Foamy viruses (FVs) are an ancient lineage of retroviruses, with an evolutionary history spanning over 450 million years. Vector systems based on Prototype Foamy Virus (PFV) are promising candidates for gene and oncolytic therapies. Structural studies of PFV contribute to the understanding of the mechanisms of FV replication, cell entry and infection, and retroviral evolution. Here we combine cryoEM and cryoET to determine high-resolution in situ structures of the PFV icosahedral capsid (CA) and envelope glycoprotein (Env), including its type III transmembrane anchor and membrane-proximal external region (MPER), and show how they are organized in an integrated structure of assembled PFV particles. The atomic models reveal an ancient retroviral capsid architecture and an unexpected relationship between Env and other class 1 fusion proteins of the Mononegavirales. Our results represent the de novo structure determination of an assembled retrovirus particle.

Retroviral integration into ancient vertebrate genomes left traces that can shed light on the early history of viruses. In this study, we explored the early evolution of retroviruses by isolating nine Spuma endogenous retroviruses (ERVs) and one Epsilon ERV from the genomes of Agnatha and Chondrichthyes. Phylogenetic analysis of protein sequences revealed a striking pattern of co-evolution between jawless fish ERV and their host, while shark ERV underwent ancient cross-class viral transmission with jawless fish, ray-finned fish, and amphibians. Nucleotide sequence analysis showed that jawless fish ERV emerged in the Palaeozoic period, relatively later than ray-finned fish ERV. Moreover, codon analysis suggested that the jawless fish ERV employed an infection strategy that mimics the host codon. The genealogical diversity of ERVs in the jawless fish genome highlights the importance of studying different viral species. Overall, our findings provide valuable insights into the evolution of retroviruses and their interactions with their hosts.

Tsushima leopard cats (TLC; Prionailurus bengalensis euptilurus) only inhabit Tsushima Island, Nagasaki, Japan and are critically endangered and threatened by infectious diseases. The feline foamy virus (FFV) is widely endemic in domestic cats. Therefore, its transmission from domestic cats to TLCs may threaten the TLC population. Thus, this study aimed to assess the possibility that domestic cats could transmit FFV to TLCs. Eighty-nine TLC samples were screened, and FFV was identified in seven (7.86%). To assess the FFV infection status of domestic cats, 199 domestic cats were screened; 14.07% were infected. The phylogenetic analysis revealed that the FFV partial sequence from domestic cats and TLC sequences clustered in one clade, suggesting that the two populations share the same strain. The statistical data minimally supported the association between increased infection rate and sex (p = 0.28), indicating that FFV transmission is not sex dependent. In domestic cats, a significant difference was observed in FFV detection in feline immunodeficiency virus (p = 0.002) and gammaherpesvirus1 infection statuses (p = 0.0001) but not in feline leukemia virus infection status (p = 0.21). Monitoring FFV infection in domestic cats and TLC populations is highly recommended as part of TLC surveillance and management strategies.

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.

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.

Many cellular and viral proteins and nucleic acids shuttle between the nucleus and the cytoplasm. It is increasingly clear that nuclear import and export involve complex and finely regulated mechanisms. Nuclear export is absolutely necessary for viral protein synthesis and particle assembly for retroviruses showing a nuclear step during their replication cycle. Nuclear export of retroviral components mainly relies on two distinct mechanisms, one involving cellular factors only, and another in which cellular and viral factors cooperate. The study of retrovirus nuclear export contributes significantly to our understanding of the molecular mechanisms of nuclear export in general, and may allow the identification of new targets to prevent retrovirus replication.

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 β2 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.

Foamy viruses (FVs) or heterologous retroviruses pseudotyped with FV glycoprotein enable transduction of a great variety of target tissues of disparate species. Specific cellular entry receptors responsible for this exceptionally broad tropism await their identification. Though, ubiquitously expressed heparan sulfate proteoglycan (HS-PG) is known to serve as an attachment factor of FV envelope (Env)-containing virus particles, greatly enhancing target cell permissiveness. Production of high-titer, FV Env-containing retroviral vectors is strongly dependent on the use of cationic polymer-based transfection reagents like polyethyleneimine (PEI). We identified packaging cell-surface HS-PG expression to be responsible for this requirement. Efficient release of FV Env-containing virus particles necessitates neutralization of HS-PG binding sites by PEI. Remarkably, remnants of PEI in FV Env-containing vector supernatants, which are not easily removable, negatively impact target cell transduction, in particular those of myeloid and lymphoid origin. To overcome this limitation for production of FV Env-containing retrovirus supernatants, we generated 293T-based packaging cell lines devoid of HS-PG by genome engineering. This enabled, for the first, time production of inhibitor-free, high-titer FV Env-containing virus supernatants by non-cationic polymer-mediated transfection. Depending on the type of virus, produced titers were 2- to 10-fold higher compared with those obtained by PEI transfection.

The retroviral surface envelope protein subunit (SU) mediates receptor binding and triggers membrane fusion by the transmembrane (TM) subunit. SU evolves rapidly under strong selective conditions, resulting in seemingly unrelated SU structures in highly divergent retroviruses. Structural modeling of the SUs of several retroviruses and related endogenous retroviral elements with AlphaFold 2 identifies a TM-proximal SU β-sandwich structure that has been conserved in the orthoretroviruses for at least 110 million years. The SU of orthoretroviruses diversified by the differential expansion of the β-sandwich core to form domains involved in virus-host interactions. The β-sandwich domain is also conserved in the SU equivalent GP1 of Ebola virus although with a significantly different orientation in the trimeric envelope protein structure relative to the β-sandwich of human immunodeficiency virus type 1 gp120, with significant evidence for divergent rather than convergent evolution. The unified structural view of orthoretroviral SU and filoviral GP1 identifies an ancient, structurally conserved, and evolvable domain underlying the structural diversity of orthoretroviral SU and filoviral GP1. IMPORTANCE The structural relationships of SUs of retroviral groups are obscured by the high rate of sequence change of SU and the deep-time divergence of retroviral lineages. Previous data showed no structural or functional relationships between the SUs of type C gammaretroviruses and lentiviruses. A deeper understanding of structural relationships between the SUs of different retroviral lineages would allow the generalization of critical processes mediated by these proteins in host cell infection. Modeling of SUs with AlphaFold 2 reveals a conserved core domain underlying the structural diversity of orthoretroviral SUs. Definition of the conserved SU structural core allowed the identification of a homologue structure in the SU equivalent GP1 of filoviruses that most likely shares an origin, unifying the SU of orthoretroviruses and GP1 of filoviruses into a single protein family. These findings will allow an understanding of the structural basis for receptor-mediated membrane fusion mechanisms in a broad range of biomedically important retroviruses.

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.