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.

DNA strand breaks and gaps target retroviral intasome binding and integration.

Retrovirus integration into a host genome is essential for productive infections. The integration strand transfer reaction is catalyzed by a nucleoprotein complex (Intasome) containing the viral integrase (IN) and the reverse transcribed (RT) copy DNA (cDNA). Previous studies suggested that DNA target-site recognition limits intasome integration. Using single molecule Förster resonance energy transfer (smFRET), we show prototype foamy virus (PFV) intasomes specifically bind to DNA strand breaks and gaps. These break and gap DNA discontinuities mimic oxidative base excision repair (BER) lesion-processing intermediates that have been shown to affect retrovirus integration in vivo. The increased DNA binding events targeted strand transfer to the break/gap site without inducing substantial intasome conformational changes. The major oxidative BER substrate 8-oxo-guanine as well as a G/T mismatch or +T nucleotide insertion that typically introduce a bend or localized flexibility into the DNA, did not increase intasome binding or targeted integration. These results identify DNA breaks or gaps as modulators of dynamic intasome-target DNA interactions that encourage site-directed integration.

PREB inhibits the replication of prototype foamy virus by affecting its transcription.

BACKGROUND: Foamy viruses (FVs) are unique nonpathogenic retroviruses, which remain latent in the host for a long time. Therefore, they may be safe, effective gene transfer vectors. In this study, were assessed FV-host cell interactions and the molecular mechanisms underlying FV latent infection. METHODS: We used the prototype FV (PFV) to infect HT1080 cells and a PFV indicator cell line (PFVL) to measure virus titers. After 48 h of infection, the culture supernatant (i.e., cell-free PFV particles) and transfected cells (i.e., cell-associated PFV particles) were harvested and incubated with PFVL. After another 48 h, the luciferase activity was used to measure virus titers. RESULTS: Through transcriptomics sequencing, we found that PREB mRNA expression was significantly upregulated. Moreover, PREB overexpression reduced PFV replication, whereas endogenous PREB knockdown increased PFV replication. PREB interacted with the Tas DNA-binding and transcriptional activation domains and interfered with its binding to the PFV long terminal repeat and internal promoter, preventing the recruitment of transcription factors and thereby inhibiting the transactivation function of Tas. PREB C-terminal 329-418 aa played a major role in inhibiting PFV replication; PREB also inhibited bovine FV replication. Therefore, PREB has a broad-spectrum inhibitory effect on FV replication. CONCLUSIONS: Our results demonstrated that PREB inhibits PFV replication by impeding its transcription.

An Outbred Calf Model for Determining Innate Immune Sensing and Evolutionary Trajectories of a Cell Culture-Adapted Bovine Foamy Virus Variant.

Bovine foamy virus (BFVbta) displays a very high degree of cell-associated replication which is unprecedented even among the other known foamy viruses. Interestingly, recent studies have shown that it can in fact adapt in vitro to high-titer (HT) cell-free transmission due to genetic changes acquired during repeated rounds of cell-free BFVbta passages in immortalized bovine MDBK cells. Molecular clones obtained from the HT BFVbta Riems cell-free variant (HT BFVbta Riems) have been thoroughly characterized in MDBK cell cultures However, during recent years, it has become increasingly clear that the source of the host cells used for virus growth and functional studies of virus replication and virus-cell interactions plays a paramount role. Established cell lines, mostly derived from tumors, but occasionally experimentally immortalized and transformed, frequently display aberrant features relating, for example. to growth, metabolism, and genetics. Even state-of-the-art organoid cultures of primary cells cannot replicate the conditions in an authentic host, especially those concerning cell diversity and the role of innate and adaptive immunity. Therefore, to determine the overall replication characteristics of the cloned wt and HT BFVbta Riems variant, we conducted a small-scale animal pilot study. The replication of the original wt BFVbta Riems isolate, as well as that of its HT variant, were analyzed. Both BFVbta variants established infection in calves, with proviruses in peripheral blood mononuclear cells and induced Gag-specific antibodies. In addition, a related pattern in the host innate immune reaction was detected in the peripheral blood leukocytes of the BFV-infected calves. Surprisingly, an analysis of the Gag sequence two weeks post-inoculation revealed that the HT BFVbta variant showed a very high level of genetic reversion to the wild type (parental BFVbta genotype).

