Prototype Foamy Virus Integrase Displays Unique Biochemical Activities among Retroviral Integrases

Integrases of different retroviruses assemble as functional complexes with varying multimers of the protein. Retroviral integrases require a divalent metal cation to perform one-step transesterification catalysis. Tetrameric prototype foamy virus (PFV) intasomes assembled from purified integrase and viral DNA oligonucleotides were characterized for their activity in the presence of different cations. While most retroviral integrases are inactive in calcium, PFV intasomes appear to be uniquely capable of catalysis in calcium. The PFV intasomes also contrast with other retroviral integrases by displaying an inverse correlation of activity with increasing manganese beginning at relatively low concentrations. The intasomes were found to be significantly more active in the presence of chloride co-ions compared to acetate. While HIV-1 integrase appears to commit to a target DNA within 20 s, PFV intasomes do not commit to target DNA during their reaction lifetime. Together, these data highlight the unique biochemical activities of PFV integrase compared to other retroviral integrases.

Physiological magnesium concentrations increase fidelity of diverse reverse transcriptases from HIV-1, HIV-2, and foamy virus, but not MuLV or AMV

Reverse transcriptases (RTs) are typically assayed using optimized Mg2+ concentrations (~5–10 mM) several-fold higher than physiological cellular free Mg2+ (~0.5 mM). Recent analyses demonstrated that HIV-1, but not Moloney murine leukaemia (MuLV) or avain myeloblastosis (AMV) virus RTs has higher fidelity in low Mg2+. In the current report, lacZα-based α-complementation assays were used to measure the fidelity of several RTs including HIV-1 (subtype B and A/E), several drug-resistant HIV-1 derivatives, HIV-2, and prototype foamy virus (PFV), all which showed higher fidelity using physiological Mg2+, while MuLV and AMV RTs demonstrated equivalent fidelity in low and high Mg2+. In 0.5 mM Mg2+, all RTs demonstrated approximately equal fidelity, except for PFV which showed higher fidelity. A Next Generation Sequencing (NGS) approach that used barcoding to determine mutation profiles was used to examine the types of mutations made by HIV-1 RT (type B) in low (0.5 mM) and high (6 mM) Mg2+ on a lacZα template. Unlike α-complementation assays which are dependent on LacZα activity, the NGS assay scores mutations at all positions and of every type. Consistent with α-complementation assays, a ~four-fold increase in mutations was observed in high Mg2+. These findings help explain why HIV-1 RT displays lower fidelity in vitro (with high Mg2+ concentrations) than other RTs (e.g. MuLV and AMV), yet cellular fidelity for these viruses is comparable. Establishing in vitro conditions that accurately represent RT’s activity in cells is pivotal to determining the contribution of RT and other factors to the mutation profile observed with HIV-1.

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. Graphical Abstract

The mouse mammary tumor virus intasome exhibits distinct dynamics on target DNA

Retroviral intasomes are complexes assembled from purified integrase (IN) and oligonucleotides mimicking viral DNA ends (vDNA). Recombinant intasomes faithfully recapitulate integration of vDNA into a target DNA. Structural studies of retroviral intasomes have revealed an array of IN oligomer forms, which appear to share a conserved intasome core coordinating the vDNA ends for strand transfer into the target DNA. Here we have explored the biochemical and dynamic properties of the mouse mammary tumor virus (MMTV) octameric intasome. We show that the MMTV intasome is remarkably stable compared to the prototype foamy virus (PFV) tetrameric intasome. MMTV integration activity peaks within the range of physiological ionic strength and is more active in the presence of manganese compared to magnesium. Single-molecule images demonstrate that the target DNA search by MMTV intasomes appears rate-limiting, similar to PFV intasomes. The time between strand transfer of the two MMTV vDNA ends into the target DNA is ~3 fold slower than PFV intasomes. MMTV intasomes can form extremely stable, largely immobile filaments on a target DNA that are comprised of multiple intasomes. This unusual property suggests that MMTV intasomes may readily form higher order oligomers that might underpin their increased stability.

Among retroviral integrases prototype foamy virus integrase displays unique biochemical activities

Integrase enzymes of different retroviruses assemble as functional complexes with varying multimers of the protein. Retroviral integrases require a divalent metal cation to perform one-step transesterification catalysis. Tetrameric prototype foamy virus (PFV) intasomes assembled from purified integrase and viral DNA oligonucleotides were characterized for their activity in the presence of different cations. While most retroviral integrases are inactive in calcium, PFV intasomes appear to be uniquely capable of catalysis in calcium. The PFV intasomes also contrast other retroviral integrases by displaying an inverse correlation of activity with increasing manganese beginning at relatively low concentrations. The intasomes were found to be significantly more active in the presence of chloride co-ions compared to acetate. While HIV-1 integrase appears to commit to a target DNA within 20 seconds, PFV intasomes do not commit to target DNA during their reaction lifetime. Together these data highlight the unique biochemical activities of PFV integrase compared to other retroviral integrases.

