Comparative analysis of human, rodent and snake deltavirus replication.

The recent discovery of Hepatitis D (HDV)-like viruses across a wide range of taxa led to the establishment of the Kolmioviridae family. Recent studies suggest that kolmiovirids can be satellites of viruses other than Hepatitis B virus (HBV), challenging the strict HBV/HDV-association dogma. Studying whether kolmiovirids are able to replicate in any animal cell they enter is essential to assess their zoonotic potential. Here, we compared replication of three kolmiovirids: HDV, rodent (RDeV) and snake (SDeV) deltavirus in vitro and in vivo. We show that SDeV has the narrowest and RDeV the broadest host cell range. High resolution imaging of cells persistently replicating these viruses revealed nuclear viral hubs with a peculiar RNA-protein organization. Finally, in vivo hydrodynamic delivery of viral replicons showed that both HDV and RDeV, but not SDeV, efficiently replicate in mouse liver, forming massive nuclear viral hubs. Our comparative analysis lays the foundation for the discovery of specific host factors controlling Kolmioviridae host-shifting.

A co-opted endogenous retroviral envelope promotes cell survival by controlling CTR1-mediated copper transport and homeostasis.

Copper is a critical element for eukaryotic life involved in numerous cellular functions, including redox balance, but is toxic in excess. Therefore, tight regulation of copper acquisition and homeostasis is essential for cell physiology and survival. Here, we identify a different regulatory mechanism for cellular copper homeostasis that requires the presence of an endogenous retroviral envelope glycoprotein called Refrex1. We show that cells respond to elevated extracellular copper by increasing the expression of Refrex1, which regulates copper acquisition through interaction with the main copper transporter CTR1. Downmodulation of Refrex1 results in intracellular copper accumulation leading to reactive oxygen species (ROS) production and subsequent apoptosis, which is prevented by copper chelator treatment. Our results show that Refrex1 has been co-opted for its ability to regulate copper entry through CTR1 in order to limit copper excess, redox imbalance, and ensuing cell death, strongly suggesting that other endogenous retroviruses may have similar metabolic functions among vertebrates.

Combined immunodeficiency caused by pathogenic variants in the ZAP70 C-terminal SH2 domain.

Introduction: ZAP-70, a protein tyrosine kinase recruited to the T cell receptor (TCR), initiates a TCR signaling cascade upon antigen stimulation. Mutations in the ZAP70 gene cause a combined immunodeficiency characterized by low or absent CD8+ T cells and nonfunctional CD4+ T cells. Most deleterious missense ZAP70 mutations in patients are located in the kinase domain but the impact of mutations in the SH2 domains, regulating ZAP-70 recruitment to the TCR, are not well understood. Methods: Genetic analyses were performed on four patients with CD8 lymphopenia and a high resolution melting screening for ZAP70 mutations was developed. The impact of SH2 domain mutations was evaluated by biochemical and functional analyses as well as by protein modeling. Results and discussion: Genetic characterization of an infant who presented with pneumocystis pneumonia, mycobacterial infection, and an absence of CD8 T cells revealed a novel homozygous mutation in the C-terminal SH2 domain (SH2-C) of the ZAP70 gene (c.C343T, p.R170C). A distantly related second patient was found to be compound heterozygous for the R170C variant and a 13bp deletion in the ZAP70 kinase domain. While the R170C mutant was highly expressed, there was an absence of TCR-induced proliferation, associated with significantly attenuated TCR-induced ZAP-70 phosphorylation and a lack of binding of ZAP-70 to TCR-ζ. Moreover, a homozygous ZAP-70 R192W variant was identified in 2 siblings with combined immunodeficiency and CD8 lymphopenia, confirming the pathogenicity of this mutation. Structural modeling of this region revealed the critical nature of the arginines at positions 170 and 192, in concert with R190, forming a binding pocket for the phosphorylated TCR-ζ chain. Deleterious mutations in the SH2-C domain result in attenuated ZAP-70 function and clinical manifestations of immunodeficiency.

The DEAD box RNA helicase DDX42 is an intrinsic inhibitor of positive-strand RNA viruses.

