RÉSUMÉ
In villous trophoblasts, DROSHA is a key ribonuclease III enzyme that processes pri-microRNAs (pri-miRNAs) into pre-miRNAs at the placenta-specific, chromosome 19 miRNA cluster (C19MC) locus. However, little is known of its other functions. We performed formaldehyde crosslinking, immunoprecipitation, and sequencing (fCLIP-seq) analysis of terminal chorionic villi to identify DROSHA-binding RNAs in villous trophoblasts. In villous trophoblasts, DROSHA predominantly generated placenta-specific C19MC pre-miRNAs, including antiviral C19MC pre-miRNAs. The fCLIP-seq analysis also identified non-miRNA transcripts with hairpin structures potentially capable of binding to DROSHA (e.g., SNORD100 and VTRNA1-1). Moreover, in vivo immunohistochemical analysis revealed DROSHA in the cytoplasm of villous trophoblasts. DROSHA was abundant in the cytoplasm of villous trophoblasts, particularly in the apical region of syncytiotrophoblast, in the full-term placenta. Furthermore, in BeWo trophoblasts infected with Sindbis virus (SINV), DROSHA translocated to the cytoplasm and recognized the genomic RNA of SINV. Therefore, in trophoblasts, DROSHA not only regulates RNA metabolism, including the biogenesis of placenta-specific miRNAs, but also recognizes viral RNAs. After SINV infection, BeWo DROSHA-binding VTRNA1-1 was significantly upregulated, and cellular VTRNA1-1 was significantly downregulated, suggesting that DROSHA soaks up VTRNA1-1 in response to viral infection. These results suggest that the DROSHA-mediated recognition of RNAs defends against viral infection in villous trophoblasts. Our data provide insight into the antiviral functions of DROSHA in villous trophoblasts of the human placenta.
Sujet(s)
microARN , Maladies virales , Humains , Ribonuclease III/génétique , Ribonuclease III/composition chimique , Ribonuclease III/métabolisme , microARN/génétique , microARN/métabolisme , Cytoplasme/métabolisme , Trophoblastes/métabolisme , AntivirauxRÉSUMÉ
The isolation of the Koala retrovirus-like virus from Australian megabats and the identification of endogenous retroviruses in the bat genome have raised questions on bat susceptibility to retroviruses in general. To answer this, we studied the susceptibility of 12 cell lines from 11 bat species to four well-studied retroviruses (human and simian immunodeficiency viruses [HIV and SIV] and murine leukemia viruses [B- and N-MLV]). Systematic comparison of retroviral susceptibility among bats revealed that megabat cell lines were overall less susceptible to the four retroviruses than microbat cell lines, particularly to HIV-1 infection, whereas lineage-specific differences were observed for MLV susceptibility. Quantitative PCR of reverse transcription (RT) products, infection in heterokaryon cells, and point mutation analysis of the capsid (CA) revealed that (i) HIV-1 and MLV replication were blocked at the nuclear transport of the pre-integration complexes and before and/or during RT, respectively, and (ii) the observed lineage-specific restriction can be attributed to a dominant cellular factor constrained by specific positions in CA. Investigation of bat homologs of the three previously reported post-entry restriction factors constrained by the same residues in CA, tripartite motif-protein 5α (TRIM5α), myxovirus resistance 2/B (Mx2/MxB), and carboxy terminus-truncated cleavage and polyadenylation factor 6 (CPSF6-358), demonstrated poor anti-HIV-1 activity in megabat cells, whereas megabat TRIM5α restricted MLV infection, suggesting that the major known CA-dependent restriction factors were not dominant in the observed lineage-specific susceptibility to HIV-1 in bat cells. Therefore, HIV-1 susceptibility of megabat cells may be determined in a manner distinct from that of primate cells. IMPORTANCE Recent studies have demonstrated the circulation of gammaretroviruses among megabats in Australia and the bats' resistance to HIV-1 infection; however, the origins of these viruses in megabats and the contribution of bats to retrovirus spread to other mammalian species remains unclear. To determine the intrinsic susceptibility of bat cells to HIV-1 infection, we investigated 12 cell lines isolated from 11 bat species. We report that lineage-specific retrovirus restriction in the bat cell lines can be attributed to CA-dependent factors. However, in the megabat cell lines examined, factors known to bind capsid and block infection in primate cell culture, including homologs of TRIM5α, Mx2/MxB, and CPSF6, failed to exhibit significant anti-HIV-1 activities. These results suggested that the HIV-1 susceptibility of megabat cells occurs in a manner distinct from that of primate cells, where cellular factors, other than major known CA-dependent restriction factors, with lineage-specific functions could recognize retroviral proteins in megabats.
