The piRNA Response to Retroviral Invasion of the Koala Genome.

Antisense Piwi-interacting RNAs (piRNAs) guide silencing of established transposons during germline development, and sense piRNAs drive ping-pong amplification of the antisense pool, but how the germline responds to genome invasion is not understood. The KoRV-A gammaretrovirus infects the soma and germline and is sweeping through wild koalas by a combination of horizontal and vertical transfer, allowing direct analysis of retroviral invasion of the germline genome. Gammaretroviruses produce spliced Env mRNAs and unspliced transcripts encoding Gag, Pol, and the viral genome, but KoRV-A piRNAs are almost exclusively derived from unspliced genomic transcripts and are strongly sense-strand biased. Significantly, selective piRNA processing of unspliced proviral transcripts is conserved from insects to placental mammals. We speculate that bypassed splicing generates a conserved molecular pattern that directs proviral genomic transcripts to the piRNA biogenesis machinery and that this "innate" piRNA response suppresses transposition until antisense piRNAs are produced, establishing sequence-specific adaptive immunity.

Second site reversion of HIV-1 envelope protein baseplate mutations maps to the matrix protein.

The HIV-1 Envelope (Env) protein cytoplasmic tail (CT) recently has been shown to assemble an unusual trimeric baseplate structure that locates beneath Env ectodomain trimers. Mutations at linchpin residues that help organize the baseplate impair virus replication in restrictive T cell lines but not in permissive cell lines. We have identified and characterized a second site suppressor of these baseplate mutations, located at residue 34 in the viral matrix (MA) protein, that rescues viral replication in restrictive cells. The suppressor mutation was dependent on the CT to exert its activity and did not appear to affect Env protein traffic or fusion functions in restrictive cells. Instead, the suppressor mutation increased Env incorporation into virions 3-fold and virus infectivity in single-round infections 10-fold. We also found that a previously described suppressor of Env-incorporation defects that stabilizes the formation of MA trimers was ineffective at rescuing Env baseplate mutations. Our results support an interpretation in which changes at MA residue 34 induce conformational changes that stabilize MA lattice trimer-trimer interactions and/or direct MA-CT associations.IMPORTANCEHow HIV-1 Env trimers assemble into virus particles remains incompletely understood. In restrictive cells, viral incorporation of Env is dependent on the Env CT and on the MA protein, which assembles lattices composed of hexamers of trimers in immature and mature viruses. Recent evidence indicates that CT assembles trimeric baseplate structures that require membrane-proximal residues to interface with trimeric transmembrane domains and C-terminal helices in the CT. We found that mutations of these membrane-proximal residues impaired replication in restrictive cells. This defect was countered by a MA mutation that does not localize to any obvious interprotein regions but was only inefficiently suppressed by a MA mutation that stabilizes MA trimers and has been shown to suppress other CT-dependent Env defects. Our results suggest that efficient suppression of baseplate mutations involves stabilization of MA inter-trimer contacts and/or direct MA-CT associations. These observations shed new light on how Env assembles into virions.

FeLIX is a restriction factor for mammalian retrovirus infection.

Endogenous retroviruses (ERVs) are remnants of ancestral viral infections. Feline leukemia virus (FeLV) is an exogenous and endogenous retrovirus in domestic cats. It is classified into several subgroups (A, B, C, D, E, and T) based on viral receptor interference properties or receptor usage. ERV-derived molecules benefit animals, conferring resistance to infectious diseases. However, the soluble protein encoded by the defective envelope (env) gene of endogenous FeLV (enFeLV) functions as a co-factor in FeLV subgroup T infections. Therefore, whether the gene emerged to facilitate viral infection is unclear. Based on the properties of ERV-derived molecules, we hypothesized that the defective env genes possess antiviral activity that would be advantageous to the host because FeLV subgroup B (FeLV-B), a recombinant virus derived from enFeLV env, is restricted to viral transmission among domestic cats. When soluble truncated Env proteins from enFeLV were tested for their inhibitory effects against enFeLV and FeLV-B, they inhibited viral infection. Notably, this antiviral machinery was extended to infection with the Gibbon ape leukemia virus, Koala retrovirus A, and Hervey pteropid gammaretrovirus. Although these viruses used feline phosphate transporter 1 (fePit1) and phosphate transporter 2 as receptors, the inhibitory mechanism involved competitive receptor binding in a fePit1-dependent manner. The shift in receptor usage might have occurred to avoid the inhibitory effect. Overall, these findings highlight the possible emergence of soluble truncated Env proteins from enFeLV as a restriction factor against retroviral infection and will help in developing host immunity and antiviral defense by controlling retroviral spread.IMPORTANCERetroviruses are unique in using reverse transcriptase to convert RNA genomes into DNA, infecting germ cells, and transmitting to offspring. Numerous ancient retroviral sequences are known as endogenous retroviruses (ERVs). The soluble Env protein derived from ERVs functions as a co-factor that assists in FeLV-T infection. However, herein, we show that the soluble Env protein exhibits antiviral activity and provides resistance to mammalian retrovirus infection through competitive receptor binding. In particular, this finding may explain why FeLV-B transmission is not observed among domestic cats. ERV-derived molecules can benefit animals in an evolutionary arms race, highlighting the double-edged-sword nature of ERVs.