Modulation of the functional interfaces between retroviral intasomes and the human nucleosome.

Infection by retroviruses as HIV-1 requires the stable integration of their genome into the host cells. This process needs the formation of integrase (IN)-viral DNA complexes, called intasomes, and their interaction with the target DNA wrapped around nucleosomes within cell chromatin. To provide new tools to analyze this association and select drugs, we applied the AlphaLISA technology to the complex formed between the prototype foamy virus (PFV) intasome and nucleosome reconstituted on 601 Widom sequence. This system allowed us to monitor the association between both partners and select small molecules that could modulate the intasome/nucleosome association. Using this approach, drugs acting either on the DNA topology within the nucleosome or on the IN/histone tail interactions have been selected. Within these compounds, doxorubicin and histone binders calixarenes were characterized using biochemical, in silico molecular simulations and cellular approaches. These drugs were shown to inhibit both PFV and HIV-1 integration in vitro. Treatment of HIV-1-infected PBMCs with the selected molecules induces a decrease in viral infectivity and blocks the integration process. Thus, in addition to providing new information about intasome-nucleosome interaction determinants, our work also paves the way for further unedited antiviral strategies that target the final step of intasome/chromatin anchoring. IMPORTANCE In this work, we report the first monitoring of retroviral intasome/nucleosome interaction by AlphaLISA. This is the first description of the AlphaLISA application for large nucleoprotein complexes (>200 kDa) proving that this technology is suitable for molecular characterization and bimolecular inhibitor screening assays using such large complexes. Using this system, we have identified new drugs disrupting or preventing the intasome/nucleosome complex and inhibiting HIV-1 integration both in vitro and in infected cells. This first monitoring of the retroviral/intasome complex should allow the development of multiple applications including the analyses of the influence of cellular partners, the study of additional retroviral intasomes, and the determination of specific interfaces. Our work also provides the technical bases for the screening of larger libraries of drugs targeting specifically these functional nucleoprotein complexes, or additional nucleosome-partner complexes, as well as for their characterization.

ZNF219, a novel transcriptional repressor, inhibits transcription of the prototype foamy virus by interacting with the viral LTR promoter.

Prototype foamy virus (PFV) is an ancient retrovirus that infects humans with persistent latent infections and non-pathogenic consequences. Lifelong latent PFV infections can be caused by restrictive factors in the host. However, the molecular mechanisms underlying host cell regulation during PFV infection are not fully understood. The aim of the study was to investigate whether a zinc finger protein (ZFP), ZNF219, as a transcription factor, can regulate the transcriptional activity of the viral promoter. Here, using transcriptome sequencing, we found that ZNF219, is downregulated in PFV infected cells and that ZNF219 suppresses viral replication by targeting the viral 5'LTR promoter region to repress its transcription. We also found that PFV infection induced abnormal expression of miRNAs targeting the ZNF219-3'UTR to downregulate ZNF219 expression. These findings indicated that ZNF219 may be a potent antiviral factor for suppressing PFV infection, and may shed light on the mechanism of virus-host interactions.

Comparison of a Genotype 1 and a Genotype 2 Macaque Foamy Virus env Gene Indicates Distinct Infectivity and Cell-Cell Fusion but Similar Tropism and Restriction of Cell Entry by Interferon-Induced Transmembrane Proteins.