Sujet(s)
Capside , Chiroptera , Prédisposition aux maladies , Retroviridae , Animaux , Humains , Souris , Australie , Capside/métabolisme , Protéines de capside/génétique , Protéines de capside/métabolisme , Chiroptera/virologie , Retroviridae/classification , Retroviridae/métabolisme , Infections à Retroviridae/métabolisme , Infections à Retroviridae/virologie , Prédisposition aux maladies/métabolisme , Prédisposition aux maladies/virologie , Lignée cellulaire , Spécificité d'espèce , Facteurs de restriction antiviraux/métabolismeRÉSUMÉ
Langerhans cells (LCs), a subset of dendritic cells (DCs), reside in body surface presenting antigens from various pathogens and activate immune system after migrating to vicinal lymph nodes. We recently demonstrated that the E-cadherin interaction allowed peripheral blood (PB) CD14+ cells to differentiate into LC-like cells that closely resemble primary LCs. Here, with a combination of GM-CSF, TGF-ß, and TNF-α, we induced LC-like cells from umbilical cord blood (UCB)derived CD34+ cells and compared them with those induced from PB CD14+ cells. In contrast to PB CD14+ cell-derived LC-like cells with an undetectable surface level of toll-like receptor (TLR)4 and an unresponsiveness feature to bacterial lipopolysaccharide (LPS), CB CD34+ cellsderived LC like cells expressed a low, but apparent, surface level of TLR4 and a reduced level of intracellular TLR3. Consistent with this result, they responded to bacterial LPS, but poorly to poly(I:C) reflecting viral RNA. These findings suggest that LC-precursors from circulating PB CD14+ cells seem to be arranged in the outer barrier of skin, while LC-precursors from local undifferentiated UCB-derived CD34+ cells may be arranged in the inner barrier of mucosal tissues and work together to combat against external pathogens as well as internal malignancies throughout body surface.
Sujet(s)
Antigènes CD/métabolisme , Sang foetal/cytologie , Facteur de stimulation des colonies de granulocytes et de macrophages/pharmacologie , Cellules souches hématopoïétiques/effets des médicaments et des substances chimiques , Cellules souches hématopoïétiques/métabolisme , Lectines de type C/métabolisme , Lectines liant le mannose/métabolisme , Récepteur de type Toll-3/génétique , Facteur de croissance transformant bêta-1/pharmacologie , Facteur de nécrose tumorale alpha/pharmacologie , Cadhérines/métabolisme , Cytokines/biosynthèse , Femelle , Humains , Immunophénotypage , Antigènes CD14/métabolisme , Phénotype , Grossesse , Liaison aux protéines , Récepteur de type Toll-3/agonistesRÉSUMÉ
To better understand the binding mechanism of TRIM5α to retrovirus capsid, we had previously selected N-tropic murine leukemia virus (N-MLV) mutants escaping from rhesus macaque TRIM5α (rhTRIM5α) by passaging the virus in rhTRIM5α-expressing cells and selecting for nonrestricted variants. To test the commonality of the findings from the rhTRIM5α study, we have now employed a similar genetic approach using human TRIM5α (huTRIM5α). Consistent with the rhTRIM5α study, the mapped huTRIM5α escape mutations were distributed across the capsid exterior, confirming the extended binding surface between virus and restriction factor. Compared to the results of the previous study, fewer escape mutations were identified, with particular mutants being repeatedly selected. Three out four huTRIM5α escape variants showed resistance to all primate TRIM5αs tested, but two of them sacrificed viral fitness, observations that were not made in the rhTRIM5α study. Moreover, differences in amino acid changes associated with escape from hu- and rhTRIM5αs suggested a charge dependence of the restriction by different TRIM5αs. Taken together, these results suggest that the recognition of the entire capsid surface is a general strategy for TRIM5α to restrict MLV but that significantly different specific interactions are involved in the binding of TRIM5α from different species to the MLV capsid core.