Genome-wide screens are powerful approaches to unravel regulators of viral infections. Here, a CRISPR screen identifies the RNA helicase DDX42 as an intrinsic antiviral inhibitor of HIV-1. Depletion of endogenous DDX42 increases HIV-1 DNA accumulation and infection in cell lines and primary cells. DDX42 overexpression inhibits HIV-1 infection, whereas expression of a dominant-negative mutant increases infection. Importantly, DDX42 also restricts LINE-1 retrotransposition and infection with other retroviruses and positive-strand RNA viruses, including CHIKV and SARS-CoV-2. However, DDX42 does not impact the replication of several negative-strand RNA viruses, arguing against an unspecific effect on target cells, which is confirmed by RNA-seq analysis. Proximity ligation assays show DDX42 in the vicinity of viral elements, and cross-linking RNA immunoprecipitation confirms a specific interaction of DDX42 with RNAs from sensitive viruses. Moreover, recombinant DDX42 inhibits HIV-1 reverse transcription in vitro. Together, our data strongly suggest a direct mode of action of DDX42 on viral ribonucleoprotein complexes. Our results identify DDX42 as an intrinsic viral inhibitor, opening new perspectives to target the life cycle of numerous RNA viruses.

Identification of Copper Transporter 1 as a Receptor for Feline Endogenous Retrovirus ERV-DC14.

Vertebrates harbor hundreds of endogenous retroviral (ERV) sequences in their genomes, which are considered signs of past infections that occurred during evolution. On rare occasions, ERV genes like env are maintained and coopted by hosts for physiological functions, but they also participate in recombination events with exogenous retroviruses to generate rearranged viruses with novel tropisms. In domestic cats, feline leukemia virus type D (FeLV-D) has been described as a recombinant virus between the infectious FeLV-A and likely the ERV-DC14 env gene that resulted in an extended tropism due to the usage of a new uncharacterized retroviral receptor. Here, we report the identification of SLC31A1 encoding the copper transporter 1 (CTR1) as a susceptibility gene for ERV-DC14 infection. Expression of human CTR1 into nonpermissive cells was sufficient to confer sensitivity to ERV-DC14 pseudotype infection and to increase the binding of an ERV-DC14 Env ligand. Moreover, inactivation of CTR1 by genome editing or cell surface downmodulation of CTR1 by a high dose of copper dramatically decreased ERV-DC14 infection and binding, while magnesium treatment had no effect. We also investigated the role of CTR1 in the nonpermissivity of feline and hamster cells. While feline CTR1 was fully functional for ERV-DC14, we found that binding was strongly reduced upon treatment with conditioned medium of feline cells, suggesting that the observed resistance to infection was a consequence of CTR1 saturation. In contrast, hamster CTR1 was inactive due to the presence of a N-linked glycosylation site at position 27, which is absent in the human ortholog. These results provide evidence that CTR1 is a receptor for ERV-DC14. Along with chimpanzee endogenous retrovirus type 2, ERV-DC14 is the second family of endogenous retrovirus known to have used CTR1 during past infections of vertebrates. IMPORTANCE Receptor usage is an important determinant of diseases induced by pathogenic retroviruses. In the case of feline leukemia viruses, three subgroups (A, B, and C) based on their ability to recognize different cell host receptors, respectively, the thiamine transporter THTR1, the phosphate transporter PiT1, and the heme exporter FLVCR1, are associated with distinct feline diseases. FeLV-A is horizontally transmitted and found in all naturally infected cats, while FeLV-B and FeLV-C have emerged from FeLV-A, respectively, by recombination with endogenous retroviral env sequences or by mutations in the FeLV-A env gene, both leading to a switch in receptor usage and in subsequent in vivo tropism. Here, we set up a genetic screen to identify the retroviral receptor of ERV-DC14, a feline endogenous provirus whose env gene has been captured by infectious FeLV-A to give rise to FeLV-D in a process similar to FeLV-B. Our results reveal that the copper transporter CTR1 was such a receptor and provide new insights into the acquisition of an expanded tropism by FeLV-D.

Interplay between primary familial brain calcification-associated SLC20A2 and XPR1 phosphate transporters requires inositol polyphosphates for control of cellular phosphate homeostasis.