Cross-species transmission of an ancient endogenous retrovirus and convergent co-option of its envelope gene in two mammalian orders.

Endogenous retroviruses (ERVs) found in vertebrate genomes are remnants of retroviral invasions of their ancestral species. ERVs thus represent molecular fossil records of ancient retroviruses and provide a unique opportunity to study viral-host interactions, including cross-species transmissions, in deep time. While most ERVs contain the mutated remains of the original retrovirus, on rare occasions evolutionary selection pressures lead to the co-option/exaptation of ERV genes for a host function. Here, we report the identification of two ancient related non-orthologous ERV env genes, ARTenvV and CARenvV, that are preserved with large open reading frames (ORFs) in the mammalian orders Artiodactyla and Carnivora, respectively, but are not found in other mammals. These Env proteins lack a transmembrane motif, but phylogenetic analyses show strong sequence preservation and positive selection of the env surface ORF in their respective orders, and transcriptomic analyses show a broad tissue expression pattern for both ARTenvV and CARenvV, suggesting that these genes may be exapted for a host function. Multiple lines of evidence indicate that ARTenvV and CARenvV were derived from an ancient ancestral exogenous gamma-like retrovirus that was independently endogenized in two mammalian orders more than 60 million years ago, which roughly coincides with the K-Pg mass extinction event and subsequent mammalian diversification. Thus, these findings identify the oldest known retroviral cross-ordinal transmission of a gamma-like retrovirus with no known extant infectious counterpart in mammals, and the first discovery of the convergent co-option of an ERV gene derived from the same ancestral retrovirus in two different mammalian orders.

Correlation between antibodies against the pathogenic pHERV-W envelope protein and the inflammatory phase of multiple sclerosis.

The role of retroviral envelope proteins belonging to the Human Endogenous Retroviral family 'W' (HERV-W), specifically syncytin-1 and pathogenic HERV-W (pHERV-W), as potential risk factors in multiple sclerosis (MS) has been established. This study aimed to investigate the humoral response to syncytin-1 and pHERV-W-derived peptides in a group of relapsing remitting MS patients categorized as having acute or stable disease. Furthermore, an inhibition assay was conducted to assess the extent of cross-reactivity between the two epitopes. The findings revealed that MS patients in the acute phase exhibited a higher specific antibody response to the pHERV-W env epitope compared to syncytin-1. This suggests a potential pathogenic role for pHERV-W env during the inflammatory stages of central nervous system involvement, and these antibody responses could serve as useful biomarkers for monitoring the progression of the disease.

Dynamic Evolution of Retroviral Envelope Genes in Egg-Laying Mammalian Genomes.