Sujet(s)
Protéines de capside/métabolisme , Virus de la leucémie murine/métabolisme , Infections à Retroviridae/métabolisme , Animaux , Facteurs de restriction antiviraux , Protéines de capside/génétique , Protéines de transport/génétique , Protéines de transport/métabolisme , Humains , Virus de la leucémie murine/génétique , Macaca mulatta , Modèles moléculaires , Liaison aux protéines , Infections à Retroviridae/génétique , Infections à Retroviridae/virologie , Protéines à motif tripartite , Ubiquitin-protein ligasesRÉSUMÉ
After entry into target cells, retroviruses encounter the host restriction factors such as Fv1 and TRIM5α. While it is clear that these factors target retrovirus capsid proteins (CA), recognition remains poorly defined in the absence of structural information. To better understand the binding interaction between TRIM5α and CA, we selected a panel of novel N-tropic murine leukaemia virus (N-MLV) escape mutants by a serial passage of replication competent N-MLV in rhesus macaque TRIM5α (rhTRIM5α)-positive cells using a small percentage of unrestricted cells to allow multiple rounds of virus replication. The newly identified mutations, many of which involve changes in charge, are distributed over the outer 'top' surface of N-MLV CA, including the N-terminal ß-hairpin, and map up to 29 A(o) apart. Biological characterisation with a number of restriction factors revealed that only one of the new mutations affects restriction by human TRIM5α, indicating significant differences in the binding interaction between N-MLV and the two TRIM5αs, whereas three of the mutations result in dual sensitivity to Fv1(n) and Fv1(b). Structural studies of two mutants show that no major changes in the overall CA conformation are associated with escape from restriction. We conclude that interactions involving much, if not all, of the surface of CA are vital for TRIM5α binding.
Sujet(s)
Protéines de capside/métabolisme , Capside/métabolisme , Protéines de transport/métabolisme , Virus de la leucémie murine/métabolisme , Animaux , Facteurs de restriction antiviraux , Séquence nucléotidique , Sites de fixation , Protéines de capside/génétique , Lignée cellulaire , Humains , Virus de la leucémie murine/génétique , Macaca mulatta , Souris , Mutation , Motifs et domaines d'intéraction protéique , Analyse de séquence d'ADN , Protéines à motif tripartite , Ubiquitin-protein ligasesRÉSUMÉ
Foamy viruses (FVs) are unconventional retroviruses with a replication strategy that is significantly different from orthoretroviruses and bears some homology to that of hepadnaviruses. Although some cellular proteins, such as APOBEC3, have been reported to block FVs, no restriction by Trim5alpha has been described to date. The sensitivity of three FV isolates of human-chimpanzee or prototypic (PFV), macaque (SFVmac), and feline (FFV) origin to a variety of primate Trim5alphas was therefore tested. PFV and SFVmac were restricted by Trim5alphas from most New World monkeys, but not from other primates, whereas FFV-based vectors were restricted by Trim5alphas from the great apes gorilla and orangutan. Trim5alphas from Old World monkeys did not restrict any FV isolate tested. Capuchin Trim5alpha was unique, as it restricted SFVmac and FFV but not PFV. Trim5alpha specificity for FVs was determined by the B30.2 domain, interestingly involving, in some instances, the same residues of the variable regions previously implicated as major determinants for human immunodeficiency virus type 1 restriction. FVs with chimeric Gags were made to map the viral determinants of sensitivity to restriction. The N-terminal half of the Gag molecule was found to contain the regions that control susceptibility. This region most likely corresponds to the capsid of conventional retroviruses. Due to their unique replication strategy, FVs should provide a valuable new system to examine the mechanism of retroviral restriction by Trim5alpha.
