Takayuki Shojima

Identification of a Novel Subgroup of Koala Retrovirus from Koalas in Japanese Zoos.

ABSTRACT We identified a new subgroup of koala retrovirus (KoRV), named KoRV-J, which utilizes thiamine transport protein 1 as a receptor instead of the Pit-1 receptor used by KoRV (KoRV-A). By subgroup-specific PCR, KoRV-J and KoRV-A were detected in 67.5 and 100% of koalas originating from koalas from northern Australia, respectively. Altogether, our results indicate that the invasion of the koala population by KoRV-J may have occurred more recently than invasion by KoRV-A.

Identification of Novel Endogenous Betaretroviruses Which Are Transcribed in the Bovine Placenta

ABSTRACT Sequences of retroviral origin occupy approximately 10% of mammalian genomes. Various infectious endogenous retroviruses (ERVs) and functional retroviral elements have been reported for several mammals but not cattle. Here, we identified two proviruses, designated bovine endogenous retrovirus K1 (BERV-K1) and BERV-K2, containing full-length envelope (env) genes in the bovine genome. Phylogenetic analysis revealed that they belong to the genus Betaretrovirus. By reverse transcription (RT)-PCR, both BERV-K1 and -K2 env mRNAs were detected in the placenta and cultured bovine trophoblast cells. Real-time RT-PCR analysis using RNAs isolated from various bovine tissues revealed that BERV-K1 env mRNA was preferentially expressed in the placenta. Moreover, we also found the expression of doubly spliced transcripts, named the REBK1 and REBK2 genes. Both the REBK1 and REBK2 proteins have motifs for a putative nuclear localization signal and a nuclear export signal. REBK1 and REBK2 fused with green fluorescent proteins were localized mainly in the nuclei when they were expressed in bovine and porcine cells. In the env and 3′ long terminal repeats of BERV-K1 and -K2, we found regulatory elements responsible for the splicing and transport of viral RNAs and/or translation of the env genes. Although we have not identified the expressed Env proteins in bovine tissues, these data suggest that both BERV-K1 and BERV-K2 express Env proteins and that these proteins may have physiological functions in vivo.

Construction and characterization of an infectious molecular clone of koala retrovirus

ABSTRACT Koala retrovirus (KoRV) is a gammaretrovirus that is currently endogenizing into koalas. Studies on KoRV infection have been hampered by the lack of a replication-competent molecular clone. In this study, we constructed an infectious molecular clone, termed plasmid pKoRV522, of a KoRV isolate (strain Aki) from a koala reared in a Japanese zoo. The virus KoRV522, derived from pKoRV522, grew efficiently in human embryonic kidney (HEK293T) cells, attaining 106 focus-forming units/ml. Several mutations in the Gag (L domain) and Env regions reported to be involved in reduction in viral infection/production in vitro are found in pKoRV522, yet KoRV522 replicated well, suggesting that any effects of these mutations are limited. Indeed, a reporter virus pseudotyped with pKoRV522 Env was found to infect human, feline, and mink cell lines efficiently. Analyses of KoRV L-domain mutants showed that an additional PPXY sequence, PPPY, in Gag plays a critical role in KoRV budding. Altogether, our results demonstrate the construction and characterization of the first infectious molecular clone of KoRV. The infectious clone reported here will be useful for elucidating the mechanism of endogenization of the virus in koalas and screening for antiretroviral drugs for KoRV-infected koalas.

Heterogeneity of koala retrovirus isolates

Koala retrovirus (KoRV) is a gammaretrovirus which may induce immune suppression, leukemia and lymphoma in koalas. Currently three KoRV subgroups (A, B, and J) have been reported. Our phylogenetic analysis suggests that KoRV‐B and KoRV‐J should be classified as the same subgroup. In long terminal repeat (LTR), a KoRV‐B isolate has four 17 bp tandem repeats named direct repeat (DR)‐1, while a KoRV‐J isolate (strain OJ‐4) has three 37 bp tandem repeats named DR‐2. We also found that the promoter activity of the KoRV‐J strain OJ‐4 is stronger than that of original KoRV‐A, suggesting that KoRV‐J may replicate more efficiently than KoRV‐A.

