Establishment of a Dual-Vector System for Gene Delivery Utilizing Prototype Foamy Virus.

Foamy viruses (FVs) are generally recognized as non-pathogenic, often causing asymptomatic or mild symptoms in infections. Leveraging these unique characteristics, FV vectors hold significant promise for applications in gene therapy. This study introduces a novel platform technology using a pseudo-virus with single-round infectivity. In contrast to previous vector approaches, we developed a technique employing only two vectors, pcHFV lacking Env and pCMV-Env, to introduce the desired genes into target cells. Our investigation demonstrated the efficacy of the prototype foamy virus (PFV) dual-vector system in producing viruses and delivering transgenes into host cells. To optimize viral production, we incorporated the codon-optimized Env (optEnv) gene in pCMV-Env and the Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) at the 3' end of the transgene in the transfer vector. Consequently, the use of optEnv led to a significant enhancement in transgene expression in host cells. Additionally, the WPRE exhibited an enhancing effect. Furthermore, the introduced EGFP transgene was present in host cells for a month. In an effort to expand transgene capacity, we further streamlined the viral vector, anticipating the delivery of approximately 4.3 kbp of genes through our PFV dual-vector system. This study underscores the potential of PFVs as an alternative to lentiviruses or other retroviruses in the realm of gene therapy.

The Novel Benzothiazole Derivative PB11 Induces Apoptosis via the PI3K/AKT Signaling Pathway in Human Cancer Cell Lines.

Among several anti-cancer therapies, chemotherapy can be used regardless of the stage of the disease. However, development of anti-cancer agents from potential chemicals must be executed very cautiously because of several problems, such as safety, drug resistance, and continuous administration. Most chemotherapeutics selectively cause cancer cells to undergo apoptosis. In this study, we tested the effects of a novel chemical, the benzothiazole derivative N-[2-[(3,5-dimethyl-1,2-oxazol-4-yl)methylsulfanyl]-1,3-benzothiazol-6-yl]-4-oxocyclohexane-1-carboxamide (PB11) on the human cell lines U87 (glioblastoma), and HeLa (cervix cancer). It was observed that this chemical was highly cytotoxic for these cells (IC50s < 50 nM). In addition, even 40 nM PB11 induced the classical apoptotic symptoms of DNA fragmentation and nuclear condensation. The increase of caspase-3 and -9 activities also indicated an increased rate of apoptosis, which was further confirmed via Western blotting analysis of apoptosis-associated proteins. Accordingly, PB11 treatment up-regulated the cellular levels of caspase-3 and cytochrome-c, whereas it down-regulated PI3K and AKT. These results suggest that PB11 induces cytotoxicity and apoptosis in cancer cells by suppressing the PI3K/AKT signaling pathways and, thus, may serve as an anti-cancer therapeutic.

IFITM proteins inhibit the late step of feline foamy virus replication.

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.

Foamy Virus Integrase in Development of Viral Vector for Gene Therapy.

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.

Structural Insights on Retroviral DNA Integration: Learning from Foamy Viruses.

Foamy viruses (FV) are retroviruses belonging to the Spumaretrovirinae subfamily. They are non-pathogenic viruses endemic in several mammalian hosts like non-human primates, felines, bovines, and equines. Retroviral DNA integration is a mandatory step and constitutes a prime target for antiretroviral therapy. This activity, conserved among retroviruses and long terminal repeat (LTR) retrotransposons, involves a viral nucleoprotein complex called intasome. In the last decade, a plethora of structural insights on retroviral DNA integration arose from the study of FV. Here, we review the biochemistry and the structural features of the FV integration apparatus and will also discuss the mechanism of action of strand transfer inhibitors.

Single residue mutation in integrase catalytic core domain affects feline foamy viral DNA integration.

DD(35)E motif in catalytic core domain (CCD) of integrase (IN) is extremely involved in retroviral integration step. Here, nine single residue mutants of feline foamy virus (FFV) IN were generated to study their effects on IN activities and on viral replication. As expected, mutations in the highly conserved D107, D164, and E200 residues abolished all IN catalytic activities (3'-end processing, strand transfer, and disintegration) as well as viral infectivity by blocking viral DNA integration into cellular DNA. However, Q165, Y191, and S195 mutants, which are located closely to DDE motif were observed to have diverse levels of enzymatic activities, compared to those of the wild type IN. Their mutant viruses produced by one-cycle transfection showed different infectivity on their natural host cells. Therefore, it is likely that effects of single residue mutation at DDE motif is critical on viral replication depending on the position of the residues.

