Structures of Substrate Complexes of Foamy Viral Protease-Reverse Transcriptase.

Reverse transcriptases (RTs) use their DNA polymerase and RNase H activities to catalyze the conversion of single-stranded RNA to double-stranded DNA (dsDNA), a crucial process for the replication of retroviruses. Foamy viruses (FVs) possess a unique RT, which is a fusion with the protease (PR) domain. The mechanism of substrate binding by this enzyme has been unknown. Here, we report a crystal structure of monomeric full-length marmoset FV (MFV) PR-RT in complex with an RNA/DNA hybrid substrate. We also describe a structure of MFV PR-RT with an RNase H deletion in complex with a dsDNA substrate in which the enzyme forms an asymmetric homodimer. Cryo-electron microscopy reconstruction of the full-length MFV PR-RT-dsDNA complex confirmed the dimeric architecture. These findings represent the first structural description of nucleic acid binding by a foamy viral RT and demonstrate its ability to change its oligomeric state depending on the type of bound nucleic acid. IMPORTANCE Reverse transcriptases (RTs) are intriguing enzymes converting single-stranded RNA to dsDNA. Their activity is essential for retroviruses, which are divided into two subfamilies differing significantly in their life cycles: Orthoretrovirinae and Spumaretrovirinae. The latter family is much more ancient and comprises five genera. A unique feature of foamy viral RTs is that they contain N-terminal protease (PR) domains, which are not present in orthoretroviral enzymes. So far, no structural information for full-length foamy viral PR-RT interacting with nucleic substrates has been reported. Here, we present crystal and cryo-electron microscopy structures of marmoset foamy virus (MFV) PR-RT. These structures revealed the mode of binding of RNA/DNA and dsDNA substrates. Moreover, unexpectedly, the structures and biochemical data showed that foamy viral PR-RT can adopt both a monomeric configuration, which is observed in our structures in the presence of an RNA/DNA hybrid, and an asymmetric dimer arrangement, which we observed in the presence of dsDNA.

Coelacanth SERINC2 Inhibits HIV-1 Infectivity and Is Counteracted by Envelope Glycoprotein from Foamy Virus.

SERINC5 restricts nef-defective HIV-1 by affecting early steps of the virus life cycle. Distantly related retroviruses with a wide host range encode virulent factors in response to challenge by SERINC5. However, the evolutionary origins of this antiretroviral activity, its prevalence among the paralogs, and its ability to target retroviruses remain understudied. In agreement with previous studies, we found that four human SERINC paralogs inhibit nef-defective HIV-1, with SERINC2 being an exception. Here, we demonstrate that this lack of activity in human SERINC2 is associated with its post-whole-genome duplication (post-WGD) divergence, as evidenced by the ability of pre-WGD orthologs from Saccharomyces cerevisiae and flies and a post-WGD-proximate SERINC2 from coelacanths to inhibit the virus. Intriguingly, Nef is unable to counter coelacanth SERINC2, indicating that such activity was directed toward other retroviruses found in coelacanths (like foamy viruses). However, foamy virus-derived vectors are intrinsically resistant to the action of SERINC2, and we show that the foamy virus envelope confers this resistance by affecting its steady-state levels. Our study highlights an ancient origin of antiretroviral activity in SERINCs and a hitherto-unknown interaction with a foamy virus. IMPORTANCESERINC5 constitutes a critical barrier to the propagation of retroviruses, as highlighted by parallel emergence of anti-SERINC5 activities among distant retroviral lineages. Therefore, understanding the origin and evolution of these host factors will provide key information about virus-host relationships that can be exploited for future drug development. Here, we show that SERINC5-mediated nef-defective HIV-1 infection inhibition is evolutionarily conserved. SERINC2 from coelacanth restricts HIV-1, and it was functionally adapted to target foamy viruses. Our findings provide insights into the evolutionary origin of antiretroviral activity in the SERINC gene family and uncover the role of SERINCs in shaping the long-term conflicts between retroviruses and their hosts.

Is there any relationship between human foamy virus infections and familial Mediterranean fever?