Independently acquired envelope (env) genes from endogenous retroviruses have contributed to the placental trophoblast cell-cell fusion in therian mammals. Egg-laying mammals (monotremes) are an important sister clade for understanding mammalian placental evolution, but the env genes in their genomes have yet to be investigated. Here, env-derived open reading frames (env-ORFs) encoding more than 400 amino acid lengths were searched in the genomes of two monotremes: platypus and echidna. Only two env-ORFs were present in the platypus genome, whereas 121 env-ORFs were found in the echidna genome. The echidna env-ORFs were phylogenetically classified into seven groups named env-Tac1 to -Tac7. Among them, the env-Tac1 group contained only a single gene, and its amino acid sequence showed high similarity to those of the RD114/simian type D retroviruses. Using the pseudotyped virus assay, we demonstrated that the Env-Tac1 protein utilizes echidna sodium-dependent neutral amino acid transporter type 1 and 2 (ASCT1 and ASCT2) as entry receptors. Moreover, the Env-Tac1 protein caused cell-cell fusion in human 293T cells depending on the expression of ASCT1 and ASCT2. These results illustrate that fusogenic env genes are not restricted to placental mammals, providing insights into the evolution of retroviral genes and the placenta.

A cellular trafficking signal in the SIV envelope protein cytoplasmic domain is strongly selected for in pathogenic infection.

The HIV/SIV envelope glycoprotein (Env) cytoplasmic domain contains a highly conserved Tyr-based trafficking signal that mediates both clathrin-dependent endocytosis and polarized sorting. Despite extensive analysis, the role of these functions in viral infection and pathogenesis is unclear. An SIV molecular clone (SIVmac239) in which this signal is inactivated by deletion of Gly-720 and Tyr-721 (SIVmac239ΔGY), replicates acutely to high levels in pigtail macaques (PTM) but is rapidly controlled. However, we previously reported that rhesus macaques and PTM can progress to AIDS following SIVmac239ΔGY infection in association with novel amino acid changes in the Env cytoplasmic domain. These included an R722G flanking the ΔGY deletion and a nine nucleotide deletion encoding amino acids 734-736 (ΔQTH) that overlaps the rev and tat open reading frames. We show that molecular clones containing these mutations reconstitute signals for both endocytosis and polarized sorting. In one PTM, a novel genotype was selected that generated a new signal for polarized sorting but not endocytosis. This genotype, together with the ΔGY mutation, was conserved in association with high viral loads for several months when introduced into naïve PTMs. For the first time, our findings reveal strong selection pressure for Env endocytosis and particularly for polarized sorting during pathogenic SIV infection in vivo.

Evolution and Genetic Diversity of the Retroviral Envelope in Anamniotes.

Retroviruses are widely distributed in all vertebrates, as are their endogenous forms, endogenous retroviruses (ERV), which serve as "fossil" evidence to trace the ancient origins and history of virus-host interactions over millions of years. The retroviral envelope (Env) plays a significant role in host range determination, but major information on their genetic diversification and evolution in anamniotes is lacking. Here, by incorporating multiple-round in silico similarity search and phylogenomic analysis, more than 30,000 copies of ERV lineages with gamma-type Env (GTE), covalently associated Env, were discovered by searching against all fish and amphibian genomes and transcriptomic assemblies, but no beta-type Env (BTE), noncovalently associated Env, was found. Furthermore, a nine-type classification system of anamniote GTE was proposed by combining phylogenetic and domain/motif analyses. The elastic genomic organization and overall phylogenetic incongruence between anamniotic Env and its neighboring polymerase (Pol) implied that early retroviral diversification in anamniotic vertebrates was facilitated by frequent recombination. At last, host cellular opioid growth factor receptor (OGFr) gene capturing by anamniotic ERVs with GTE was reported for the first time. Overall, our findings overturn traditional Pol genotyping and reveal a complex evolutionary history of anamniotic retroviruses inferred by Env evolution. IMPORTANCE Although the retroviral envelope (Env) protein in amniotes has been well studied, its evolutionary history in anamniotic vertebrates is ambiguous. By analyzing more than 30,000 copies of ERV lineages with gamma-type Env (GTE) in anamniotes, several important evolutionary features were identified. First, GTE was found to be widely distributed among different amphibians and fish. Second, nine types of GTE were discovered and defined, revealing their great genetic diversity. Third, the incongruence between the Env and Pol phylogenies suggested that frequent recombination shaped the early evolution of anamniote retroviruses. Fourth, an ancient horizontal gene transfer event was discovered from anamniotes to ERVs with GTE. These findings reveal a complex evolution pattern for retroviral Env in anamniotes.

The important biological roles of Syncytin-1 of human endogenous retrovirus W (HERV-W) and Syncytin-2 of HERV-FRD in the human placenta development.