Sujet(s)
Protéines de transport/métabolisme , Cercopithecidae/métabolisme , Spumavirus/métabolisme , Séquence d'acides aminés , Animaux , Protéines de transport/composition chimique , Protéines de transport/génétique , Lignée cellulaire , Cercopithecidae/génétique , Produits du gène gag/génétique , Produits du gène gag/métabolisme , Vecteurs génétiques/génétique , Humains , Données de séquences moléculaires , Mutation/génétique , Phylogenèse , Alignement de séquences , Spumavirus/génétique , Doigts de zincRÉSUMÉ
Recent studies have revealed the contribution of TRIM5alpha to retrovirus restriction in cells from a variety of primate species. TRIM5alpha consists of a tripartite motif (the RBCC domain) followed by a B30.2 domain. The B30.2 domain is thought to be involved in determination of restriction specificity and contains three variable regions. To investigate the relationship between the phylogeny of primate TRIM5alpha and retrovirus restriction specificity, a series of chimeric TRIM5alpha consisting of the human RBCC domain followed by the B30.2 domain from various primates was constructed. These constructs showed restriction profiles largely consistent with the origin of the B30.2 domain. Restriction specificity was further investigated with a variety of TRIM5alphas containing mixed or mutated B30.2 domains. This study revealed the importance of all three variable regions for determining restriction specificity. Based on the molecular structures of other PRYSPRY domains solved recently, a model for the molecular structure of the B30.2 domain of TRIM5alpha was developed. The model revealed that the variable regions of the B30.2 domain are present as loops located on one side of the B30.2 core structure. It is hypothesized that these three loops form a binding surface for virus and that evolutionary changes in any one of the loops can alter restriction specificity.
Sujet(s)
Maladies des grands singes/virologie , Protéines de transport/génétique , Variation génétique , Haplorhini/virologie , Maladies des singes/virologie , Infections à Retroviridae/médecine vétérinaire , Retroviridae/pathogénicité , Séquence d'acides aminés , Animaux , Facteurs de restriction antiviraux , Protéines de transport/composition chimique , Exons/génétique , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/pathogénicité , Humains , Virus de la leucémie murine/pathogénicité , Souris , Données de séquences moléculaires , Phylogenèse , Infections à Retroviridae/virologie , Virus de l'immunodéficience simienne/pathogénicité , Spécificité d'espèce , Protéines à motif tripartite , Ubiquitin-protein ligasesRÉSUMÉ
We previously reported that a nef-deleted SHIV (SHIV-NI) is nonpathogenic and gave macaques protection from challenge infection with pathogenic SHIV-C2/1. To investigate whether IFN-gamma augments the immune response induced by this vaccination, we examined the antiviral and adjuvant effect of recombinant human IFN-gamma (rIFN-gamma) in vaccinated and unvaccinated monkeys. Nine monkeys were vaccinated with nef-deleted nonpathogenic SHIV-NI. Four of them were administered with rIFN-gamma and the other five monkeys were administered with placebo. After the challenge with pathogenic SHIV-C2/1, CD4(+) T-cell counts were maintained similarly in monkeys of both groups, while those of the unvaccinated monkeys decreased dramatically at 2 weeks after challenge. However, the peaks of plasma viral load were reduced to 100-fold in SHIV-NI vaccinated monkeys combined with rIFN-gamma compared with those in SHIV-NI vaccinated monkeys without rIFN-gamma. The peaks of plasma viral load were inversely correlated with the number of SIV Gag-specific IFN-gamma-producing cells. In SHIV-NI-vaccinated monkeys with rIFN-gamma, the number of SIV Gag-specific IFN-gamma-producing cells of PBMCs increased 2-fold compared with those in SHIV-NI-vaccinated monkeys without rIFN-gamma, and the NK activity and MIP-1alpha production of PBMCs were also enhanced. Thus, vaccination of SHIV-NI in combination with rIFN-gamma was more effective in modulating the antiviral immune system into a Th1 type response than SHIV-NI vaccination alone. These results suggest that IFN-gamma augmented the anti-viral effect by enhancing innate immunity and shifting the immune response to Th1.