BACKGROUND: Familial Mediterranean fever (FMF) is generally defined as an autosomal recessive disease, characterized by the automatic activation of the innate immune system in the absence of a detectable pathogenic stimulant. We hypothesize that the pathogenic factors, besides the genetic causes, may affect the development of FMF symptoms. To test this hypothesis, we examined the effects of human foamy virus (HFV) positivity on the occurrence of the clinical symptoms of FMF. MATERIALS AND METHODS: Two hundred and twenty-two FMF patients with definitive diagnosis according to Tel Hashomer criteria (study group 1 [SG1]), 205 symptomatic FMF patients who had definitive diagnosis according to the same criteria but did not carry any of the 12 most commonly occurring MEFV gene mutations (study group 2 [SG2]), and 200 healthy individuals were included as control group (study group 3 [SG3]) in the study. The genetic analysis was applied in the Molecular Genetics Laboratory of the Department of Medical Biology, Faculty of Medicine, Ondokuz Mayis University. This study was designed as a case-control study. HFV positivity was tested by amplifying the HFV bel1 gene sequence with polymerase chain reaction technique. Statistical analyses were conducted using SPSS version 23.0 software. RESULTS: HFV positivity showed significant differences between the study groups (P = 0.002). While 43 (19.02%) of the 222 SG1 patients were positive for the HFV bel1 gene sequence, 33 (16.09%) of the 205 SG2 patients were positive for the same sequence. Only 15 (7.5%) of the SG3 participants were positive for the presence of HFV bel1 gene sequence. CONCLUSION: The results of our study suggested that HFV positivity can be a stimulant pathogenic factor of natural immune system which can cause the emergence of FMF symptoms.

A purine-rich element in foamy virus pol regulates env splicing and gag/pol expression.

BACKGROUND: The foamy viral genome encodes four central purine-rich elements localized in the integrase-coding region of pol. Previously, we have shown that the first two of these RNA elements (A and B) are required for protease dimerization and activation. The D element functions as internal polypurine tract during reverse transcription. Peters et al., described the third element (C) as essential for gag expression suggesting that it might serve as an RNA export element for the unspliced genomic transcript. RESULTS: Here, we analysed env splicing and demonstrate that the described C element composed of three GAA repeats known to bind SR proteins regulates env splicing, thus balancing the amount of gag/pol mRNAs. Deletion of the C element effectively promotes a splice site switch from a newly identified env splice acceptor to the intrinsically strong downstream localised env 3' splice acceptor permitting complete splicing of almost all LTR derived transcripts. We provide evidence that repression of this env splice acceptor is a prerequisite for gag expression. This repression is achieved by the C element, resulting in impaired branch point recognition and SF1/mBBP binding. Separating the branch point from the overlapping purine-rich C element, by insertion of only 20 nucleotides, liberated repression and fully restored splicing to the intrinsically strong env 3' splice site. This indicated that the cis-acting element might repress splicing by blocking the recognition of essential splice site signals. CONCLUSIONS: The foamy viral purine-rich C element regulates splicing by suppressing the branch point recognition of the strongest env splice acceptor. It is essential for the formation of unspliced gag and singly spliced pol transcripts.

Retroviral RNA Processing.

This review is an accompaniment to a Special Issue on "Retroviral RNA Processing". It discusses post-transcriptional regulation of retroviruses, ranging from the ancient foamy viruses to more modern viruses, such as HIV-1, HTLV-1, Rous sarcoma virus, murine leukemia virus, mouse mammary tumor virus, and Mason-Pfizer monkey virus. This review is not comprehensive. However, it tries to address some of the major questions in the field with examples of how different retroviruses express their genes. It is amazing that a single primary RNA transcript can have so many possible fates: genomic RNA, unspliced mRNA, and up to 50 different alternatively spliced mRNAs. This review will discuss the sorting of RNAs for packaging or translation, RNA nuclear export mechanisms, splicing, translation, RNA modifications, and avoidance of nonsense-mediated RNA decay.

Infection with Foamy Virus in Wild Ruminants-Evidence for a New Virus Reservoir?

Foamy viruses (FVs) are widely distributed and infect many animal species including non-human primates, horses, cattle, and cats. Several reports also suggest that other species can be FV hosts. Since most of such studies involved livestock or companion animals, we aimed to test blood samples from wild ruminants for the presence of FV-specific antibodies and, subsequently, genetic material. Out of 269 serum samples tested by ELISA with the bovine foamy virus (BFV) Gag and Bet antigens, 23 sera showed increased reactivity to at least one of them. High reactive sera represented 30% of bison samples and 7.5% of deer specimens. Eleven of the ELISA-positives were also strongly positive in immunoblot analyses. The peripheral blood DNA of seroreactive animals was tested by semi-nested PCR. The specific 275 bp fragment of the pol gene was amplified only in one sample collected from a red deer and the analysis of its sequence showed the highest homology for European BFV isolates. Such results may suggest the existence of a new FV reservoir in bison as well as in deer populations. Whether the origin of such infections stems from a new FV or is the result of BFV inter-species transmission remains to be clarified.

Identification and evolution of avian endogenous foamy viruses.