BACKGROUND:  Human endogenous retroviruses (HERVs) entered the germ line by retroviral infection from a distant ancestor over 30 million years ago and constitute 8% of the human genome. The majorities of HERVs are non-protein coding and lack function because of the accumulation of mutations, insertions, deletions, and/or truncations. However, a small number of HERV genes carried ORFs with beneficial functions for the host. METHODS & RESULTS: In this review, we summarize the structural and important biological roles of two HERV gene products termed Syncytin-1 and Syncytin-2 in human placenta development. Indeed, two retroviral gene products that have important roles in mammalian development, Syncytin-1 (HERV-W) and Syncytin-2 (HERV-FRD), are prime examples encoded by env genes and expressed in the placental trophoblasts. Several pivotal studies revealed that Syncytins are fundamental genes implicated in regulating trophoblast fusion and placenta morphogenesis. CONCLUSION:  Interestingly, it has been suggested that syncytins may also be implicated in non-fusogenic activities leading to apoptosis, proliferation, and immunosuppressive activities.

Syncytin-1 nonfusogenic activities modulate inflammation and contribute to preeclampsia pathogenesis.

Maternal cellular and humoral immune responses to the allogeneic fetoplacental unit are a normal part of pregnancy adaptation. Overactive or dysregulated immune responses that often manifest as inflammation are considered a key element for the development of preeclampsia. Infiltration and activation of macrophages, nature killer cells, and T lymphocytes are frequently observed in the decidua and placenta associated with preeclampsia. In addition to local inflammation, systemic inflammatory changes including increased levels of TNF-α and interleukins (ILs) are detected in the maternal circulation. Syncytin-1 is an endogenous retroviral envelope protein that mediates the fusion of trophoblasts to form syncytiotrophoblasts, a cellular component carrying out most of placental barrier, exchange, and endocrine functions. In addition to these well-defined fusogenic functions that are known for their close association with preeclampsia, multiple studies indicated that syncytin-1 possesses nonfusogenic activities such as those for cell cycle and apoptosis regulation. Moreover, syncytin-1 expressed by trophoblasts and various types of immune cells may participate in regulation of inflammation in preeclamptic placenta and decidua. This review concentrates on the triangular relationship among inflammation, syncytin-1 nonfusogenic functions, and preeclampsia pathogenesis. Data regarding the reciprocal modulations of inflammation and poor vascularization/hypoxia are summarized. The impacts of syncytin-A (the mouse counterpart of human syncytin-1) gene knockout on placental vascularization and their implications for preeclampsia are discussed. Syncytin-1 expression in immune cells and its significance for inflammation are analyzed in the context of preeclampsia development. Finally, the involvements of syncytin-1 nonfusogenic activities in neuroinflammation and multiple sclerosis are compared to findings from preeclampsia.

The Role of Human Endogenous Retrovirus (HERV)-K119 env in THP-1 Monocytic Cell Differentiation.

Human endogenous retrovirus (HERV)-K was reportedly inserted into the human genome millions of years ago and is closely related to various diseases, including cancer and immune regulation. In our previous studies, CRISPR-Cas9-enabled knockout (KO) of the HERV-K env gene was found to potentially reduce cell proliferation, cell migration, and invasion in colorectal and ovarian cancer cell lines. The immune response involves the migration and invasion of cells and is similar to cancer; however, in certain ways, it is completely unlike cancer. Therefore, we induced HERV-K119 env gene KO in THP-1, a monocytic cell that can be differentiated into a macrophage, to investigate the role of HERV-K119 env in immune regulation. Cell migration and invasion were noted to be significantly increased in HERV-K119 env KO THP-1 cells than in MOCK, and these results were contrary to those of cancer cells. To identify the underlying mechanism of HERV-K119 env KO in THP-1 cells, transcriptome analysis and cytokine array analysis were conducted. Semaphorin7A (SEMA7A), which induces the production of cytokines in macrophages and monocytic cells and plays an important role in immune effector cell activation during an inflammatory immune response, was significantly increased in HERV-K119 env KO THP-1 cells. We also found that HERV-K119 env KO THP-1 cells expressed various macrophage-specific surface markers, suggesting that KO of HERV-K119 env triggers the differentiation of THP-1 cells from monocytic cells into macrophages. In addition, analysis of the expression of M1 and M2 macrophage markers showed that M1 macrophage marker cluster of differentiation 32 (CD32) was significantly increased in HERV-K119 env KO cells. These results suggest that HERV-K119 env is implicated in the differentiation of monocytic cells into M1 macrophages and plays important roles in the immune response.