Solute carrier family 20 member 2 (SLC20A2) and xenotropic and polytropic retrovirus receptor 1 (XPR1) are transporters with phosphate uptake and efflux functions, respectively. Both are associated with primary familial brain calcification (PFBC), a genetic disease characterized by cerebral calcium-phosphate deposition and associated with neuropsychiatric symptoms. The association of the two transporters with the same disease suggests that they jointly regulate phosphate fluxes and cellular homeostasis, but direct evidence is missing. Here, we found that cross-talk between SLC20A2 and XPR1 regulates phosphate homeostasis, and we identified XPR1 as a key inositol polyphosphate (IP)-dependent regulator of this process. We found that overexpression of WT SLC20A2 increased phosphate uptake, as expected, but also unexpectedly increased phosphate efflux, whereas PFBC-associated SLC20A2 variants did not. Conversely, SLC20A2 depletion decreased phosphate uptake only slightly, most likely compensated for by the related SLC20A1 transporter, but strongly decreased XPR1-mediated phosphate efflux. The SLC20A2-XPR1 axis maintained constant intracellular phosphate and ATP levels, which both increased in XPR1 KO cells. Elevated ATP levels are a hallmark of altered inositol pyrophosphate (PP-IP) synthesis, and basal ATP levels were restored after phosphate efflux rescue with WT XPR1 but not with XPR1 harboring a mutated PP-IP-binding pocket. Accordingly, inositol hexakisphosphate kinase 1-2 (IP6K1-2) gene inactivation or IP6K inhibitor treatment abolished XPR1-mediated phosphate efflux regulation and homeostasis. Our findings unveil an SLC20A2-XPR1 interplay that depends on IPs such as PP-IPs and controls cellular phosphate homeostasis via the efflux route, and alteration of this interplay likely contributes to PFBC.

Cyclophilins and nucleoporins are required for infection mediated by capsids from circulating HIV-2 primary isolates.

HIV-2 groups have emerged from sooty mangabey SIV and entered the human population in Africa on several separate occasions. Compared to world pandemic HIV-1 that arose from the chimpanzee SIVcpz virus, the SIVsm-derived HIV-2, largely confined to West Africa, is less replicative, less transmissible and less pathogenic. Here, we evaluated the interactions between host cellular factors, which control HIV-1 infection and target the capsid, and HIV-2 capsids obtained from primary isolates from patients with different disease progression status. We showed that, like HIV-1, all HIV-2 CA we tested exhibited a dependence on cyclophilin A. However, we observed no correlation between HIV-2 viremia and susceptibility to hu-TRIM5alpha or dependence to CypA. Finally, we found that all CA from HIV-2 primary isolates exploit Nup358 and Nup153 for nucleus transposition. Altogether, these findings indicate that the ability to use the two latter nucleoporins is essential to infection of human cells for both HIV-1 and HIV-2. This dependence provides another molecular target that could be used for antiviral strategies against both HIV-1 and 2, based on both nucleoporins.

Searching for Common Mammalian Retroviruses in Pediatric Idiopathic Diseases.

Mammalian retroviruses cause a variety of diseases in their hosts, including hematological and immunodeficiency disorders. Both human T-cell leukemia (HTLV) and human immunodeficiency (HIV) viruses originated from several independent zoonotic transmissions, indicating that cross-species transmissions from animal to humans may still occur. Thus, as the risk for retroviral transmissions from animals to humans increase, we investigated whether mammalian retroviruses are involved in selected pediatric idiopathic diseases whose symptoms evoke retroviral infections. Blood samples, sera, and synovial fluids, or bone marrow cells were collected from pediatric patients under 18 years of age with different autoimmune idiopathic diseases. Overall, we screened clinical samples from 110 children using sensitive nested and semi-nested PCR strategies targeting env genes, and a C-type retrovirus reverse transcriptase (RT) activity kit. All clinical samples were free of retroviral signatures, indicating the unlikelihood of an etiological role of the retroviruses we assessed in the pediatric diseases we tested.

Comparison of different extraction techniques to profile microRNAs from human sera and peripheral blood mononuclear cells.

BACKGROUND: microRNAs (miRNAs) play crucial roles in major biological processes and their deregulations are often associated with human malignancies. As such, they represent appealing candidates as targets of innovative therapies. Another interesting aspect of their biology is that they are present in various biological fluids where, advantageously, they appear to be very stable. A plethora of studies have now reported their potential as biomarkers that can be used in diagnosis, prognosis and/or theranostic issues. However, the application of circulating miRNAs in clinical practices still requires the identification of highly efficient, robust and reproducible methods for their isolation from biological samples.In that context, we performed an independent cross-comparison of three commercially available RNA extraction kits for miRNAs isolation from human blood samples (Qiagen and Norgen kits as well as the new NucleoSpin miRNAs Plasma kit from Macherey-Nagel). miRNAs were further profiled using the Taqman Low Density Array technology. RESULTS: We found that, although these 3 kits had equal performances in extracting miRNAs from peripheral blood mononuclear cells, the Macherey-Nagel kit presented several advantages when isolating miRNAs from sera. Besides, our results have indicated that, depending on the quantity of the biological samples used, the extraction procedure directly impacted on the G/C composition of the miRNAs detected. CONCLUSION: Overall, our study contributes to the definition of a reliable framework for profiling circulating miRNAs.

Heterogeneous susceptibility of circulating SIV isolate capsids to HIV-interacting factors.

