Characterization of Prototype Foamy Virus Infectivity in Transportin 3 Knockdown Human 293t Cell Line

( Faysal Bin Hamid ),( Jinsun Kim ),( Cha-gyun Shin ) 한국미생물 · 생명공학회 2017 Journal of microbiology and biotechnology Vol.27 No.2

The foamy viruses are currently considered essential for development as vectors for gene delivery. Previous studies demonstrated that prototype foamy virus (PFV) can infect and replicate prevalently in a variety of cell types for its exclusive replication strategy. However, the virus-host interaction, especially PFV-transportin3 (TNPO3), is still poorly understood. In our investigation of the role of TNPO3 in PFV infection, we found lower virus production in TNPO3 knockdown (KD) cells compared with wild-type 293T cells. PCR analysis revealed that viral DNAs were mostly altered to circular forms: both 1-long terminal repeat (1-LTR) and 2-LTR in TNPO3 KD cells. We therefore suggest that TNPO3 is required for successful PFV replication, at least at/after the nuclear entry step of viral DNA. These findings highlight the obscure mysteries of PFV-host interaction and the requirement of TNPO3 for productive infection of PFV in 293T cells.

IFITM proteins inhibit the late step of feline foamy virus replication

김지선,신차균 한국통합생물학회 2020 Animal cells and systems Vol.24 No.5

Interferon-induced transmembrane (IFITM) proteins as host restriction factors are known to inhibit the replication of several viruses. In this study, transient IFITM expression vectors were used to investigate whether IFITMs inhibit feline foamy viral (FFV) replication and which step of viral replication is inhibited. In our studies, viral production was significantly reduced when cells were infected with FFV at almost same times such as −3, 0, or 3 h post-transfection with IFITM vector. However viral production was not reduced even though cells were infected with FFV at 3 or 6 days post-transfection when production of IFITM proteins was maximized. Considering that IFITM expression was maximized at 3 days post-transfection, the stage of viral replication inhibited by IFITM appears to be the late step of viral replication. Moreover, the viral Gag proteins detected in the virus-infected cell lysates were proportionally correlated with viral titer of the culture supernatants. Therefore, it is likely that IFITMs can restrict production of FFV at the late step of viral replication.

Antiviral activities of hydroxylated flavones on feline foamy viral proliferation

이가은,김진선,신차균 한국응용생명화학회 2017 Applied Biological Chemistry (Appl Biol Chem) Vol.60 No.4

Many hydroxylated flavones are reported to have antibacterial, anticancer, or antiviral activities. In this study, eleven hydroxylated flavones including 3,30,40,5,7-pentahydroxyflavone (quercetin), three polymethoxyflavones, two polyethoxyflavones, two polypropoxyflavones, one butoxyflavone, and two benzoxyflavones were tested for antiviral activity using feline foamy virus. Most of the compounds tested showed that they did not have significant cytotoxic effect on the crandell-Ress feline kidney cells. However, four compounds, including quercetin, 5,30-dihydroxy- 3,7,40-trimethoxyflavone, 5-hydroxy-3,7,30,40-tetramethoxyflavone, and 3,5,7,30,40-pentahydroxyflavone, showed a strong inhibitory effect on feline foamy virus (FFV) proliferation, by reducing viral production to 7–24% of that in the un-treated control when they were added to the cells at a final concentration of 10 lM. These results were supported by western blot detecting viral protein in the infected cell lysate. In further analysis, quercetin was observed to have a direct inhibitory effect on reverse transcriptase and integrase in vitro, which can explain the mechanism by which quercetin inhibits FFV proliferation. These preliminary results suggest that hydroxylated flavones such as quercetin, 5,30-dihydroxy-3,7,40-trimethoxyflavone, 5-hydroxy-3,7,30,40-tetramethoxyflavone, and 3,5,7,30,40- pentahydroxyflavone have strong antiretroviral activities.