Influence of Pretreatment with Immunosuppressive Drugs on Viral Proliferation.

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 immune-compromised individuals who are using immune-suppressive drugs may be especially vulnerable to viral infection originated from pets.

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

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 in vitro 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.

Distribution and fate of HIV-1 unintegrated DNA species: a comprehensive update.

Reverse transcription of viral RNA and the subsequent integration of reverse transcripts are the classical early events of the HIV-1 life-cycle. Simultaneously, abundant unintegrated DNAs (uDNAs), are formed in cells ubiquitously. The uDNAs either undergo recombination or degradation or persist inactively for long periods in the nucleus as future resources. Among them, 2-LTR circles are considered a dead-end for viral spread. Their contribution to the HIV-1 infection is still poorly understood. Nevertheless, the preintegration transcription of the aberrant DNAs and the consequent alterations of cellular factors have already been reported. Since the major fate of the viral genome is to persist as episomal DNA, precise characterization is required for studying the biology of HIV-1. This review compiles the biochemical and genetic updates on uDNA in the HIV-1 life cycle and could provide direction to further study of their roles in HIV-1 replication and application in HIV-1 pathogenesis.

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

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.

Cellular and viral determinants of retroviral nuclear entry.

Retroviruses must integrate their cDNA into the host genome to generate proviruses. Viral DNA-protein complexes interact with cellular proteins and produce pre-integration complexes, which carry the viral genome and cross the nuclear pore channel to enter the nucleus and integrate viral DNA into host chromosomal DNA. If the reverse transcripts fail to integrate, linear or circular DNA species such as 1- and 2-long terminal repeats are generated. Such complexes encounter numerous cellular proteins in the cytoplasm, which restrict viral infection and protect the nucleus. To overcome host cell defenses, the pathogens have evolved several evasion strategies. Viral proteins often contain nuclear localization signals, allowing entry into the nucleus. Among more than 1000 proteins identified as required for HIV infection by RNA interference screening, karyopherins, cleavage and polyadenylation specific factor 6, and nucleoporins have been predominantly studied. This review discusses current opinions about the synergistic relationship between the viral and cellular factors involved in nuclear import, with focus on the unveiled mysteries of the host-pathogen interaction, and highlights novel approaches to pinpoint therapeutic targets.

Knockdown of the host cellular protein transportin 3 attenuates prototype foamy virus infection.

Transportin 3 (TNPO3) is a member of the importin-ß superfamily proteins. Despite numerous studies, the exact molecular mechanism of TNPO3 in retroviral infection is still controversial. Here, we provide evidence for the role and mechanism of TNPO3 in the replication of prototype foamy virus (PFV). Our findings revealed that PFV infection was reduced 2-fold by knockdown (KD) of TNPO3. However, late stage of viral replication including transcription, translation, viral assembly, and release was not influenced. The differential cellular localization of PFV integrase (IN) in KD cells pinpointed a remarkable reduction of viral replication at the nuclear import step. We also found that TNPO3 interacted with PFV IN but not with Gag, suggesting that IN-TNPO3 interaction is important for nuclear import of PFV pre-integration complex. Our report enlightens the mechanism of PFV interaction with TNPO3 and support ongoing research on PFV as a promising safe vector for gene therapy.

Nuclear localization signals in prototype foamy viral integrase for successive infection and replication in dividing cells.