BACKGROUND: Many species of non-human primates in Africa are naturally infected by simian immunodeficiency viruses (SIV) and humans stand at the forefront of exposure to these viruses in Sub-Saharan Africa. Cross-species transmission and adaptation of SIV to humans have given rise to human immunodeficiency viruses (HIV-1 and HIV-2) on twelve accountable, independent occasions. However, the determinants contributing to a simian-to-human lasting transmission are not fully understood. Following entry, viral cores are released into the cytoplasm and become the principal target of host cellular factors. Here, we evaluated cellular factors likely to be involved in potential new SIV cross-species transmissions. We investigated the interactions of capsids from naturally circulating SIV isolates with both HIV-1 restricting (i.e. TRIM5 proteins) and facilitating (i.e. cyclophilin A and nucleopore-associated Nup358/RanBP2 and Nup153) factors in single-round infectivity assays that reproduce early stages of the viral life-cycle. RESULTS: We show that human TRIM5α is unlikely to prevent cross-species transmission of any SIV we tested and observed that the SIV CA-CypA interaction is a widespread but not a universal feature. Moreover, entry in the nucleus of different SIV appeared to follow pathways that do not necessarily recruit Nup358/RanBP2 or Nup153, and this regardless of their interaction with CypA. Nevertheless, we found that, like HIV-1, human-adapted HIV-2 infection was dependent on Nup358/RanBP2 and Nup153 interactions for optimal infection. Furthermore, we found that, unlike HIV CA, SIV CA did not require a direct interaction with the Cyp-like domain of Nup358/RanBP2 to carry out successful infection. CONCLUSIONS: Circulating SIV present a variety of phenotypes with regard to CA-interacting restricting or facilitating factors. Altogether, we unveiled unidentified pathways for SIV CA, which could also be exploited by HIV in different cellular contexts, to drive entry into the nucleus. Our findings warrant a closer evaluation of other potential defenses against circulating SIV.

Retroviral GAG proteins recruit AGO2 on viral RNAs without affecting RNA accumulation and translation.

Cellular micro(mi)RNAs are able to recognize viral RNAs through imperfect micro-homologies. Similar to the miRNA-mediated repression of cellular translation, this recognition is thought to tether the RNAi machinery, in particular Argonaute 2 (AGO2) on viral messengers and eventually to modulate virus replication. Here, we unveil another pathway by which AGO2 can interact with retroviral mRNAs. We show that AGO2 interacts with the retroviral Group Specific Antigen (GAG) core proteins and preferentially binds unspliced RNAs through the RNA packaging sequences without affecting RNA stability or eliciting translation repression. Using RNAi experiments, we provide evidences that these interactions, observed with both the human immunodeficiency virus 1 (HIV-1) and the primate foamy virus 1 (PFV-1), are required for retroviral replication. Taken together, our results place AGO2 at the core of the retroviral life cycle and reveal original AGO2 functions that are not related to miRNAs and translation repression.

Detection of Merkel cell polyomavirus on environmental surfaces.

The Merkel cell polyomavirus (MCPyV) is a human virus identified recently which is associated with the Merkel cell carcinoma. This virus is also detected frequently in the skin of healthy individuals. The presence of MCPyV has been investigated on environmental surfaces in contact with human skin. Various surfaces in four laboratories, public places, and individual homes were swabbed. Human DNA and MCPyV DNA were detected in swabs by real-time PCR. MCPyV DNA levels were measured before and after DNase treatment in a set of 12 MCPyV DNA-positive samples. A total of 60 environmental surface samples were collected. Fifty-one (85.0%) were positive for human DNA detection and 45 (75.0%) were positive for MCPyV DNA detection. All samples positive for MCPyV DNA were positive for human DNA detection. After DNase treatment, a 1.3 log decrease in MCPyV DNA level was observed indicating that about 5% of viral DNA is protected from DNase degradation and might be associated with infectious virus. These results indicate that MCPyV DNA may be detected on environmental surfaces in contact with human skin. Detection of viral DNA might reflect the presence of infectious viral particles and transmission from environmental source to humans cannot be ruled out.

No evidence for XMRV association in pediatric idiopathic diseases in France.