Characterization of the membrane penetration-enhancing peptide S19 derived from human syncytin-1 for the intracellular delivery of TAT-fused proteins.

Although cell-penetrating peptides such as the HIV-derived TAT peptide have been used as tools for the intracellular delivery of therapeutic peptides and proteins, a problem persists: the endosomal escape efficiency is low. Previously, we found that the fusogenic peptide S19, derived from the human protein syncytin-1, enhance the endosomal escape efficiency of proteins that incorporated by endocytosis via TAT. In this study, we first performed Ala-scanning mutagenesis of S19, and found that all Ile, Val, Leu and Phe with high ß-sheet forming propensities in S19 are important for the intracellular uptake of S19-TAT-fused proteins. In a secondary structure analysis of the mutated S19-TAT peptides in the presence of liposomes mimicking late endosomes (LEs), the CD spectra of V3A and I4A mutants with low uptake activity showed the appearance of an α-helix structure, whereas the mutant G5A retained both the uptake activity and the ß-structure. In addition, we investigated the appropriate linking position and order of the S19 and TAT peptides to a cargo protein including an apoptosis-induced peptide and found that both the previous C-terminal S19-TAT tag and the N-terminal TAT-S19 tag promote the cytoplasmic delivery of the fusion protein. These results and previous results suggest that the interaction of TAT with the LE membrane causes a structural change in S19 from a random coil to a ß-strand and that the subsequent parallel ß-sheet formation between two S19 peptides may promote adjacent TAT dimerization, resulting in endosomal escape from the LE membrane.

Autoantibodies against the envelope proteins of endogenous retroviruses K102 and K108 in patients with systemic lupus erythematosus correlate with active disease.

OBJECTIVES: To determine if patients with systemic lupus erythematosus (SLE), a disease characterised by elevated type I interferons reminiscent of anti-viral immunity, have expression of human endogenous retrovirus K (HERV-K) proviruses capable of producing envelope (Env) protein, as well as associated autoantibodies against the Env protein. METHODS: ELISAs were conducted with recombinant Env protein and sera from SLE patients with active (n=60) or inactive (n=49) disease, healthy controls (n=47), other rheumatic disorders (n=59), as well as plasma from paediatric lupus patients with active (n=30) or inactive (n=30) disease, and 17 healthy children. Antibody reactivity was evaluated for correlations with clinical and laboratory parameters of the patients. Expression of HERV-K transcripts were profiled in SLE leukocytes by RNA-Seq. RESULTS: Both adult and paediatric SLE patients had autoantibodies against HERV-K Env with higher titres than healthy controls or patients with Sjögren's syndrome, small- or large-vessel vasculitis, or psoriatic arthritis. Transcripts from only two HERV-K loci capable of producing Env, HERV-K102 and -K108, were detected among the 10 expressed loci in SLE patients. CONCLUSIONS: Our data reveal that HERV-K proviruses are expressed in SLE and that the HERV-K-encoded Env protein elicits an immune response in patients, particularly during active disease.

Mutations in the HIV-1 envelope glycoprotein can broadly rescue blocks at multiple steps in the virus replication cycle.

The p6 domain of HIV-1 Gag contains highly conserved peptide motifs that recruit host machinery to sites of virus assembly, thereby promoting particle release from the infected cell. We previously reported that mutations in the YPXnL motif of p6, which binds the host protein Alix, severely impair HIV-1 replication. Propagation of the p6-Alix binding site mutants in the Jurkat T cell line led to the emergence of viral revertants containing compensatory mutations not in Gag but in Vpu and the envelope (Env) glycoprotein subunits gp120 and gp41. The Env compensatory mutants replicate in Jurkat T cells and primary human peripheral blood mononuclear cells, despite exhibiting severe defects in cell-free particle infectivity and Env-mediated fusogenicity. Remarkably, the Env compensatory mutants can also rescue a replication-delayed integrase (IN) mutant, and exhibit reduced sensitivity to the IN inhibitor Dolutegravir (DTG), demonstrating that they confer a global replication advantage. In addition, confirming the ability of Env mutants to confer escape from DTG, we performed de novo selection for DTG resistance and observed resistance mutations in Env. These results identify amino acid substitutions in Env that confer broad escape from defects in virus replication imposed by either mutations in the HIV-1 genome or by an antiretroviral inhibitor. We attribute this phenotype to the ability of the Env mutants to mediate highly efficient cell-to-cell transmission, resulting in an increase in the multiplicity of infection. These findings have broad implications for our understanding of Env function and the evolution of HIV-1 drug resistance.