Sujet(s)
VIH (Virus de l'Immunodéficience Humaine)/immunologie , Interféron gamma/immunologie , Virus recombinants/immunologie , Vaccins contre le SIDA simien/administration et posologie , Syndrome d'immunodéficience acquise du singe/prévention et contrôle , Virus de l'immunodéficience simienne/immunologie , Vaccination , Animaux , Délétion de gène , Gènes nef/génétique , Injections musculaires , Injections veineuses , Interféron gamma/administration et posologie , Macaca mulatta , Protéines recombinantes , Vaccins contre le SIDA simien/génétique , Virus de l'immunodéficience simienne/génétique , Vaccins antivirauxRÉSUMÉ
In order to understand primate lentivirus evolution, characterization of additional simian immunodeficiency virus (SIV) strains is essential. Here, an SIV from a black mangabey (Lophocebus aterrimus) originating from the Democratic Republic of Congo was analysed phylogenetically. The monkey had cross-reactive antibodies against human immunodeficiency virus type 1 (HIV-1) and HIV-2. The viral pol region sequence was amplified by nested PCR and sequence analysis confirmed that it was related to known SIV sequences. This is the first report to characterize genetically an SIV from the monkey genus Lophocebus. Phylogenetic analysis of the pol region revealed that this novel SIV, designated SIVbkm, fell into the SIVsyk and SIVgsn virus group, containing viruses isolated from the genus Cercopithecus, and suggests that cross-species transmission has occurred between species of the genera Lophocebus and Cercopithecus.
Sujet(s)
Cercocebus/virologie , Phylogenèse , Syndrome d'immunodéficience acquise du singe/virologie , Virus de l'immunodéficience simienne/classification , Animaux , Cercopithecus/virologie , République démocratique du Congo , Humains , Données de séquences moléculaires , Analyse de séquence d'ADN , Virus de l'immunodéficience simienne/génétique , Virus de l'immunodéficience simienne/isolement et purificationRÉSUMÉ
Seven isolates of human T cell leukemia virus type 1 (HTLV-1) were taken in southern India and phylogenetically analyzed to gain new insights into the origin and dissemination of HTLV-1 in the subcontinent. The new Indian HTLV-1s were found to be members of subgroup A (Transcontinental subgroup) of the Cosmopolitan group. They formed three different clusters (South African/Caribbean, Middle Eastern, and East Asian clusters). These results demonstrate that Indian HTLV-1s are genetically heterogeneous and include the most divergent strain of subgroup A. On the basis of these results, we speculate that subgroup A HTLV- 1s may have been present for thousands of years in India.
Sujet(s)
Infections à HTLV-I/virologie , Virus T-lymphotrope humain de type 1/génétique , Polymorphisme génétique , Adulte , Enfant , ADN viral/composition chimique , Femelle , Virus T-lymphotrope humain de type 1/isolement et purification , Humains , Inde , Mâle , Adulte d'âge moyen , Données de séquences moléculaires , Phylogenèse , Analyse de séquence d'ADN , Séquences répétées terminales/génétiqueRÉSUMÉ
To clarify the relationship between the amino acid variations of the gp120 of human immunodeficiency virus type 1 (HIV-1) and the chemokine receptors that are used as the second receptor for HIV, we evaluated amino acid site variation of gp120 between the X4 strains (use CXCR4) and the R5 strains (use CCR5) from 21 sequences of subtype B. Our analysis showed that residues 306 and 322 in the V3 loop and residue 440 in the C4 region were associated with usage of the second receptor. The polymorphism at residue 440 is clearly associated with the usage of the second receptor: The amino acid at position 440 was a basic amino acid in the R5 strains, and a nonbasic and smaller amino acid in the X4 strains, while the V3 loop of the X4 strains was more basic than that of the R5 strains. This suggests that residue 440 in the C4 region, which is close to the V3 loop in the three-dimensional structure, is critical in determining which second receptor is used. Analysis of codon frequency suggests that, in almost all cases, the difference at residue 440 between basic amino acids in the R5 strains and nonbasic amino acids in the X4 strains could be due to a single nucleotide change. These findings predict that the evolutionary changes in amino acid residue 440 may be correlated with evolutionary changes in the V3 loop. One possibility is that a change in electric charge at residue 440 compensates for a change in electric charge in the V3 loop. The amino acid polymorphism at position 440 can be useful to predict the cell tropism of a strain of HIV-1 subtype B.