Foamy viruses (FVs) are naturally found in many different animals and also in primates with the notable exception of humans, but zoonotic infections are common. In several species, two different envelope (env) gene sequence clades or genotypes exist. We constructed a simian FV (SFV) clone containing a reporter gene cassette. In this background, we compared the env genes of the SFVmmu-DPZ9524 (genotype 1) and of the SFVmmu_R289hybAGM (genotype 2) isolates. SFVmmu_R289hybAGM env-driven infection was largely resistant to neutralization by SFVmmu-DPZ9524-neutralizing sera. While SFVmmu_R289hybAGM env consistently effected higher infectivity and cell-cell fusion, we found no differences in the cell tropism conferred by either env across a range of different cells. Infection by both viruses was weakly and non-significantly enhanced by simultaneous knockout of interferon-induced transmembrane proteins (IFITMs) 1, 2, and 3 in A549 cells, irrespective of prior interferon stimulation. Infection was modestly reduced by recombinant overexpression of IFITM3, suggesting that the SFV entry step might be weakly restricted by IFITM3 under some conditions. Overall, our results suggest that the different env gene clades in macaque foamy viruses induce genotype-specific neutralizing antibodies without exhibiting overt differences in cell tropism, but individual env genes may differ significantly with regard to fitness.

Identification of Cartilaginous Fish Endogenous Foamy Virus Rooting to Vertebrate Counterparts.

Foamy viruses (FVs) are ideal models for studying the long-term evolutionary history between viruses and their hosts. Currently, FVs have been documented in nearly all major taxa of vertebrates, but evidence is lacking for true FV infiltration in cartilaginous fish, the most basal living vertebrates with jaws. Here, we screened 11 available genomes and 10 transcriptome sequence assemblies of cartilaginous fish and revealed a novel endogenous foamy virus, termed cartilaginous fish endogenous foamy virus (CFEFV), in the genomes of sharks and rays. Genomic analysis of CFEFVs revealed feature motifs that were retained among canonical FVs. Phylogenetic analysis using polymerase sequences revealed the rooting nature of CFEFVs to vertebrate FVs, indicating their deep origin. Interestingly, three viral lineages were found in a shark (Scyliorhinus torazame), one of which was clustered with ray-finned fish foamy-like viruses, indicating that multiple episodes of viral infiltrations had occurred in this species. These findings fill a major gap in the Spumaretrovirinae taxon and reveal the aquatic origin of FVs found in terrestrial vertebrates. IMPORTANCE Although foamy viruses (FVs) have been found in major branches of vertebrates, the presence of these viruses in cartilaginous fish, the most basal living vertebrates with jaws, remains to be explored. This study revealed a collection of cartilaginous endogenous FVs in sharks and rays through in silico genomic mining. These viruses were rooted in the polymerase (POL) phylogeny, indicating the ancient aquatic origin of FVs. However, their envelope (ENV) protein grouped with those of amphibian FVs, suggesting different evolutionary histories of different FV genes. Overall, we provide the last missing gap for the taxonomic investigation of Spumaretrovirinae and provide concrete support for the aquatic origin of FVs.

Antiviral role of IFITM3 in prototype foamy virus infection.

BACKGROUND: Foamy viruses (FVs) are retroviruses with unique replication strategies that cause lifelong latent infections in their hosts. FVs can also produce foam-like cytopathic effects in vitro. However, the effect of host cytokines on FV replication requires further investigation. Although interferon induced transmembrane (IFITMs) proteins have become the focus of antiviral immune response research due to their broad-spectrum antiviral ability, it remains unclear whether IFITMs can affect FV replication. METHOD: In this study, the PFV virus titer was characterized by measuring luciferase activity after co-incubation of PFVL cell lines with the cell culture supernatants (cell-free PFV) or the cells transfected with pcPFV plasmid/infected with PFV (cell-associated PFV). The foam-like cytopathic effects of PFV infected cells was observed to reflect the virus replication. The total RNA of PFV infected cells was extracted, and the viral genome was quantified by Quantitative reverse transcription PCR to detect the PFV entry into target cells. RESULTS: In the present study, we demonstrated that IFITM1-3 overexpression inhibited prototype foamy virus (PFV) replication. In addition, an IFITM3 knockdown by small interfering RNA increased PFV replication. We further demonstrated that IFITM3 inhibited PFV entry into host cells. Moreover, IFITM3 also reduced the number of PFV envelope proteins, which was related to IFITM3 promoted envelope degradation through the lysosomal pathway. CONCLUSIONS: Taken together, these results demonstrate that IFITM3 inhibits PFV replication by inhibiting PFV entry into target cells and reducing the number of PFV envelope.