Heterologous avian system for quantitative analysis of Syncytin-1 interaction with ASCT2 receptor.

BACKGROUND: Human Syncytin-1 is a placentally-expressed cell surface glycoprotein of retroviral origin. After interaction with ASCT2, its cellular receptor, Syncytin-1 triggers cell-cell fusion and formation of a multinuclear syncytiotrophoblast layer of the placenta. The ASCT2 receptor is a multi-spanning membrane protein containing a protruding extracellular part called region C, which has been suggested to be a retrovirus docking site. Precise identification of the interaction site between ASCT2 and Syncytin-1 is challenging due to the complex structure of ASCT2 protein and the background of endogenous ASCT2 gene in the mammalian genome. Chicken cells lack the endogenous background and, therefore, can be used to set up a system with surrogate expression of the ASCT2 receptor. RESULTS: We have established a retroviral heterologous chicken system for rapid and reliable assessment of ectopic human ASCT2 protein expression. Our dual-fluorescence system proved successful for large-scale screening of mutant ASCT2 proteins. Using this system, we demonstrated that progressive deletion of region C substantially decreased the amount of ASCT2 protein. In addition, we implemented quantitative assays to determine the interaction of ASCT2 with Syncytin-1 at multiple levels, which included binding of the soluble form of Syncytin-1 to ASCT2 on the cell surface and a luciferase-based assay to evaluate cell-cell fusions that were triggered by Syncytin-1. Finally, we restored the envelope function of Syncytin-1 in a replication-competent retrovirus and assessed the infection of chicken cells expressing human ASCT2 by chimeric Syncytin-1-enveloped virus. The results of the quantitative assays showed that deletion of the protruding region C did not abolish the interaction of ASCT2 with Syncytin-1. CONCLUSIONS: We present here a heterologous chicken system for effective assessment of the expression of transmembrane ASCT2 protein and its interaction with Syncytin-1. The system profits from the absence of endogenous ASCT2 background and implements the quantitative assays to determine the ASCT2-Syncytin-1 interaction at several levels. Using this system, we demonstrated that the protruding region C was essential for ASCT2 protein expression, but surprisingly, not for the interaction with Syncytin-1 glycoprotein.

An ancient retroviral RNA element hidden in mammalian genomes and its involvement in co-opted retroviral gene regulation.

BACKGROUND: Retroviruses utilize multiple unique RNA elements to control RNA processing and translation. However, it is unclear what functional RNA elements are present in endogenous retroviruses (ERVs). Gene co-option from ERVs sometimes entails the conservation of viral cis-elements required for gene expression, which might reveal the RNA regulation in ERVs. RESULTS: Here, we characterized an RNA element found in ERVs consisting of three specific sequence motifs, called SPRE. The SPRE-like elements were found in different ERV families but not in any exogenous viral sequences examined. We observed more than a thousand of copies of the SPRE-like elements in several mammalian genomes; in human and marmoset genomes, they overlapped with lineage-specific ERVs. SPRE was originally found in human syncytin-1 and syncytin-2. Indeed, several mammalian syncytin genes: mac-syncytin-3 of macaque, syncytin-Ten1 of tenrec, and syncytin-Car1 of Carnivora, contained the SPRE-like elements. A reporter assay revealed that the enhancement of gene expression by SPRE depended on the reporter genes. Mutation of SPRE impaired the wild-type syncytin-2 expression while the same mutation did not affect codon-optimized syncytin-2, suggesting that SPRE activity depends on the coding sequence. CONCLUSIONS: These results indicate multiple independent invasions of various mammalian genomes by retroviruses harboring SPRE-like elements. Functional SPRE-like elements are found in several syncytin genes derived from these retroviruses. This element may facilitate the expression of viral genes, which were suppressed due to inefficient codon frequency or repressive elements within the coding sequences. These findings provide new insights into the long-term evolution of RNA elements and molecular mechanisms of gene expression in retroviruses.