Retroviruses have been linked to a variety of diseases such as neoplastic and immunodeficiency disorders and neurologic and respiratory diseases. Recently, a novel infectious human retrovirus, the xenotropic murine leukemia virus-related virus (XMRV), has been identified in cohorts of patients with either a familial type of prostate cancer or chronic fatigue syndrome. The apparent unrelatedness of these diseases raised the question of the potential involvement of XMRV in other diseases.Here, we investigated the presence of XMRV in a selection of pediatric idiopathic infectious diseases with symptoms that are suggestive of a retroviral infection, as well as in children with respiratory diseases and in adult patients with spondyloarthritis (SpA). Using a XMRV env-nested PCR, we screened 72 DNA samples obtained from 62 children hospitalized in the Montpellier university hospital (France) for hematological, neurological or inflammatory pathologies, 80 DNA samples from nasopharyngeal aspirates from children with respiratory diseases and 19 DNA samples from SpA. None of the samples tested was positive for XMRV or MLV-like env sequences, indicating that XMRV is not involved in these pathologies.

Spontaneous control of viral replication during primary HIV infection: when is "HIV controller" status established?

Eight patients in the ANRS PRIMO cohort experienced early spontaneous viral control. Viral control was established a median of 6.2 months after primary human immunodeficiency virus type 1 infection and lasted a median of 4.1 years. Seven of the patients initially had detectable viral replication. For 4 patients, viral control was lost during follow-up.

High HIV Type 1 prevalence and wide genetic diversity with dominance of recombinant strains but low level of antiretroviral drug-resistance mutations in untreated patients in northeast Gabon, Central Africa.

The northeast of Gabon, central Africa is characterized by high population density and a high rate of immigration from the surrounding countries. To determine the prevalence, circulating subtypes, and antiretroviral resistance mutations of HIV-1, 810 blood samples were collected from the general population of the two main cities (Oyem and Makokou) of this region. Of these, 61 (7.5%) were found to be positive for HIV-1. Analysis of the env (gp120), pol, and gag (p24) sequences as well as phylogenetic analyses showed at least eight different viral lineages. The most prevalent strains were CRF02 recombinants, followed by subtypes A, D, and C. The remaining strains were found to be F, J, G, and also, for the first time in Gabon, the recombinant form CRF11cpx. Analysis of antiretroviral drug-resistance mutations in protease and reverse transcriptase from this untreated population showed a low level of specific mutations. These mutations were associated with subtype polymorphism rather than with resistance to antiretroviral drugs. The wide diversity and the emergence of recombinant strains are in accordance with the rapid spread of new HIV strains in the population and, thus, the dynamic evolution of the epidemic.

Full-length genome characterization of a novel simian immunodeficiency virus lineage (SIVolc) from olive Colobus (Procolobus verus) and new SIVwrcPbb strains from Western Red Colobus (Piliocolobus badius badius) from the Tai Forest in Ivory Coast.

Simian immunodeficiency viruses (SIVs) are found in an extensive number of African primates and humans continue to be exposed to these viruses by hunting and handling of primate bushmeat. Full-length genome sequences were obtained from SIVs derived from two Colobinae species inhabiting the Taï forest, Ivory Coast, each belonging to a different genus: SIVwrc from western red colobus (Piliocolobus badius badius) (SIVwrcPbb-98CI04 and SIVwrcPbb-97CI14) and SIVolc (SIVolc-97CI12) from olive colobus (Procolobus verus). Phylogenetic analysis showed that western red colobus are the natural hosts of SIVwrc, and SIVolc is also a distinct species-specific lineage, although distantly related to the SIVwrc lineage across the entire length of its genome. Overall, both SIVwrc and SIVolc, are also distantly related to the SIVlho/sun lineage across the whole genome. Similar to the group of SIVs (SIVsyk, SIVdeb, SIVden, SIVgsn, SIVmus, and SIVmon) infecting members of the Cercopithecus genus, SIVs derived from western red and olive colobus, L'Hoest and suntailed monkeys, and SIVmnd-1 from mandrills form a second group of viruses that cluster consistently together in phylogenetic trees. Interestingly, the divergent SIVcol lineage, from mantled guerezas (Colobus guereza) in Cameroon, is also closely related to SIVwrc, SIVolc, and the SIVlho/sun lineage in the 5' part of Pol. Overall, these results suggest an ancestral link between these different lentiviruses and highlight once more the complexity of the natural history and evolution of primate lentiviruses.

HIV-1 co-infection prevalence in two cohorts of early HIV-1 seroconverters in France.

Despite anecdotal reports of HIV-1 co-infections and super-infections, few large-scale prevalence studies are available on multiple HIV-1 infection. We systematically searched for HIV-1 co-infections by means of a heteroduplex mobility assay in 660 HIV-1 seroconverters from the two ANRS SEROCO and PRIMO cohorts. Our results strongly suggest that HIV-1 co-infection remains a rare phenomenon in HIV-1 seroconverters infected in France between 1986 and 2004.