Shared and cell type-specific adaptation strategies of Gag and Env yield high titer bovine foamy virus variants.

During in vitro selection and evolution screens to adapt the tightly cell-associated bovine foamy virus BFV to high titer cell-free transmission, common, cell-type specific and concurrent adaptive changes in Gag and Env, the major players of foamy virus particle assembly and release, were detected. Upon early establishment of cell type-independent pioneering mutations in Env and, subsequently in Gag, a diverse virus pool emerged that was characterized by the occurrence of shared and additional cell type-specific exchanges. At late passages and saturated titers, remarkably homogeneous virus populations characterized by functionally important mutations developed which may be partly due to stochastic evolutionary events that occurred earlier during adaptation. Reverse genetics showed that defined mutations were functionally important for high titer cell-free transmission.

The chromatin binding domain, including the QPQRYG motif, of feline foamy virus Gag is required for viral DNA integration and nuclear accumulation of Gag and the viral genome.

The retroviral Gag protein, the major component of released particles, plays different roles in particle assembly, maturation or infection of new host cells. Here, we characterize the Gag chromatin binding site including the highly conserved QPQRYG motif of feline foamy virus, a member of the Spumaretrovirinae. Mutagenesis of critical residues in the chromatin binding site/QPQRYG motif almost completely abrogates viral DNA integration and reduces nuclear accumulation of Gag and viral DNA. Genome packaging, reverse transcription, particle release and uptake into new target cells are not affected. The integrity of the QPQRYG motif appears to be important for processes after cytosolic entry, likely influencing incoming virus capsids or disassembly intermediates but not Gag synthesized de novo in progeny virus-producing cells. According to our data, chromatin binding is a shared feature among foamy viruses but further work is needed to understand the mechanisms involved.

Mutagenesis of N-terminal residues of feline foamy virus Gag reveals entirely distinct functions during capsid formation, particle assembly, Gag processing and budding.

BACKGROUND: Foamy viruses (FVs) of the Spumaretrovirinae subfamily are distinct retroviruses, with many features of their molecular biology and replication strategy clearly different from those of the Orthoretroviruses, such as human immunodeficiency, murine leukemia, and human T cell lymphotropic viruses. The FV Gag N-terminal region is responsible for capsid formation and particle budding via interaction with Env. However, the critical residues or motifs in this region and their functional interaction are currently ill-defined, especially in non-primate FVs. RESULTS: Mutagenesis of N-terminal Gag residues of feline FV (FFV) reveals key residues essential for either capsid assembly and/or viral budding via interaction with the FFV Env leader protein (Elp). In an in vitro Gag-Elp interaction screen, Gag mutations abolishing particle assembly also interfered with Elp binding, indicating that Gag assembly is a prerequisite for this highly specific interaction. Gradient sedimentation analyses of cytosolic proteins indicate that wild-type Gag is mostly assembled into virus capsids. Moreover, proteolytic processing of Gag correlates with capsid assembly and is mostly, if not completely, independent from particle budding. In addition, Gag processing correlates with the presence of packaging-competent FFV genomic RNA suggesting that Pol encapsidation via genomic RNA is a prerequisite for Gag processing. Though an appended heterogeneous myristoylation signal rescues Gag particle budding of mutants unable to form capsids or defective in interacting with Elp, it fails to generate infectious particles that co-package Pol, as evidenced by a lack of Gag processing. CONCLUSIONS: Changes in proteolytic Gag processing, intracellular capsid assembly, particle budding and infectivity of defined N-terminal Gag mutants highlight their essential, distinct and only partially overlapping roles during viral assembly and budding. Discussion of these findings will be based on a recent model developed for Gag-Elp interactions in prototype FV.

In Vitro Evolution of Bovine Foamy Virus Variants with Enhanced Cell-Free Virus Titers and Transmission.

Virus transmission is essential for spreading viral infections and is a highly coordinated process which occurs by cell-free transmission or cell-cell contact. The transmission of Bovine Foamy Virus (BFV) is highly cell-associated, with undetectable cell-free transmission. However, BFV particle budding can be induced by overexpression of wild-type (wt) BFV Gag and Env or artificial retargeting of Gag to the plasma membrane via myristoylation membrane targeting signals, closely resembling observations in other foamy viruses. Thus, the particle release machinery of wt BFV appears to be an excellent model system to study viral adaption to cell-free transmission by in vitro selection and evolution. Using selection for BFV variants with high cell-free infectivity in bovine and non-bovine cells, infectivity dramatically increased from almost no infectious units to about 105-106 FFU (fluorescent focus forming units)/mL in both cell types. Importantly, the selected BFV variants with high titer (HT) cell-free infectivity could still transmit via cell-cell contacts and were neutralized by serum from naturally infected cows. These selected HT-BFV variants will shed light into virus transmission and potential routes of intervention in the spread of viral infections. It will also allow the improvement or development of new promising approaches for antiretroviral therapies.

Identification of novel, highly expressed retroviral microRNAs in cells infected by bovine foamy virus.