Genealogical Diversity of Endogenous Retrovirus in the Jawless Fish Genome

Jing Song,Jie Wei,Yongping Ma,Yan Sun,Zhi Li 한국미생물·생명공학회 2023 Journal of microbiology and biotechnology Vol.33 No.11

Retroviral integration into ancient vertebrate genomes left traces that can shed light on the early history of viruses. In this study, we explored the early evolution of retroviruses by isolating nine Spuma endogenous retroviruses (ERVs) and one Epsilon ERV from the genomes of Agnatha and Chondrichthyes. Phylogenetic analysis of protein sequences revealed a striking pattern of coevolution between jawless fish ERV and their host, while shark ERV underwent ancient cross-class viral transmission with jawless fish, ray-finned fish, and amphibians. Nucleotide sequence analysis showed that jawless fish ERV emerged in the Palaeozoic period, relatively later than ray-finned fish ERV. Moreover, codon analysis suggested that the jawless fish ERV employed an infection strategy that mimics the host codon. The genealogical diversity of ERVs in the jawless fish genome highlights the importance of studying different viral species. Overall, our findings provide valuable insights into the evolution of retroviruses and their interactions with their hosts.

Apoptotic events induced by prototype foamy virus infection

김진선,Faysal Bin Hamid,신차균 한국통합생물학회 2016 Animal cells and systems Vol.20 No.1

Foamy virus infection induces cytopathology in several cell types from different species. But the exact mechanism is still unknown. In this study, we have investigated the mechanism of cell death induced by prototype foamy virus (PFV) infection in baby hamster kidney (BHK 21) cell lines. PFV induces apoptosis by exhibiting morphological alterations such as chromatin condensation, blebbing, and nuclear fragmentation. In addition, PFV infection causes chromosomal DNA fragmentation, up-regulation of Bax, and activation of caspase-3, but not caspase-8. Up-regulation of Bax initiates the translocation of cytochrome-c from mitochondria to the cytoplasm, suggesting predominantly to the mitochondrial-mediated pathway. Blocking apoptosis using caspase inhibitors increased PFV-infected BHK 21 cell viability. Although blocking apoptosis resulted in reduced progeny release, maximal accumulation of PFV was found in apoptosis-blocked cells. This report provides the first experimental evidence of apoptosis induced by PFV infection, which will provide valuable insights for foamy viral pathology.

Foamy Virus Integrase in Development of Viral Vector for Gene Therapy

김지선,이가은,신차균 한국미생물·생명공학회 2020 Journal of microbiology and biotechnology Vol.30 No.9

Due to the broad host suitability of viral vectors and their high gene delivery capacity, many researchers are focusing on viral vector-mediated gene therapy. Among the retroviruses, foamy viruses have been considered potential gene therapy vectors because of their non-pathogenicity. To date, the prototype foamy virus is the only retrovirus that has a high-resolution structure of intasomes, nucleoprotein complexes formed by integrase, and viral DNA. The integration of viral DNA into the host chromosome is an essential step for viral vector development. This process is mediated by virally encoded integrase, which catalyzes unique chemical reactions. Additionally, recent studies on foamy virus integrase elucidated the catalytic functions of its three distinct domains and their effect on viral pathogenicity. This review focuses on recent advancements in biochemical, structural, and functional studies of foamy virus integrase for gene therapy vector research.