Preclinical Evaluation of Foamy Virus Vector-Mediated Gene Addition in Human Hematopoietic Stem/Progenitor Cells for Correction of Leukocyte Adhesion Deficiency Type 1.

Ex vivo gene therapy procedures targeting hematopoietic stem and progenitor cells (HSPCs) predominantly utilize lentivirus-based vectors for gene transfer. We provide the first pre-clinical evidence of the therapeutic utility of a foamy virus vector (FVV) for the genetic correction of human leukocyte adhesion deficiency type 1 (LAD-1), an inherited primary immunodeficiency resulting from mutation of the ß2 integrin common chain, CD18. CD34+ HSPCs isolated from a severely affected LAD-1 patient were transduced under a current good manufacturing practice-compatible protocol with FVV harboring a therapeutic CD18 transgene. LAD-1-associated cellular chemotactic defects were ameliorated in transgene-positive, myeloid-differentiated LAD-1 cells assayed in response to a strong neutrophil chemoattractant in vitro. Xenotransplantation of vector-transduced LAD-1 HSPCs in immunodeficient (NSG) mice resulted in long-term (∼5 months) human cell engraftment within murine bone marrow. Moreover, engrafted LAD-1 myeloid cells displayed in vivo levels of transgene marking previously reported to ameliorate the LAD-1 phenotype in a large animal model of the disease. Vector insertion site analysis revealed a favorable vector integration profile with no overt evidence of genotoxicity. These results coupled with the unique biological features of wild-type foamy virus support the development of FVVs for ex vivo gene therapy of LAD-1.

Occurrence of Equine Foamy Virus Infection in Horses from Poland.

Equine foamy virus (EFVeca) is a foamy virus of non-primate origin and among the least-studied members of this retroviral subfamily. By sequence comparison, EFVeca shows the highest similarity to bovine foamy virus. In contrast to simian, bovine or feline foamy viruses, knowledge about the epidemiology of EFVeca is still limited. Since preliminary studies suggested EFVeca infections among horses in Poland, we aimed to expand the diagnostics of EFVeca infections by developing specific diagnostic tools and apply them to investigate its prevalence. An ELISA test based on recombinant EFVeca Gag protein was developed for serological investigation, while semi-nested PCR for the detection of EFVeca DNA was established. 248 DNA and serum samples from purebred horses, livestock and saddle horses, Hucul horses and semi-feral Polish primitive horses were analyzed in this study. ELISA was standardized, and cut off value, sensitivity and specificity of the test were calculated using Receiver Operating Characteristic and Bayesian estimation. Based on the calculated cut off, 135 horses were seropositive to EFVeca Gag protein, while EFVeca proviral DNA was detected in 85 animals. The rate of infected individuals varied among the horse groups studied; this is the first report confirming the existence of EFVeca infections in horses from Poland using virus-specific tools.

SGK1, a Serine/Threonine Kinase, Inhibits Prototype Foamy Virus Replication.