UNLABELLED: While numerous viral microRNAs (miRNAs) expressed by DNA viruses, especially herpesvirus family members, have been reported, there have been very few reports of miRNAs derived from RNA viruses. Here we describe three miRNAs expressed by bovine foamy virus (BFV), a member of the spumavirus subfamily of retroviruses, in both BFV-infected cultured cells and BFV-infected cattle. All three viral miRNAs are initially expressed in the form of an ∼ 122-nucleotide (nt) pri-miRNA, encoded within the BFV long terminal repeat U3 region, that is subsequently cleaved to generate two pre-miRNAs that are then processed to yield three distinct, biologically active miRNAs. The BFV pri-miRNA is transcribed by RNA polymerase III, and the three resultant mature miRNAs were found to contribute a remarkable ∼ 70% of all miRNAs expressed in BFV-infected cells. These data document the second example of a retrovirus that is able to express viral miRNAs by using embedded proviral RNA polymerase III promoters. IMPORTANCE: Foamy viruses are a ubiquitous family of nonpathogenic retroviruses that have potential as gene therapy vectors in humans. Here we demonstrate that bovine foamy virus (BFV) expresses high levels of three viral microRNAs (miRNAs) in BFV-infected cells in culture and also in infected cattle. The BFV miRNAs are unusual in that they are initially transcribed by RNA polymerase III as a single, ∼ 122-nt pri-miRNA that is subsequently processed to release three fully functional miRNAs. The observation that BFV, a foamy virus, is able to express viral miRNAs in infected cells adds to emerging evidence that miRNA expression is a common, albeit clearly not universal, property of retroviruses and suggests that these miRNAs may exert a significant effect on viral replication in vivo.

A novel glucagon-like peptide 1 peptide identified from Ophisaurus harti.

Glucagon-like peptide 1 receptor (GLP1R) is a promising target for the treatment of type 2 diabetes. Because of the short half-life of endogenous GLP1 peptide, other GLP1R agonists are considered to be appealing therapeutic candidates. A high-throughput assay has been established to screen for GLP1R agonists in a 60 000-well natural product compound library fractionated from 670 different herbs/materials widely used in traditional Chinese medicines (TCMs). The screening is based on primary screen of GLP1R⁺ reporter gene assay with the counter screen in GLP1R⁻ cell line. An active fraction, A089-147, was identified from the screening. Fraction A089-147 was isolated from dried Ophisaurus harti, and the fact that its GLP1R agonist activity was sensitive to trypsin treatment indicates its peptidic nature. The active ingredient of A089-147 was later identified as O. harti GLP1 through transcriptome analysis. Chemically synthesized O. harti GLP1 showed GLP1R agonist activity and sensitivity to dipeptidase IV digestion. This study illustrated a comprehensive screening strategy to identify novel GLP1R agonists from TCMs libraries and at the same time underlined the difficulty of identifying a non-peptidic GLP1R agonist. The novel O. harti GLP1 peptide yielded from this study confirmed broader application of TCMs libraries in active peptide identification.

Feline foamy virus-based vectors: advantages of an authentic animal model.

New-generation retroviral vectors have potential applications in vaccination and gene therapy. Foamy viruses are particularly interesting as vectors, because they are not associated to any disease. Vector research is mainly based on primate foamy viruses (PFV), but cats are an alternative animal model, due to their smaller size and the existence of a cognate feline foamy virus (FFV). The potential of replication-competent (RC) FFV vectors for vaccination and replication-deficient (RD) FFV-based vectors for gene delivery purposes has been studied over the past years. In this review, the key achievements and functional evaluation of the existing vectors from in vitro cell culture systems to out-bred cats will be described. The data presented here demonstrate the broad application spectrum of FFV-based vectors, especially in pathogen-specific prophylactic and therapeutic vaccination using RD vectors in cats and in classical gene delivery. In the cat-based system, FFV-based vectors provide an advantageous platform to evaluate and optimize the applicability, efficacy and safety of foamy virus (FV) vectors, especially the understudied aspect of FV cell and organ tropism.

Cytotoxic triterpenoids and steroids from the bark of Melia azedarach.

Two new triterpenoids (1, 2) and two new steroids (3, 4) along with twelve related known compounds (5-16) were isolated from the bark of Melia azedarach. The new structures were elucidated by means of spectroscopic methods and molecular modeling studies and found to be 21,24-cycloeupha-7-ene-3 ß,16 ß,21 α,25-tetrol (1), 3 ß-acetoxy-12 ß-hydroxy-eupha-7,24-dien-21,16 ß-olide (2), 29-hydroperoxy-stigmasta-7,24(28) E-dien-3 ß-ol (3), and 24 ξ-hydroperoxy-24-vinyl-lathosterol (4). All isolated compounds were tested for their cytotoxic activity against three human cancer cell lines (A549, H460, HGC27) using the CellTiter Glo™ luminescent cell viability assay. Among them, compounds 2- 4, 24 ξ-hydroperoxy-24-vinyl-cholesterol (6), kulinone (7), meliastatin 3 ( 8), 3-oxo-olean-12-en-28-oic acid (10), and (22 E,24 S)-5 α,8 α-epidioxy-24-methyl-cholesta-6,22-dien-3 ß-ol (12) were found to have cytotoxic effects, with IC50 values of 5.6-21.2 µg/mL.