A history of long-term co-divergence means that foamy viruses (family Retroviridae) provide an ideal framework to understanding virus-host evolution over extended time periods. Endogenous foamy viruses (EndFVs) are rare, and to date have only been described in a limited number of mammals, amphibians, reptiles and fish genomes. By screening 414 avian genomes we identified EndFVs in two bird species: the Maguari Stork (Ciconia maguari) and the Oriental Stork (Ciconia boyciana). Analyses of phylogenetic relationships, genome structures and flanking sequences revealed a single origin of EndFVs in Ciconia species. In addition, the marked incongruence between the virus and host phylogenies suggested that this integration event occurred independently in birds. In sum, by providing evidence that birds can be infected with foamy viruses, we fill the last major gap in the taxonomic distribution of foamy viruses and their animal hosts.

Prospects for Foamy Viral Vector Anti-HIV Gene Therapy.

Stem cell gene therapy approaches for Human Immunodeficiency Virus (HIV) infection have been explored in clinical trials and several anti-HIV genes delivered by retroviral vectors were shown to block HIV replication. However, gammaretroviral and lentiviral based retroviral vectors have limitations for delivery of anti-HIV genes into hematopoietic stem cells (HSC). Foamy virus vectors have several advantages including efficient delivery of transgenes into HSC in large animal models, and a potentially safer integration profile. This review focuses on novel anti-HIV transgenes and the potential of foamy virus vectors for HSC gene therapy of HIV.

Interferon but not MxB inhibits foamy retroviruses.

Foamy viruses (FV) are retroviruses that are widely distributed in primate and non-primate animal species. We tested here FV with capsids of simian and non-simian origin for sensitivity to interferon-ß (IFN-ß). Our data show significant inhibition of FV by IFN-ß early in infection of human HOS and THP-1 but not of HEK293T cells. The post-entry restriction of FV was not mediated by the interferon-induced MxB protein that was recently identified as a capsid-interacting restriction factor targeting Human immunodeficiency virus (HIV) before integration. Neither the ectopic expression of MxA or MxB in HEK293T cells nor the lack of MxB expression in CRISPR/CAS MxB THP-1 knockout cells impacted the infection of the tested FV. IFN-ß treated THP-1 and THP-1 KO MxB cells showed the same extend of restriction to FV. Together, the data demonstrate that IFN-ß inhibits FV early in infection and that MxB is not a restriction factor of FV.

A new sensitive indicator cell line reveals cross-transactivation of the viral LTR by gorilla and chimpanzee simian foamy viruses.

The majority of currently identified simian foamy virus (SFV)-infected Cameroonian and Gabonese individuals harbor SFV from the gorilla lineage. We constructed an indicator cell line for the quantification of gorilla SFVs, in which the U3 sequence of a gorilla SFV directs the expression of the ß-galactosidase protein. The gorilla foamy virus activated ß-galactosidase (GFAB) cells efficiently quantified two zoonotic primary gorilla isolates and SFVs from three chimpanzee subspecies. Primary gorilla SFVs replicated more slowly and at lower levels than primary chimpanzee SFVs. Analysis of previously described motifs of Tas proteins and U3 LTRs involved in viral gene synthesis revealed conservation of such motifs in Tas proteins from gorilla and chimpanzee SFVs, but little sequence homology in the LTR regions previously shown to interact with viral and cellular factors.

Construction and characterisation of replicating foamy viral vectors expressing HIV-1 epitopes recognised by broadly neutralising antibodies.

With the aim to develop a replicating vector system for the delivery of HIV-1 antigens on the basis of an apathogenic foamy virus we recently showed that immunisation with purified recombinant hybrid antigens composed of the feline foamy virus Bet protein and parts of the transmembrane envelope protein of HIV-1 induced antibodies with an epitope specificity identical to that of the broadly neutralising antibody 2F5 (Mühle et al., Immunol Res., 2013, 56:61-72). Here we set out to further improve the HIV-1 inserts consisting of the membrane proximal external region (MPER) and the fusion peptide proximal region (FPPR) by stepwise shortening distinct linker residues between both domains. In a subset of these antigens, enhanced recognition by 2F5 and 4E10 was observed, indicating that a specific positioning of FPPR and MPER domains is critical for improved antibody binding. Introduction of these optimised inserts as well as of the MPER domain alone into the feline foamy virus backbone was compatible with virus replication, giving viral titres similar to wild-type virus after extended passaging. Most importantly, expression of the HIV-1 transgenes in infected feline CRFK cells remained stable in three out of four constructs and was detectable after serial passages for several weeks. These data encourage further testing of these vectors in vivo, which may allow insights into the necessity of affinity maturation for the induction of broadly reactive HIV-1 antibodies.