We identified four basic amino acid residues as nuclear localization signals (NLS) in the C-terminal domain of the prototype foamy viral (PFV) integrase (IN) protein that were essential for viral replication. We constructed seven point mutants in the C-terminal domain by changing the lysine and arginine at residues 305, 308, 313, 315, 318, 324, and 329 to threonine or proline, respectively, to identify residues conferring NLS activity. Our results showed that mutation of these residues had no effect on expression assembly, release of viral particles, or in vitro recombinant IN enzymatic activity. However, mutations at residues 305 (R → T), 313(R → T), 315(R → P), and 329(R → T) lead to the production of defective viral particles with loss of infectivity, whereas non-defective mutations at residues 308(R → T), 318(K → T), and 324(K → T) did not show any adverse effects on subsequent production or release of viral particles. Sub-cellular fractionation and immunostaining for viral protein PFV-IN and PFV-Gag localization revealed predominant cytoplasmic localization of PFV-IN in defective mutants, whereas cytoplasmic and nuclear localization of PFV-IN was observed in wild type and non-defective mutants. However sub-cellular localization of PFV-Gag resulted in predominant nuclear localization and less presence in the cytoplasm of the wild type and non-defective mutants. But defective mutants showed only nuclear localization of Gag. Therefore, we postulate that four basic arginine residues at 305, 313, 315 and 329 confer the karyoplilic properties of PFV-IN and are essential for successful viral integration and replication.

Structural and functional insights into foamy viral integrase.

Successful integration of retroviral DNA into the host chromosome is an essential step for viral replication. The process is mediated by virally encoded integrase (IN) and orchestrated by 3'-end processing and the strand transfer reaction. In vitro reaction conditions, such as substrate specificity, cofactor usage, and cellular binding partners for such reactions by the three distinct domains of prototype foamy viral integrase (PFV-IN) have been described well in several reports. Recent studies on the three-dimensional structure of the interacting complexes between PFV-IN and DNA, cofactors, binding partners, or inhibitors have explored the mechanistic details of such interactions and shown its utilization as an important target to develop anti-retroviral drugs. The presence of a potent, non-transferable nuclear localization signal in the PFV C-terminal domain extends its use as a model for investigating cellular trafficking of large molecular complexes through the nuclear pore complex and also to identify novel cellular targets for such trafficking. This review focuses on recent advancements in the structural analysis and in vitro functional aspects of PFV-IN.

Biochemical characteristics of functional domains using feline foamy virus integrase mutants.

We constructed deletion mutants and seven point mutants by polymerase chain reaction to investigate the specificity of feline foamy virus integrase functional domains. Complementation reactions were performed for three enzymatic activities such as 3'-end processing, strand transfer, and disintegration. The complementation reactions with deletion mutants showed several activities for 3'-end processing and strand transfer. The conserved central domain and the combination of the N-terminal or C-terminal domains increased disintegration activity significantly. In the complementation reactions between deletion and point mutants, the combination between D107V and deletion mutants revealed 3'-end processing activities, but the combination with others did not have any activity, including strand transfer activities. Disintegration activity increased evenly, except the combination with glutamic acid 200. These results suggest that an intact central domain mediates enzymatic activities but fails to show these activities in the absence of the N-terminal or C-terminal domains.

Development of puffed ginseng-rice snack from ginseng powder and map rice flour using steam and compression process.

A new manufacturing method for producing a puffed ginseng-rice snack (PGRS) was developed using ginseng powder and map rice flour through a steam and compression process (SCP). The physical and sensory properties of the PGRS were characterized. The pellets for puffing were prepared from ginseng powder and map rice flour. The pellets were subjected to 16, 18, and 20% moisture contents and were puffed at 225, 235, and 245° C. The specific volumes of the PGRSs increased with heating temperature and moisture content. However, the breaking strength of the PGRSs decreased. In addition, the SCP imposed special features in the PGRSs that made them more acceptable. The Hunter L-value increased with heating temperature and moisture content. These results indicate that a PGRS with functional additives could be effectively developed into a functional food with the use of a puffing machine, and that the PGRS shows potential as a new snack product.

Characterization of biochemical properties of feline foamy virus integrase.