Foamy viruses (FVs) are complex retroviruses belonging to the Spumaretrovirinae subfamily of the Retroviridae family. In contrast to human immunodeficiency virus (HIV), another member of the Retroviridae family, FVs are nonpathogenic in their natural hosts or in experimentally infected animals. Prototype foamy virus (PFV) is the only foamy virus that can infect humans through cross-species transmission and does not show any pathogenicity after infection. Consequently, PFV is considered a safe and efficient gene transfer vector. Understanding the host proteins involved in the replication of PFV and the mechanism of interaction between the host and the virus might lead to studies to improve the efficiency of gene transfer. To date, only a few host factors have been identified that affect PFV replication. In the present study, we report that PFV infection enhances the promoter activity of SGK1 (encoding serum/glucocorticoid regulated kinase 1) via the Tas protein signaling pathway, and then upregulates the mRNA and protein levels of SGK1. Overexpression of SGK1 reduced PFV replication, whereas its depletion using small interfering RNA increased PFV replication. SGK1 inhibits PFV replication by impairing the function of the PFV Tas activation domain in a kinase-independent manner and reducing the stability of the Gag protein in a kinase-dependent manner. In addition, both human and bovine SGK1 proteins inhibit the replication of bovine foamy virus (BFV) and PFV. These findings not only improved our understanding of the function of SGK1 and its relationship with foamy viruses, but also contributed to determining the antiviral mechanism of the host. IMPORTANCE Foamy viruses can integrate into the host chromosome and are nonpathogenic in natural hosts or in experimentally infected animals. Therefore, foamy viruses are considered to be safe and efficient gene transfer vectors. Persistent infection of foamy viruses is partly caused by the restrictive effect of host factors on the virus. However, only a few cellular proteins are known to influence the replication of foamy viruses. In this study, we report that SGK1 inhibits the replication of prototype foamy virus by affecting the function of the transcription activator, Tas, and reducing the stability of the structural protein, Gag. These results will increase our understanding of the interaction between the virus and host factors, deepening our perception of host antiviral defenses and the function of SGK1, and could improve the gene transfer efficiency of foamy viruses.

Characterization of Bovine Foamy Virus Gag Late Assembly Domain Motifs and Their Role in Recruiting ESCRT for Budding.

A large number of retroviruses, such as human immunodeficiency virus (HIV) and prototype foamy virus (PFV), recruit the endosomal sorting complex required for transport (ESCRT) through the late domain (L domain) on the Gag structural protein for virus budding. However, little is known about the molecular mechanism of bovine foamy virus (BFV) budding. In the present study, we report that BFV recruits ESCRT for budding through the L domain of Gag. Specifically, knockdown of VPS4 (encoding vacuolar protein sorting 4), ALIX (encoding ALG-2-interacting protein X), and TSG101 (encoding tumor susceptibility 101) indicated that BFV uses ESCRT for budding. Mutational analysis of BFV Gag (BGag) showed that, in contrast to the classical L domain motifs, BGag contains two motifs, P56LPI and Y103GPL, with L domain functions. In addition, the two L domains are necessary for the cytoplasmic localization of BGag, which is important for effective budding. Furthermore, we demonstrated that the functional site of Alix is V498 in the V domain and the functional site of Tsg101 is N69 in the UBC-like domain for BFV budding. Taken together, these results demonstrate that BFV recruits ESCRT for budding through the PLPI and YGPL L domain motifs in BGag.

Evaluation of the stability and intratumoral delivery of foreign transgenes encoded by an oncolytic Foamy Virus vector.

Foamy Viruses are cell cycle-dependent retroviruses capable of persisting unintegrated in quiescent cells until cell division occurs. This unique ability allows them to target slowly dividing human tumor cells which remains an unmet need in oncolytic virotherapy. We have previously reported the generation of oncolytic Foamy Virus (oFV) vector system and demonstrated its superiority over oncolytic Murine Leukemia Virus vectors in infecting slowly dividing cancer cells. In the present study we evaluated (i) the ability of oFV to carry foreign transgenes and (ii) the genetic stability of these vectors upon serial passage. The thymidine kinase (TK) and inducible caspase 9 (iCasp9) cDNAs could be detected in the oFV backbone for up to 3 in vitro passages. In vivo, GFP-, TK- and iCasp9- carrying oFV vectors propagated efficiently in subcutaneous xenograft glioblastoma tumors and drove transgene expression for up to 66 days. However, in vivo oFV vector spread eventually resulted in complete loss of the iCasp9 cDNA, minor loss of the TK cDNA and negligible loss of the GFP. Our results suggest that oFV is a promising gene delivery platform and that transgenes smaller than 1 kb might be most suitable for oFV arming.