Influence of Pretreatment with Immunosuppressive Drugs on Viral Proliferation

( Ga-eun Lee ),( Cha-gyun Shin ) 한국미생물생명공학회(구 한국산업미생물학회) 2018 Journal of microbiology and biotechnology Vol.28 No.10

Immunosuppressive drugs are used to make the body less likely to reject transplanted organs or to treat autoimmune diseases. In this study, five immunosuppressive drugs including two glucocorticoids (dexamethasone and prednisolone), one calcineurin inhibitor (cyclosporin A), one non-steroid anti-inflammatory drug (aspirin), and one antimetabolite (methotrexate) were tested for their effects on viral proliferation using feline foamy virus (FFV). The five drugs had different cytotoxic effects on the Crandell-Ress feline kidney (CRFK) cells, the natural host cell of FFV. Dexamethasone-pretreated CRFK cells were susceptible to FFV infection, but pretreatment with prednisolone, cyclosporin A, aspirin, and methotrexate showed obvious inhibitory effects on FFV proliferation, by reducing viral production to 29.8-83.8% of that of an untreated control. These results were supported by western blot, which detected viral Gag structural protein in the infected cell lysate. As our results showed a correlation between immunosuppressive drugs and susceptibility to viral infections, it is proposed that immunecompromised individuals who are using immune-suppressive drugs may be especially vulnerable to viral infection originated from pets.

원조포미바이러스 U5 LTR 말단의 보존적인 잔기의 돌연변이에 대한 인테그라제의 반응성

현우석,이동현,고현탁,신차균,Hyun, U-Sok,Lee, Dong-Hyun,Ko, Hyun-Tak,Shin, Cha-Gyun 대한약학회 2008 약학회지 Vol.52 No.2

The long terminal repeat (LTR) of retroviral DNA genome plays an important role in the integration process by providing substrate recognition site for viral integrase (IN). The dinucleotide CA near the 3'-end of the LTR termini is completely conserved among retoviruses. In order to study specificity of interaction between prototype foamy virus (PFV) IN and its U5 LTR DNA, the effect of mutagenesis of the CA sequence was investigated by studying reactivity of PFV IN to the mutant LTR substrates. Replacement of only the C or the A allowed 60 to 100% of the reactivity of the wild type LTR substrate. In addition, replacement of the C and the A showed 50 to 80% of the reactivity of the wild type LTR substrate, indicating that PFV IN has less specificity on the conserved CA sequence when it is compared to the other retroviral INs. Therefore it is suggested that PFV IN is less dependent on the conserved sequence of LTR termini for its enzymatic reaction.

Integrase C-terminal residues determine the efficiency of feline foamy viral DNA integration

Kim, Jinsun,Lee, Ga-Eun,Lochelt, Martin,Shin, Cha-Gyun 3M Company 2018 Virology Vol.514 No.-

<P><B>Abstract</B></P> <P>Integrase (IN) is an essential enzyme in retroviral life cycle. It mediates viral cDNA integration into host cellular DNA. Feline foamy virus (FFV) is a member of the Spumavirus subfamily of Retroviridae. Recently, its life cycle has been proposed to be different from other retroviruses. Despite this important finding, FFV IN is not understood clearly. Here, we constructed point mutations in FFV IN C-terminal domain (CTD) to obtain a clear understanding of its integration mechanism. Mutation of the amino acid residues in FFV IN CTD interacting with target DNA reduced both IN enzymatic activities <I>in vitro</I> and viral productions in infected cells. Especially, the mutants, R307 and K340, made viral DNA integration less efficient and allowed accumulation of more unintegrated viral DNA, thereby suppressing viral replication. Therefore, we suggest that the CTD residues interacting with the target DNA play a significant role in viral DNA integration and replication.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Mutation in FFV IN CTD reduced three representative enzymatic activities <I>in vitro</I>. </LI> <LI> Viruses with a mutation in CTD showed a significant reduction in viral infectivity. </LI> <LI> The mutation at R307 and K340 made viral DNA integration less efficient. </LI> <LI> The mutation at R307 and K340 leaded to accumulation of unintegrated viral DNA. </LI> </UL> </P>

Characterization of Biochemical Properties of Feline Foamy Virus Integrase

( Dong Hyun Lee ),( U Sok Hyun ),( Ji Ye Kim ),( Cha Gyun Shin ) 한국미생물 · 생명공학회 2010 Journal of microbiology and biotechnology Vol.20 No.6