Seroprevalence of feline foamy virus in domestic cats in Poland

Introduction Feline foamy virus (FFVfca) is widespread and its prevalence in naturally infected domestic cats ranges between 30% and 80% worldwide. The infection is persistent, with a sustained antibody response in FFVfca-positive cats; however to date, no defined disease or clinical symptoms have been proved to be associated with it. The goal of the presented study was to determine the prevalence of FFVfca infection in domestic cats in Poland. Material and Methods A total of 223 serum samples collected from domestic cats were tested with a glutathione S-transferase capture ELISA test to detect antibodies specific to capsid (Gag), accessory (Bet) and envelope (Env) FFVfca antigens. A Western blot test was used to confirm the ELISA results. Results The cut-off value for the Gag antigen was established by calculation and evaluation with the immunoblotting assay. The cut-off values for Bet and Env were calculated from the reactivity of Gag-negative samples. The sera of 99 cats (44%) showed reactivity to Gag, those of 80 did so (35.9 %) to Bet, while only 56 samples (25%) were reactive to Env. Only 51 (22.9%) sera were positive for all antigens. The main diagnostic antigen was selected to be Gag. A statistically significant association was found between FFVfca status and the age of the cat. Conclusions This study proved the high seroprevalence of FFVfca in domestic cats in Poland for the first time and confirmed that adult cats are at higher FFVfca infection risk than preadult cats. Its results correspond to those reported from other countries.

Viral Sequences Recovered From Puma Tooth DNA Reconstruct Statewide Viral Phylogenies

Monitoring pathogens in wildlife populations is imperative for effective management, and for identifying locations for pathogen spillover among wildlife, domestic species and humans. Wildlife pathogen surveillance is challenging, however, as sampling often requires the capture of a significant proportion of the population to understand host pathogen dynamics. To address this challenge, we assessed the ability to use hunter-collected teeth from puma across Colorado to recover genetic data of two feline retroviruses, feline foamy virus (FFV) and feline immunodeficiency virus (FIVpco) and show they can be utilized for this purpose. Comparative phylogenetic analyses of FIVpco and FFV from tooth and blood samples to previous analyses conducted with blood samples collected over a nine-year period from two distinct areas was undertaken highlighting the value of tooth derived samples. We found less FIVpco phylogeographic structuring than observed from sampling only two regions and that FFV data confirmed previous findings of endemic infection, minimal geographic structuring, and supported frequent cross-species transmission from domestic cats to pumas. Viral analysis conducted using intentionally collected blood samples required extensive financial, capture and sampling efforts. This analysis illustrates that viral genomic data can be cost effectively obtained using tooth samples incidentally-collected from hunter harvested pumas, taking advantage of samples collected for morphological age identification. This technique should be considered as an opportunistic method to provide broad geographic sampling to define viral dynamics more accurately in wildlife.

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

<b><i>Background:</i></b> 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. <b><i>Methods:</i></b> 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&amp;group=programs&amp;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. <b><i>Results:</i></b> 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. <b><i>Conclusion:</i></b> 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.

Triple Viral Infections in The Same Cats: Feline Coronavirus, Feline Parvovirus, Feline Foamy Virus

Objective. Several studies from different countries have been performed about the viral diseases of domestic cats, and detailed information has been provided on their transmission, prevalence/incidence, virulence, origins/molecular characteristics and pathogenesis so far. Multiple- or co-infections in domestic and wild cats have been described by many papers. However, viral co-infections have been reported on a limited basis. In this study, three domestic clinically diseased cats have been found to be positive with feline coronavirus (FCoV), feline parvovirus (FPV) and feline foamy virus (FFoV). We aimed to examine triple viral infections circumstances in Turkish cats. Material and method. Ascites and blood samples were collected from diseased cats. Different polymerase chain reaction protocols for each virus were performed. After PCRs, all products were run in agarose gel and visualized under a blue-light transilluminator. Results. We found FCoV, FPV and FFoV as triple infection in three cats. Conclusions. We think that the results indicating the presence of multiple infections will ease the work of veterinary clinicians concerning infection treatment options, especially when animals show multiple clinical findings due to co-infections. It should be not forgotten the presence of multi-systemic co-infections in early routine laboratory diagnosis.

Physiological Magnesium Concentrations Increase Fidelity of Diverse Reverse Transcriptases from HIV-1, HIV-2, and Foamy Virus, but not MuLV or AMV

Reverse transcriptases (RTs) are typically assayed in vitro using optimized Mg2+ concentrations (~ 5-10 mM) that are several-fold higher than physiological cellular free Mg2+ (~ 0.5 mM). Analysis of fidelity using lacZα-based α-complementation assays showed that tested HIV RTs, including HIV-1 from subtype B (HXB2-derived), HIV-2, subtype A/E, and several drug-resistant HXB2 derivatives all showed significantly higher fidelity using physiological Mg2+. This also occurred with prototype foamy virus (PFV) RT. In contrast, Moloney murine leukemia virus (MuLV) and avian myeloblastosis virus (AMV) RTs demonstrated equivalent fidelity in both low and high Mg2+. In 0.5 mM Mg2+, all RTs demonstrated ≈ equal fidelity, except for PFV RT which showed higher fidelity. A Next Generation Sequencing (NGS) approach that used barcoding to accurately determine mutation rates and profiles was used to examine the types of mutations made by HIV-1 (subtype B, wild type) in low (0.5 mM) and high (6 mM) Mg2+ with DNA or RNA that coded for lacZα. Unlike the α-complementation assay, which is dependent on LacZα activity, the NGS assay scores mutations at all positions and of every type. A ~ 4-fold increase in substitution mutations was observed in high Mg2+. The general trend was an exacerbation in high Mg2+ of more common mutation in low Mg2+, rather than the creation of new mutation hotspots. These findings help explain why HIV RT displays lower fidelity in vitro (with high Mg2+ concentrations) than other RTs (e.g., MuLV and AMV), yet cellular fidelity for these viruses is comparable.