFP). We also established a new CC81-derived reporter cell line harboring the GRE-mutated LTR-U3 promoter (CC81-GREMG); this line enabled direct visualization of BLV infectivity, leading to development of a more sensitive LuSIA for detection of both cell-to-cell and cell-free BLV infection. Moreover, co-culture with bovine immunodeficiency-like virus (BIV)- and bovine foamy virus (BFV)-infected cells confirmed that the LuSIA is BLV-specific. Finally, we established a new LuSIA based on CC81-GREMG cells in conjunction with white blood cells (WBCs) from BLV-infected cows. To test clinical applicability of the new assay, we examined the activity of neutralizing antibodies on plasma collected from BLV-infected cows.

The BLV-LTR U3 region plays a critical role in regulating BLV-Tax–dependent viral replication. Previously, we reported a method for BLV infectivity analysis, which we named the LuSIA [5]. This assay uses the established reporter cell line CC81-BLU3G, stably transfected with a reporter plasmid in which the BLV-LTR U3 region serves as the promoter. LuSIA with CC81-BLU3G cells could measure the cell-to-cell BLV infectivity clearly and quantitatively. However, the BLV-LTR U3 region also contains other transcription factor response elements that regulate viral replication [7, 10‐16]. Therefore, in this study, we constructed a new plasmid, which is a variant of the BLV-LTR U3 promoter harboring a mutation in the GRE sequence (Fig. 1). The mutated GRE promoter generated a significantly weaker background signal than promoters harboring the wild-type GRE sequence, resulting in a higher signal-to-background ratio (data not shown). This result suggested that reporter cells bearing a GRE-mutated U3 promoter would be more suitable for our BLV infectivity assay than previously established cell lines. Accordingly, we established a stably transfected cell line called CC81-GREMG. CC81-GREMG cells expressed EGFP efficiently in response to co-culture with FLK-BLV cells, which constitutively express BLV (Fig. 2a), and as expected, they also exhibited lower background fluorescence. Additionally, significantly more syncytia expressing EGFP were detected in assays using CC81-GREMG than in those using our previously established cell line, CC81-BLU3G (Fig. 2b). The reduction in background fluorescence made a major contribution to identification of samples with lower infectivity, likely because the decrease in background fluorescence made it easier to detect fluorescent syncytia with lower intensities.

Because LuSIA using CC81-GREMG was more sensitive, we attempted to use it to measure cell-free BLV infectivity. BLV was naturally and mainly transmitted via cell-to-cell but not cell-free, because it is extremely lower extent and less their infectivity of virions in the blood of BLV-infected cattle [23]. In addition, it is considered that cell-free infection occurs only limited artificial condition like a vaccination using culture supernatant of infected cell lines. Therefore, we used culture supernatant from FLK-BLV cells bearing infectious BLV virions, which is sufficient to infect the reporter cell line [24]. When CC81-GREMG cells were cultured with FLK-BLV culture supernatant containing virus particles, we successfully observed EGFP-expressing syncytia (Fig. 3a). Thus, we could quantitatively assess BLV cell-free infectivity, which was clearly correlated with the amount of BLV p24 protein in the supernatant (Fig. 3b).

Co-culture of BIV and BFV with CC81 cells results in syncytium formation (as observed for BLV) [17, 18]. Therefore, we confirmed that the LuSIA based on CC81-GREMG cells is specific for BLV (Fig. 4). CC81-GREMG expressed EGFP in the presence of the BLV viral transcriptional protein Tax, but not in the presence of BIV Tat and BFV Tas proteins, suggesting that LuSIA based on CC81-GREMG cells is highly specific for BLV. This result is very important when assays of BLV infectivity are carried out on farms with multiple/mixed infections. Indeed, mixed infection of a single cow with BLV and other viruses has been reported [25]; Recently, we constructed luciferase-based reporter plasmids pBLU3-Luc and pBLU3_GREM-Luc, which have no requirement for syncytium formation to assess the BLV infectivity, and established stably transfectant cell lines CC81-BLU3L and CC81-GREML, respectively (unpublished data). This appears to be one way of solving the problem of BLV infectivity assay on the mix-infected cow and will be a useful tool experimentally; however, it is expensive and more complicated than the system based on EGFP visualization. Taken together, these results suggest that LuSIA based on CC81-GREMG is highly sensitive and specific, and as such will be useful for developing analytical methods such as titration of anti–BLV-neutralizing antibodies.

In this study, we clearly demonstrated that our new LuSIA with CC81-GREMG is more useful than LuSIA with CC81-BLU3G for measurement of BLV infectivity. In our previous report, we used peripheral blood mononuclear cells as the infected bovine samples for co-cultivation; however, WBCs are easier to isolate and can also be used for co-cultivation (data not shown). Therefore, to confirm that our new protocol for LuSIA using CC81-GREMG could be used to assess the BLV infectivity of infected cows, we co-cultured CC81-GREMG with WBCs collected from BLV-infected cows. LuSIA using the CC81-GREMG protocol could also detect BLV infectivity (Fig. 5a), and as expected had higher resolution than our previous protocol using CC81-BLU3G (Fig. 5b). Furthermore, the number of syncytia expressing EGFP was strongly correlated with BLV provirus loads (Fig. 6b).

We also successfully assessed the titer of neutralizing antibodies in plasma collected from BLV-infected cows (Fig. 7). The plasma which collected from BLV-infected cattle includes the specific antibodies toward against BLV viral proteins, but not virions itself [23]. We could clearly discriminate the plasma of BLV-infected cows, which has BLV-neutralizing activity, from the plasma of uninfected cows. The number of fluorescence syncytia caused by co-culture with FLK-BLV was clearly decreased by plasma collected from BLV-infected cows (at 12–48-fold dilutions). By the way, it increased by presence of 12- to 768-fold diluted plasma collected from BLV-uninfected cow and over 96-fold diluted plasma collected from BLV-infected cow compared with control wells without plasma. This phenomenon suggests that plasma act as a nutrient, analogous to FBS, for CC81-GREMG and/or FLK-BLV cells. Accordingly, this result suggests that LuSIA using the CC81-GREMG protocol can not only be used to assess infectivity, but could also be adapted to other assays.

We developed a new protocol for LuSIA using CC81-GREMG cells to measure BLV infectivity. The new method is quantitative and more sensitive than the previous protocol using CC81-BLU3G cells. The new LuSIA is adaptable to several types of assay, such as BLV neutralization by plasma or serum, BLV contamination contradiction assay of bovine vaccines, and screening of anti-viral drugs.