Sujet(s)
Acides aminés/génétique , Variation génétique , Protéine d'enveloppe gp120 du VIH/génétique , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/génétique , Phylogenèse , Séquence d'acides aminés , Codon/génétique , Bases de données d'acides nucléiques , Protéine d'enveloppe gp120 du VIH/métabolisme , Fonctions de vraisemblance , Modèles génétiques , Données de séquences moléculaires , Récepteurs CCR5/métabolisme , Récepteurs CXCR4/métabolisme , Alignement de séquencesRÉSUMÉ
Simian T-cell leukemia viruses (STLVs) are the simian counterparts of human T-cell leukemia viruses (HTLVs). A novel, divergent type of STLV (STLV-L) from captive baboons was reported in 1994, but its natural prevalence remained unclear. We investigated the prevalence of STLV-L in 519 blood samples from wild-living nonhuman primates in Ethiopia. Seropositive monkeys having cross-reactive antibodies against HTLV were found among 22 out of 40 hamadryas baboons, 8 of 96 anubis baboons, 24 of 50 baboons that are hybrids between hamadryas and anubis baboons, and 41 of 177 grivet monkeys, but not in 156 gelada baboons. A Western blotting assay showed that sera obtained from seropositive hamadryas and hybrid baboons exhibited STLV-L-like reactivity. A PCR assay successfully amplified STLV sequences, which were subsequently sequenced and confirmed as being closely related to STLV-L. Surprisingly, further PCR showed that nearly half of the hamadryas (20 out of 40) and hybrid (19 out of 50) baboons had STLV-L DNA sequences. In contrast, most of the seropositive anubis baboons and grivet monkeys carried typical STLV-1 but not STLV-L. These observations demonstrate that STLV-L naturally prevails among hamadryas and hybrid baboons at significantly high rates. STLV-1 and -2, the close relative of STLV-L, are believed to have jumped across simian-human barriers, which resulted in widespread infection of HTLV-1 and -2. Further studies are required to know if STLV-L is spreading into human populations.
Sujet(s)
Anticorps antiviraux/sang , Infections à deltarétrovirus/médecine vétérinaire , Maladies des singes/épidémiologie , Papio , Virus T-lymphotrope simien de type 1/immunologie , Animaux , Animaux sauvages , Séquence nucléotidique , Réactions croisées , Infections à deltarétrovirus/épidémiologie , Infections à deltarétrovirus/virologie , Anticorps anti-HTLVI/sang , Anticorps anti-HTLVII/sang , Humains , Données de séquences moléculaires , Maladies des singes/virologie , Phylogenèse , Prévalence , Analyse de séquence d'ADN , Virus T-lymphotrope simien de type 1/génétique , Virus T-lymphotrope simien de type 1/isolement et purificationRÉSUMÉ
Human T-cell leukaemia virus type I (HTLV-I) is endemic in Melanesia, one of the three ethnogeographic regions of the Pacific; in the other two regions, Polynesia and Micronesia, the incidence of the virus is relatively low. In an effort to gain new insights into the prevalence of HTLV-I in the Pacific region, we did a seroepidemiological survey on Easter Island, which is located on the eastern edge of Polynesia. Of 138 subjects surveyed, including 108 Rapa Nui (the native inhabitants of this island), we identified one HTLV-I-seropositive Rapa Nui. The new HTLV-I isolate derived from this carrier (E-12) was phylogenetically analysed to ascertain the origin and past dissemination of HTLV-I in the island. The analysis demonstrated that isolate E-12 belongs to subgroup A of the Cosmopolitan group, and that it differs from HTLV-Is found in Melanesia, which are highly divergent variants. In subgroup A, E-12 grouped with South American HTLV-Is including those from Amerindians. This result suggests that this isolate originated in South America rather than in Melanesia.