In order to study biochemical properties, the integrase (IN) protein of feline foamy virus (FFV) was over-expressed from Escherichia coli, purified by two-step chromatography; Talon column and heparin column, and characterized in biochemical aspects. For the three enzymatic reactions of the 3' -processing, strand transfer, and disintegration activities, Mn2+ ion was essentially required as a cofactor. Interestingly, Co2+ and Zn2+ ions were found to act as effective cofactors, while other transition elements such as Ni2+, Cu2+, La3+, Y3+, Cd2+, Li1+, Ba2+, Sr2+, V3+, and so on were not. Regarding to the substrate specificity, FFV IN has low substrate specificities as it cleaved in a significant level prototype foamy virus (PFV) U5 LTR substrate as well as FFV U5 LTR substrate, while PFV IN did not. Finally, the 3' -processing activity was observed in the high concentrations of several solvents such as CHAPS, Glycerol, Tween 20 and Triton X-100, which are generally used for dissolution of chemicals in inhibitor-screening. Therefore, as it is the first report showing biochemical properties, FFV IN is proposed to have low specificities on the use of cofactor and substrate for enzymatic reaction when it is compared with other retroviral INs.

Beauvericin and enniatins H, I and MK1688 are new potent inhibitors of human immunodeficiency virus type-1 integrase.

Some enniatins (ENs) reportedly exhibit antiretroviral activities in vivo. The potential inhibitory activities of cyclic hexadepsipeptides such as beauvericin (BEA) and ENs H, I and MK1688 were investigated in vitro against human immunodeficiency virus type-1 (HIV-1) integrase and Moloney murine leukemia virus reverse transcriptase. BEA, EN I and EN MK1688 exhibited strong inhibitory activities against HIV-1 integrase, whereas EN H showed relatively weak activity. None of the examined compounds showed anti-reverse transcriptase activity. BEA was the most effective inhibitor of the tested cyclic hexadepsipeptides in inhibiting HIV-1 integrase. These results indicate the potential of cyclic hexadepsipeptides as a new class of potent inhibitors of HIV-1 integrase.

Apoptosis induced by enniatins H and MK1688 isolated from Fusarium oxysporum FB1501.

Enniatins are cyclic peptides isolated from bacteria, fungi, and plants, with numerous biological effects on animal systems. Recently, we have reported that certain enniatins (ENs), such as EN H and EN MK1688, have cytotoxic effects on several adenocarcinoma cell lines. In an effort to understand the mechanism behind their cytotoxicity, we investigated whether ENs can induce apoptosis in human colorectal carcinoma cells (HCT-15 cells). Treatment with the ENs H and MK1688 resulted in an alteration of cellular and nuclear morphology, leading to an increase in the number of the cells with apoptotic nuclei (seen as condensed or fragmented nuclei). Furthermore, it was observed that cellular DNA fragmentation increased in a dose-dependent manner in EN treated cells. These cells have elevated activity levels for caspase-3, the enzyme responsible for initiating cell death, compared with the untreated cells. Normal caspase-3 activity levels were observed when Z-VAD-FMK, a caspase inhibitor, was added simultaneously with the ENs. Based on our results, we propose that the new ENs H and MK1688 induce cytotoxicity via an apoptotic pathway.

Functional nucleotides of U5 LTR determining substrate specificity of prototype foamy virus integrase.

In order to study functional nucleotides in prototype foamy virus (PFV) DNA on specific recognition by PFV integrase (IN), we designed chimeric U5 long terminal repeat (LTR) DNA substrates by exchanging comparative sequences between human immunodeficiency virus type-1 (HIV-1) and PFV U5 LTRs, and investigated the 3'-end processing reactivity using HIV-1 and PFV INs, respectively. HIV-1 IN recognized the nucleotides present in the fifth and sixth positions at the 3'-end of the substrates more specifically than any other nucleotides in the viral DNA. However, PFV IN recognized the eighth and ninth nucleotides as distinctively as the fifth and sixth nucleotides in the reactions. In addition, none of the nucleotides present in the twelfth, sixteenth, seventeenth, eighteenth, nineteenth, and twentieth positions were not differentially recognized by HIV-1 and PFV INs, respectively. Therefore, our results suggest that the functional nucleotides that are specifically recognized by its own IN in the PFV U5 LTR are different from those in the HIV-1 U5 LTR in aspects of the positions and nucleotide sequences. Furthermore, it is proposed that the functional nucleotides related to the specific recognition by retroviral INs are present inside ten nucleotides from the 3'-end of the U5 LTR.