Susceptibility and production of a feline endogenous retrovirus (RD-114 virus) in various feline cell lines.

RD-114 virus is a replication-competent feline endogenous retrovirus that has been classified as a xenotropic virus. In this study, we examined the expression of the receptors for RD-114 virus in feline cell lines by conducting a pseudotype virus infection assay. Six out of eight feline cell lines were susceptible to the RD-114 pseudotype virus and two cell lines (MCC and FER cells) were resistant. The two resistant cell lines and one cell line (CRFK cells) weakly sensitive to the RD-114 pseudotype virus were found to produce replication-competent RD114-like viruses by the LacZ marker rescue assay and the interference assay. These data strongly suggest that RD-114 virus is polytropic and resistance to RD-114 virus in certain cell lines is due to receptor interference but not polymorphism of the RD-114 receptors. In addition, we determined the amino acid sequences of the envelope region of RD-114-like viruses produced from MCC, FER and CRFK cells. The sequences were identical with the authentic RD-114 virus. Because many feline cell lines are used to manufacture live attenuated vaccines for companion animals, attention should be paid to contamination of the RD-114 virus in vaccines.

Epigenetic regulation on the 5'-proximal CpG island of human porcine endogenous retrovirus subgroup A receptor 2/GPR172B.

Porcine endogenous retroviruses (PERVs) have been considered one of the major risks of xenotransplantation from pigs to humans. PERV-A efficiently utilizes human PERV-A receptor 2 (HuPAR-2)/GPR172B to infect human cells; however, there has been no study on the regulation mechanisms of HuPAR-2/GPR172B expression. In this study, we examined the expression of HuPAR-2/GPR172B from the standpoint of epigenetic regulation and discussed the risks of PERV-A infection in xenotransplantation. Quantitative real-time RT-PCR revealed that HuPAR-2 mRNA was preferentially expressed in placental tissue, whereas it was highly suppressed in BeWo cells (a human choriocarcinoma cell line) and HEK293 cells. A CpG island containing the HuPAR-2 transcription starting site was identified by in silico analysis. The DNA methylation ratio (the relative quantity of methylcytosine to total cytosine) and histone modification (H3K9me3) levels in the CpG island measured by bisulfite genomic sequencing and ChIP assay, respectively, were inversely correlated with the mRNA levels. Both HuPAR-2 mRNA and HuPAR-2 protein were up-regulated in HEK293 cells by inhibiting DNA methylation and histone deacetylation. Additionally, promoter/enhancer activities within the CpG island were suppressed by in vitro DNA methylation. Our results demonstrated that epigenetic modification regulates HuPAR-2 expression.

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.

A soluble envelope protein of endogenous retrovirus (FeLIX) present in serum of domestic cats mediates infection of a pathogenic variant of feline leukemia virus.

T-lymphotropic feline leukemia virus (FeLV-T), a highly pathogenic variant of FeLV, induces severe immunosuppression in cats. FeLV-T is fusion defective because in its PHQ motif, a gammaretroviral consensus motif in the N terminus of an envelope protein, histidine is replaced with aspartate. Infection by FeLV-T requires FeLIX, a truncated envelope protein encoded by an endogenous FeLV, for transactivation of infectivity and Pit1 for binding FeLIX. Although Pit1 is present in most tissues in cats, the expression of FeLIX is limited to certain cells in lymphoid organs. Therefore, the host cell range of FeLV-T was thought to be restricted to cells expressing FeLIX. However, because FeLIX is a soluble factor and is expressed constitutively in lymphoid organs, we presumed it to be present in blood and evaluated its activities in sera of various mammalian species using a pseudotype assay. We demonstrated that cat serum has FeLIX activity at a functional level, suggesting that FeLIX is present in the blood and that FeLV-T may be able to infect cells expressing Pit1 regardless of the expression of FeLIX in vivo. In addition, FeLIX activities in sera were detected only in domestic cats and not in other feline species tested. To our knowledge, this is the first report to prove that a large amount of truncated envelope protein of endogenous retrovirus is circulating in the blood to facilitate the infection of a pathogenic exogenous retrovirus.

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.