Inhibition of cell proliferation by Tas of foamy viruses through cell cycle arrest or apoptosis underlines the different mechanisms of virus-host interactions.

Foamy viruses belong to the Spumaretrovirinae subfamily member of the Retroviridae family and produce nonpathogenic infection to hosts in the natural conditions. However, infections of foamy viruses can dramatically cause severe cytopathic effects in vitro. To date, the exact molecular mechanism has remained unclear which implied the tremendous importance of virus-host cell immune reactions. In this study, we found that the transactivator Tas in two foamy viruses isolated from Old World Monkey (OWM) induced obvious inhibition of cell proliferation via the upregulation of Foxo3a expression. It was mediated by the generation of ROS and the initiation of ER stress, and ultimately, the mitochondrial apoptosis pathway was triggered. Notably, PFV Tas contributed to the accumulation of G0/G1 phase cycle arrest induced by the activation of the p53 signaling pathway and the nuclear transportation of HDAC4 via upregulating PPM1E expression. Together, these results demonstrated the different survival strategies by which foamy virus can hijack host cell cytokines and regulate virus-host cell interactions.

The Regulation of Prototype Foamy Virus 5'Long Terminal Repeats and Internal Promoter by Endogenous Transcription Factors.

BACKGROUND: For foamy virus, the transactivator of spumaretrovirus (Tas) could bind directly to target DNA sequences termed as Tas responsive elements and trigger the viral internal promoter (IP) and long terminal repeat (LTR) promoters. The cellular endogenous factors also play an important role in viral gene expressions. We hypothesized that except the viral transcription factor Tas, the cellular endogenous factors also affect the viral gene expression. METHODS: The full length of the prototype foamy virus (PFV) genome (U21247) was used to predict the potential binding sites of the transcription factors by online software JASPAR (http://jaspar.genereg.net) and Softberry (http://linux1.softberry.com/berry.phtml?topic=index&group=programs&subgroup=promoter). The Dual-Luciferase® Reporter Assay System (Promega, USA) was used to confirm the relative luciferase activities of the test groups. The different representative activating agents or inhibitors of each canonical signal pathway were used to identify the impact of these pathways on PFV 5'LTR and IP promoters. RESULTS: The results showed different cellular endogenous factors might have respective effects on PFV 5'LTR and IP. It is worth mentioning that activator protein-1 and BCL2-associated athanogene 3, 2 kinds of vital proteins associated with NF-κB and PKC pathways, could activate the basal activity of 5'LTR and IP promoters but inhibit the Tas-regulated activity of both promoters. Furthermore, PFV Tas was identified to trigger the transcription of the NF-κB promoter. CONCLUSION: NF-κB had a negative effect on PFV 5'LTR and IP promoter activities, the PKC pathway might upregulate 5'LTR and IP promoter activities, and the JNK and NF-AT signal pathway could increase the Tas-regulated promoter activity of PFV 5'LTR. This study sheds light on the interaction between PFV and the host cell and may help utilize the viral promoters in retroviral vectors designed for gene transfer experiments.

Genetic and biological characterization of feline foamy virus isolated from a leopard cat (Prionailurus bengalensis) in Vietnam.

Foamy viruses have been isolated from various mammals and show long-term co-speciation with their hosts. However, the frequent inter-species transmission of feline foamy viruses (FFVs) from domestic cats to wild cats across genera has been reported. Because infectious molecular clones of FFVs derived from wild cats have not been available, whether there are specific characteristics enabling FFVs to adapt to the new host species is still unknown. Here, we obtained the complete genome sequences of two FFV isolates (strains NV138 and SV201) from leopard cats (Prionailurus bengalensis) in Vietnam and constructed an infectious molecular clone, named pLC960, from strain NV138. The growth kinetics of the virus derived from pLC960 were comparable to those of other FFVs derived from domestic cats. Phylogenetic analysis revealed that these two FFVs from leopard cats are clustered in the same clade as FFVs from domestic cats in Vietnam. Comparisons of the amino acid sequences of Env and Bet proteins showed more than 97% identity among samples and no specific amino acid substitutions between FFVs from domestic cats and ones from leopard cats. These results indicate the absence of genetic constraint of FFVs for interspecies transmission from domestic cats to leopard cats.

