Adaptation of feline immunodeficiency virus subtype B strain TM2 to a feline astrocyte cell line (G355-5 cells).

Based on receptor usage during infection, feline immunodeficiency virus (FIV) isolates can be divided into two groups; those that require feline CD134 (fCD134) as a primary receptor in addition to CXCR4 to enter the cells, and those that require CXCR4 only. Most primary isolates, including strain TM2, belong to the former group and cannot infect a feline astrocyte cell line (G355-5 cells) due to a lack of fCD134 expression. In a previous study, we found that G355-5 cells transduced with fCD134 (termed G355-5/fOX40 cells) were susceptible to strain TM2 and the inoculated cells became persistently infected. In this study, we examined the phenotype of the virus prepared from the persistently infected cells (termed strain TM2PI). Intriguingly, strain TM2PI replicated well in naïve G355-5 cells and the inoculated G355-5 cells (termed G355-5/TM2PI cells) became persistently infected. The infection of TM2PI in G355-5 cells was inhibited by CXCR4 antagonist AMD3100 and TM2PI infected other fCD134-negative, CXCR4-positive cell lines, FeTJ and 3201 cells. Four amino acid substitutions were found in the Env protein of the strain TM2PI when compared with that of the parental strain TM2. Among the substitutions, the Env amino acid position at 407 of TM2PI was substituted to lysine which has been known to be responsible for the FIV tropism for Crandell feline kidney cells. The strain TM2PI will be useful for studying the receptor switching mechanism and FIV pathogenesis in cats.

Establishment of a feline astrocyte-derived cell line (G355-5 cells) expressing feline CD134 and a rapid quantitative assay for T-lymphotropic feline immunodeficiency viruses.

Few laboratory strains of feline immunodeficiency virus (FIV) can infect Crandell feline kidney cells (an epithelial-type of cells), however, most primary isolates are T-lymphotropic. T-lymphotropic FIV requires both feline CD134 (an activation marker of helper T-lymphocytes) and CXCR4 (a chemokine receptor) in infection as primary and secondary receptors, respectively. Using feline T-lymphoblastoid cell lines, titration of primary FIV isolates was carried out, however the titration assay was laborious and time-consuming. In this study, using G355-5 cells (a feline astrocyte-derived cell line) transduced with a cDNA of feline CD134 as target cells, an assay system was developed to quantitate primary FIV isolates. With a previous method using a feline T-lymphoblastoid cell line (MYA-1 cells) highly sensitive to FIV, it took 12 days to complete the assay, however, it took only 2 days with the new method. The FIV-infected cells became in a state of persistent infection, producing a large amount of FIV, indicating that the cells will be useful for propagation of T-lymphotropic FIV strains.

Focus assay on RD114 virus in QN10S cells.

Cats harbor an infectious endogenous retrovirus, named RD114 virus. It is therefore necessary to monitor RD114 virus production in feline cells which are used for biological products as substrates. In this study, a feline sarcoma-positive leukemia-negative (S+L-) fibroblast cell line, named QN10S cells, was found to be highly susceptible to RD114 pseudotype viruses. The cells were transformed by infection with RD114 virus and the numbers of foci could be counted. The sensitivity of the focus assay was lower than that of the LacZ marker rescue assay in detecting RD114 virus. Although the assay is not suitable to detect a small amount of the virus, the assay will be useful for virological studies of RD114 virus.

Differential role of two VDR coactivators, DRIP205 and SRC-3, in keratinocyte proliferation and differentiation.

Cell programs such as proliferation and differentiation involve the selective activation and repression of gene expression. The vitamin D receptor (VDR), through 1,25(OH)(2)D(3), controls the proliferation and differentiation of keratinocytes. Previously, we have identified two VDR binding coactivator complexes. In proliferating keratinocytes VDR bound preferentially to the DRIP complex, whereas in differentiated keratinocytes the SRC complex was preferred. We proposed that different coactivators are required for sequential gene regulation in the transition from proliferation to differentiation. Here we examined the roles of DRIP205 and SRC-3 in this transition. Silencing of DRIP205 and VDR caused hyperproliferation of keratinocytes, demonstrated by increased XTT and BrdU incorporation. SRC-3 silencing, on the other hand, did not have an effect on proliferation. In contrast, SRC-3 as well as DRIP205 and VDR silencing blocked keratinocyte differentiation as shown by decreased expression of keratin 1 and filaggrin. These results are consistent with the differential localization of DRIP205 and SRC-3 in skin. These results indicate that DRIP205 is required for keratinocyte proliferation. Both DRIP205 and SRC-3 are required for the keratinocyte differentiation. These results support the concept that the selective use of coactivators by VDR underlies the selective regulation of gene expression in keratinocyte proliferation and differentiation.