Syncytin-1/HERV-W envelope is an early activation marker of leukocytes and is upregulated in multiple sclerosis patients.

Syncytin-1 is the envelope protein of the human endogenous retrovirus W (HERV-W). It has been related to multiple sclerosis (MS) but its role in cellular immunity and its pathogenic mechanism in the autoimmune context are not fully understood. We analyzed syncytin-1 levels in peripheral blood mononuclear cells (PBMC) subsets from healthy donors, MS patients in relapse or remission, and patients with acute infections by flow cytometry. PBMC cultures were also prepared to analyze protein expression kinetics. MS patients had higher levels of syncytin-1 levels than controls. We found that syncytin-1 is elevated in monocytes during MS relapses and infections. Cells expressing syncytin-1, including monocytes, T and B lymphocytes, and NKs presented mainly an activated phenotype and, upon stimulation with LPS, its levels increased rapidly on antigen-presenting cells. Syncytin-1 ligation promoted the activation of monocytes, as demonstrated by the upregulation of CD80 and the nonclassical subset CD14low CD16+ . Our results suggest an important role for syncytin-1 in the activation of leukocytes. Given that the expression of syncytin-1 is upregulated in MS patients, this protein might be contributing to the autoimmune cascade in the disease.

Interaction Interface of Mason-Pfizer Monkey Virus Matrix and Envelope Proteins.

Retroviral envelope glycoprotein (Env) is essential for the specific recognition of the host cell and the initial phase of infection. As reported for human immunodeficiency virus (HIV), the recruitment of Env into a retroviral membrane envelope is mediated through its interaction with a Gag polyprotein precursor of structural proteins. This interaction, occurring between the matrix domain (MA) of Gag and the cytoplasmic tail (CT) of the transmembrane domain of Env, takes place at the host cell plasma membrane. To determine whether the MA of Mason-Pfizer monkey virus (M-PMV) also interacts directly with the CT of Env, we mimicked the in vivo conditions in an in vitro experiment by using a CT in its physiological trimeric conformation mediated by the trimerization motif of the GCN4 yeast transcription factor. The MA protein was used at the concentration shifting the equilibrium to its trimeric form. The direct interaction between MA and CT was confirmed by a pulldown assay. Through the combination of nuclear magnetic resonance (NMR) spectroscopy and protein cross-linking followed by mass spectrometry analysis, the residues involved in mutual interactions were determined. NMR has shown that the C terminus of the CT is bound to the C-terminal part of MA. In addition, protein cross-linking confirmed the close proximity of the N-terminal part of CT and the N terminus of MA, which is enabled in vivo by their location at the membrane. These results are in agreement with the previously determined orientation of MA on the membrane and support the already observed mechanisms of M-PMV virus-like particle transport and budding.IMPORTANCE By a combination of nuclear magnetic resonance (NMR) and mass spectroscopy of cross-linked peptides, we show that in contrast to human immunodeficiency virus type 1 (HIV-1), the C-terminal residues of the unstructured cytoplasmic tail of Mason-Pfizer monkey virus (M-PMV) Env interact with the matrix domain (MA). Based on biochemical data and molecular modeling, we propose that individual cytoplasmic tail (CT) monomers of a trimeric complex bind MA molecules belonging to different neighboring trimers, which may stabilize the MA orientation at the membrane by the formation of a membrane-bound net of interlinked Gag and CT trimers. This also corresponds with the concept that the membrane-bound MA of Gag recruits Env through interaction with the full-length CT, while CT truncation during maturation attenuates the interaction to facilitate uncoating. We propose a model suggesting different arrangements of MA-CT complexes between a D-type and C-type retroviruses with short and long CTs, respectively.

Differential Outcomes following Optimization of Simian-Human Immunodeficiency Viruses from Clades AE, B, and C.