Trim28 acts as restriction factor of prototype foamy virus replication by modulating H3K9me3 marks and destabilizing the viral transactivator Tas.

BACKGROUND: Prototype foamy virus (PFV) is nonpathogenic complex retroviruses that express a transcriptional transactivator Tas, which is essential for the activity of viral long terminal repeat (LTR) promoter and internal promoter (IP). Tripartite motif-containing protein 28 (Trim28) is well known as a scaffold protein normally enriched in gene promoter region to repress transcription. We sought to determine if whether Trim28 could be enriched in PFV promoter region to participate the establishment of PFV latency infection. RESULTS: In this study, we show that Trim28 restricts Tas-dependent transactivation activity of PFV promoter and negatively regulates PFV replication. Trim28 was found to be enriched in LTR instead of IP promoter regions of PFV genome and contribute to the maintenance of histone H3K9me3 marks on the LTR promoter. Furthermore, Trim28 interacts with Tas and colocalizes with Tas in the nucleus. Besides, we found that Trim28, an E3 ubiquitin ligase, binds directly to and promotes Tas for ubiquitination and degradation. And the RBCC domain of Trim28 is required for the ubiquitination and degradation of Tas. CONCLUSIONS: Collectively, our findings not only identify a host factor Trim28 negatively inhibits PFV replication by acting as transcriptional restriction factor enriched in viral LTR promoter through modulating H3K9me3 mark here, but also reveal that Trim28 mediated ubiquitin proteasome degradation of Tas as a mechanism underlying Trim28 restricts Tas-dependent transcription activity of PFV promoter and PFV replication. These findings provide new insights into the process of PFV latency establishment.

Identification of Feline Foamy Virus-derived MicroRNAs.

MicroRNAs (miRNAs) classified as non-coding RNAs regulate various metabolic systems and viral life cycles. To date, numerous DNA viruses, many of which are members of the herpesvirus family, and a relatively small number of RNA viruses, including retroviruses, have been reported to encode and express miRNAs in infected cells. A few retroviruses have been shown to express miRNAs, and foamy viruses (FVs) were initially predicted by computational analyses to possess miRNA-coding regions. Subsequent studies on simian and bovine FVs confirmed the presence of functional and biologically active miRNA expression cassettes. We herein identified feline FV-derived miRNAs using a small RNA deep sequencing ana-lysis. We confirmed their repressive functions on gene expression by dual-luciferase reporter assays. We found that the seed sequences of the miRNAs identified in the present study were conserved among all previously reported FFV isolates. These results suggest that FFV-derived miRNAs play a pivotal role in FFV infection.

Molecular Epidemiology and Whole-Genome Analysis of Bovine Foamy Virus in Japan.

Bovine foamy virus (BFV) is a member of the foamy virus family in cattle. Information on the epidemiology, transmission routes, and whole-genome sequences of BFV is still limited. To understand the characteristics of BFV, this study included a molecular survey in Japan and the determination of the whole-genome sequences of 30 BFV isolates. A total of 30 (3.4%, 30/884) cattle were infected with BFV according to PCR analysis. Cattle less than 48 months old were scarcely infected with this virus, and older animals had a significantly higher rate of infection. To reveal the possibility of vertical transmission, we additionally surveyed 77 pairs of dams and 3-month-old calves in a farm already confirmed to have BFV. We confirmed that one of the calves born from a dam with BFV was infected. Phylogenetic analyses revealed that a novel genotype was spread in Japan. In conclusion, the prevalence of BFV in Japan is relatively low and three genotypes, including a novel genotype, are spread in Japan.