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The endosomal sorting complex required for transport (ESCRT) machinery is essential for the budding of retroviruses such as human immunodeficiency virus (HIV) and bovine foamy virus (BFV), which rely on their late domain to recruit ESCRT complexes to facilitate budding. However, the impact of intracellular host proteins on BFV budding remains poorly understood. In this study, we aimed to investigate the impact of CCL2 on BFV budding and interactions with key host proteins. Our results indicate that CCL2 promotes BFV budding in an ALG-2-interacting protein X (Alix)-dependent manner by enhancing the interaction between Alix and BFV Gag (BGag). Notably, we found a link between Alix, BGag and CCL2, with Alix mediating the interaction between the latter two. Furthermore, we observed that natural host bovine CCL2 also has a facilitating role in the budding process of BFV, similar to human CCL2. Taken together, these results demonstrate that CCL2 promotes BFV budding by enhancing the Alix-BGag association.

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).

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.

Membrane proteins of enveloped viruses have been reported to undergo palmitoylation, a post-translational modification often having a critical role in the function of these viral proteins and hence viral replication. In this study, we report that the foamy virus (FV) envelope (Env) glycoprotein is palmitoylated. Specifically, we found that bovine foamy virus (BFV) Env (BEnv) is palmitoylated at amino acid positions C58 and C59 by BDHHC3 and BDHHC20 in a DHHC motif-dependent manner. In addition, mutations C58S and C58/59S significantly decrease cell surface expression of BEnv, subviral particle (SVP) egress, and its membrane fusion activity, thus ultimately inhibiting BFV replication. The C59S mutation exerts a minor effect in this regard. Taken together, these data demonstrate that the function of BEnv in the context of BFV replication is under the regulation of palmitoylation.

In addition to regulatory or accessory proteins, some complex retroviruses gain a repertoire of micro-RNAs (miRNAs) to regulate and control virus–host interactions for efficient replication and spread. In particular, bovine and simian foamy viruses (BFV and SFV) have recently been shown to express a diverse set of RNA polymerase III-directed miRNAs, some with a unique primary miRNA double-hairpin, dumbbell-shaped structure not known in other viruses or organisms. While the mechanisms of expression and structural requirements have been studied, the functional importance of these miRNAs is still far from understood. Here, we describe the in silico identification of BFV miRNA targets and the subsequent experimental validation of bovine Ankyrin Repeat Domain 17 (ANKRD17) and Bax-interacting factor 1 (Bif1) target genes in vitro and, finally, the suppression of ANKRD17 downstream genes in the affected pathway. Deletion of the entire miRNA cassette in the non-coding part of the U3 region of the long terminal repeats attenuated replication of corresponding BFV mutants in bovine cells. This repression can be almost completely trans-complemented by the most abundant miRNA BF2-5p having the best scores for predicted and validated BFV miRNA target genes. Deletion of the miRNA cassette does not grossly affect particle release and overall particle composition.

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.

The retroviral subfamily of Spumaretrovirinae consists of five genera of foamy (spuma) viruses (FVs) that are endemic in some mammalian hosts [1]. Closely related species may be susceptible to the same or highly related FVs. FVs are not known to induce overt disease and thus do not pose medical problems to humans and livestock or companion animals. A robust lab animal model is not available or is a lab animal a natural host of a FV. Due to this, research is limited and often focused on the simian FVs with their well-established zoonotic potential. The authors of this review and their groups have conducted several studies on bovine FV (BFV) in the past with the intention of (i) exploring the risk of zoonotic infection via beef and raw cattle products, (ii) studying a co-factorial role of BFV in different cattle diseases with unclear etiology, (iii) exploring unique features of FV molecular biology and replication strategies in non-simian FVs, and (iv) conducting animal studies and functional virology in BFV-infected calves as a model for corresponding studies in primates or small lab animals. These studies gained new insights into FV-host interactions, mechanisms of gene expression, and transcriptional regulation, including miRNA biology, host-directed restriction of FV replication, spread and distribution in the infected animal, and at the population level. The current review attempts to summarize these findings in BFV and tries to connect them to findings from other FVs.

Foamy viruses (FVs) have extensive cell tropism in vitro, special replication features, and no clinical pathogenicity in naturally or experimentally infected animals, which distinguish them from orthoretroviruses. Among FVs, bovine foamy virus (BFV) has undetectable or extremely low levels of cell-free transmission in the supernatants of infected cells and mainly spreads by cell-to-cell transmission, which deters its use as a gene transfer vector. Here, using an in vitro virus evolution system, we successfully isolated high-titer cell-free BFV strains from the original cell-to-cell transmissible BFV3026 strain and further constructed an infectious cell-free BFV clone called pBS-BFV-Z1. Following sequence alignment with a cell-associated clone pBS-BFV-B, we identified a number of changes in the genome of pBS-BFV-Z1. Extensive mutagenesis analysis revealed that the C-terminus of envelope protein, especially the K898 residue, controls BFV cell-free transmission by enhancing cell-free virus entry but not the virus release capacity. Taken together, our data show the genetic determinants that regulate cell-to-cell and cell-free transmission of BFV.