Simian-human immunodeficiency virus (SHIV) infection of rhesus monkeys is an important preclinical model for human immunodeficiency virus type 1 (HIV-1) vaccines, therapeutics, and cure strategies. SHIVs have been optimized by incorporating HIV-1 Env residue 375 mutations that mimic the bulky or hydrophobic residues typically found in simian immunodeficiency virus (SIV) Env to improve rhesus CD4 binding. We applied this strategy to three SHIV challenge stocks (SHIV-SF162p3, SHIV-AE16, and SHIV-325c) and observed three distinct outcomes. We constructed six Env375 variants (M, H, W, Y, F, and S) for each SHIV, and we performed a pool competition study in rhesus monkeys to define the optimal variant for each SHIV prior to generating large-scale challenge stocks. We identified SHIV-SF162p3S/wild type, SHIV-AE16W, and SHIV-325cH as the optimal variants. SHIV-SF162p3S could not be improved, as it already contained the optimal Env375 residue. SHIV-AE16W exhibited a similar replicative capacity to the parental SHIV-AE16 stock. In contrast, SHIV-325cH demonstrated a 2.6-log higher peak and 1.6-log higher setpoint viral loads than the parental SHIV-325c stock. These data demonstrate the diversity of potential outcomes following Env375 modification in SHIVs. Moreover, the clade C SHIV-325cH challenge stock may prove useful for evaluating prophylactic or therapeutic interventions against clade C HIV-1.IMPORTANCE We sought to enhance the infectivity of three SHIV stocks by optimization of a key residue in human immunodeficiency virus type 1 (HIV-1) Env (Env375). We developed the following three new simian-human immunodeficiency virus (SHIV) stocks: SHIV-SF162p3S/wild type, SHIV-AE16W, and SHIV-325cH. SHIV-SF162p3S could not be optimized, SHIV-AE16W proved comparable to the parental virus, and SHIV-325cH demonstrated markedly enhanced replicative capacity compared with the parental virus.

Presence of Endogenous Viral Elements Negatively Correlates with Feline Leukemia Virus Susceptibility in Puma and Domestic Cat Cells.

While feline leukemia virus (FeLV) has been shown to infect felid species other than the endemic domestic cat host, differences in FeLV susceptibility among species has not been evaluated. Previous reports have noted a negative correlation between endogenous FeLV (enFeLV) copy number and exogenous FeLV (exFeLV) infection outcomes in domestic cats. Since felids outside the genus Felis do not harbor enFeLV genomes, we hypothesized absence of enFeLV results in more severe disease consequences in felid species lacking these genomic elements. We infected primary fibroblasts isolated from domestic cats (Felis catus) and pumas (Puma concolor) with FeLV and quantitated proviral and viral antigen loads. Domestic cat enFeLV env and long terminal repeat (LTR) copy numbers were determined for each individual and compared to FeLV viral outcomes. FeLV proviral and antigen levels were also measured in 6 naturally infected domestic cats and 11 naturally infected Florida panthers (P. concolor coryi). We demonstrated that puma fibroblasts are more permissive to FeLV than domestic cat cells, and domestic cat FeLV restriction was highly related to enFeLV-LTR copy number. Terminal tissues from FeLV-infected Florida panthers and domestic cats had similar exFeLV proviral copy numbers, but Florida panther tissues have higher FeLV antigen loads. Our work indicates that enFeLV-LTR elements negatively correlate with exogenous FeLV replication. Further, Puma concolor samples lacking enFeLV are more permissive to FeLV infection than domestic cat samples, suggesting that endogenization can play a beneficial role in mitigating exogenous retroviral infections. Conversely, presence of endogenous retroelements may relate to new host susceptibility during viral spillover events.IMPORTANCE Feline leukemia virus (FeLV) can infect a variety of felid species. Only the primary domestic cat host and related small cat species harbor a related endogenous virus in their genomes. Previous studies noted a negative association between the endogenous virus copy number and exogenous virus infection in domestic cats. This report shows that puma cells, which lack endogenous FeLV, produce more virus more rapidly than domestic cat fibroblasts following cell culture challenge. We document a strong association between domestic cat cell susceptibility and FeLV long terminal repeat (LTR) copy number, similar to observations in natural FeLV infections. Viral replication does not, however, correlate with FeLV env copy number, suggesting that this effect is specific to FeLV-LTR elements. This discovery indicates a protective capacity of the endogenous virus against the exogenous form, either via direct interference or indirectly via gene regulation, and may suggest evolutionary outcomes of retroviral endogenization.