RESUMO
SARS-CoV-2 is a betacoronavirus and the etiological agent of COVID-19, a devastating infectious disease. Due to its far-reaching effect on human health, there is an urgent and growing need to understand the viral molecular biology of SARS-CoV-2 and its interaction with the host cell. SARS-CoV-2 encodes 9 predicted accessory proteins, which are presumed to be dispensable for in vitro replication, most likely having a role in modulating the host cell environment to aid viral replication. Here we show that the ORF6 accessory protein interacts with cellular Rae1 to inhibit cellular protein production by blocking mRNA export. We utilised cell fractionation coupled with mRNAseq to explore which cellular mRNA species are affected by ORF6 expression and show that ORF6 can inhibit the export of many mRNA including those encoding antiviral factors such as IRF1 and RIG-I. We also show that export of these mRNA is blocked in the context of SARS-CoV-2 infection. Together, our studies identify a novel mechanism by which SARS-CoV-2 can manipulate the host cell environment to supress antiviral responses, providing further understanding to the replication strategies of a virus that has caused an unprecedented global health crisis.
Assuntos
COVID-19 , SARS-CoV-2 , Proteínas Virais/metabolismo , Antivirais , COVID-19/genética , Humanos , Imunidade Inata , Proteínas Associadas à Matriz Nuclear , Proteínas de Transporte Nucleocitoplasmático/genética , RNA Mensageiro/genéticaRESUMO
Viruses and their hosts are locked in an evolutionary race where resistance to infection is acquired by the hosts while viruses develop strategies to circumvent these host defenses. Forming one arm of the host defense armory are cell autonomous restriction factors like Fv1. Originally described as protecting laboratory mice from infection by murine leukemia virus (MLV), Fv1s from some wild mice have also been found to restrict non-MLV retroviruses, suggesting an important role in the protection against viruses in nature. We surveyed the Fv1 genes of wild mice trapped in Thailand and characterized their restriction activities against a panel of retroviruses. An extra copy of the Fv1 gene, named Fv7, was found on chromosome 6 of three closely related Asian species of mice: Mus caroli, M. cervicolor, and M. cookii. The presence of flanking repeats suggested it arose by LINE-mediated retroduplication within their most recent common ancestor. A high degree of natural variation was observed in both Fv1 and Fv7 and, on top of positive selection at certain residues, insertions and deletions were present that changed the length of the reading frames. These genes exhibited a range of restriction phenotypes, with activities directed against gamma-, spuma-, and lentiviruses. It seems likely, at least in the case of M. caroli, that the observed gene duplication may expand the breadth of restriction beyond the capacity of Fv1 alone and that one or more such viruses have recently driven or continue to drive the evolution of the Fv1 and Fv7 genes.
Assuntos
Evolução Molecular , Duplicação Gênica , Camundongos/genética , Proteínas/genética , Infecções por Retroviridae/genética , Animais , Resistência à Doença/genética , Camundongos/virologia , Retroviridae/patogenicidade , Infecções por Retroviridae/imunologia , Infecções por Retroviridae/virologiaRESUMO
Both exogenous and endogenous retroviruses have long been studied in mice, and some of the earliest mouse studies focused on the heritability of genetic factors influencing permissivity and resistance to infection. The prototypic retroviral restriction factor, Fv1, is now understood to exhibit a degree of control across multiple retroviral genera and is highly diverse within Mus To better understand the age and evolutionary history of Fv1, a comprehensive survey of the Muroidea was conducted, allowing the progenitor integration to be dated to â¼45 million years. Intact coding potential is visible beyond Mus, and sequence analysis reveals strong signatures of positive selection also within field mice, ApodemusFv1's survival for such a period implies a recurring and shifting retroviral burden imparting the necessary selective pressures-an influence likely also common to analogous factors. Regions of Fv1 adapt cooperatively, highlighting its preference for repeated structures and suggesting that this functionally constrained aspect of the retroviral capsid lattice presents a common target in the evolution of intrinsic immunity.
Assuntos
Evolução Molecular , Proteínas/genética , Animais , Camundongos , MurinaeRESUMO
SAMHD1, an analogue of the murine interferon (IFN)-γ-induced gene Mg11 (ref. 1), has recently been identified as a human immunodeficiency virus-1 (HIV-1) restriction factor that blocks early-stage virus replication in dendritic and other myeloid cells and is the target of the lentiviral protein Vpx, which can relieve HIV-1 restriction. SAMHD1 is also associated with Aicardi-Goutières syndrome (AGS), an inflammatory encephalopathy characterized by chronic cerebrospinal fluid lymphocytosis and elevated levels of the antiviral cytokine IFN-α. The pathology associated with AGS resembles congenital viral infection, such as transplacentally acquired HIV. Here we show that human SAMHD1 is a potent dGTP-stimulated triphosphohydrolase that converts deoxynucleoside triphosphates to the constituent deoxynucleoside and inorganic triphosphate. The crystal structure of the catalytic core of SAMHD1 reveals that the protein is dimeric and indicates a molecular basis for dGTP stimulation of catalytic activity against dNTPs. We propose that SAMHD1, which is highly expressed in dendritic cells, restricts HIV-1 replication by hydrolysing the majority of cellular dNTPs, thus inhibiting reverse transcription and viral complementary DNA (cDNA) synthesis.
Assuntos
HIV-1/fisiologia , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Nucleosídeo-Trifosfatase/química , Nucleosídeo-Trifosfatase/metabolismo , Regulação Alostérica , Biocatálise , Domínio Catalítico , Cristalografia por Raios X , Células Dendríticas/metabolismo , Células Dendríticas/virologia , Nucleotídeos de Desoxiadenina/metabolismo , Nucleotídeos de Desoxicitosina/metabolismo , Nucleotídeos de Desoxiguanina/metabolismo , Humanos , Hidrólise , Modelos Biológicos , Modelos Moleculares , Proteínas Monoméricas de Ligação ao GTP/genética , Células Mieloides/virologia , Nucleosídeo-Trifosfatase/genética , Estrutura Terciária de Proteína , Transcrição Reversa , Proteína 1 com Domínio SAM e Domínio HD , Nucleotídeos de Timina/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Replicação ViralRESUMO
Restriction factors (RFs) form important components of host defenses to retroviral infection. The Fv1, Trim5α, and TrimCyp RFs contain N-terminal dimerization and C-terminal specificity domains that target assembled retroviral capsid (CA) proteins enclosing the viral core. However, the molecular detail of the interaction between RFs and their CA targets is unknown. Therefore, we have determined the crystal structure of the B-box and coiled-coil (BCC) region from Trim5α and used small-angle X-ray scattering to examine the solution structure of Trim5α BCC, the dimerization domain of Fv1 (Fv1Ntd), and the hybrid restriction factor Fv1Cyp comprising Fv1NtD fused to the HIV-1 binding protein Cyclophilin A (CypA). These data reveal that coiled-coil regions of Fv1 and Trim5α form extended antiparallel dimers. In Fv1Cyp, two CypA moieties are located at opposing ends, creating a molecule with a dumbbell appearance. In Trim5α, the B-boxes are located at either end of the coiled-coil, held in place by interactions with a helical motif from the L2 region of the opposing monomer. A comparative analysis of Fv1Cyp and CypA binding to a preformed HIV-1 CA lattice reveals how RF dimerization enhances the affinity of interaction through avidity effects. We conclude that the antiparallel organization of the NtD regions of Fv1 and Trim5α dimers correctly positions C-terminal specificity and N-terminal effector domains and facilitates stable binding to adjacent CA hexamers in viral cores.
Assuntos
Capsídeo/metabolismo , HIV-1/metabolismo , Modelos Moleculares , Muramidase/química , Proteínas/química , Internalização do Vírus , Sequência de Aminoácidos , Animais , Bacteriófago T4/enzimologia , Sequência de Bases , Cromatografia em Gel , Cristalização , Dimerização , Escherichia coli , Modelos Lineares , Macaca mulatta , Microscopia Eletrônica , Dados de Sequência Molecular , Conformação Proteica , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes de Fusão/genética , Espalhamento a Baixo Ângulo , Análise de Sequência de DNA , Ressonância de Plasmônio de Superfície , Ubiquitina-Proteína Ligases , Difração de Raios XRESUMO
BACKGROUND: The mouse protein Fv1 is a factor that can confer resistance to retroviral infection. The two major Fv1 alleles from laboratory mice, Fv1 (n) and Fv1 (b) , restrict infection by different murine leukaemia viruses (MLVs). Fv1(n) restricts B-tropic MLV, but not N-tropic MLV or NB-tropic MLV. In cells expressing Fv1(b) at natural levels, only N-MLV is restricted, however restriction of NB-MLV and partial restriction of B-MLV were observed when recombinant Fv1(b) was expressed from an MLV promoter in Fv1 null Mus dunni tail fibroblast cells. To investigate the relationship between expression level and restriction specificity we have developed new retroviral delivery vectors which allow inducible expression of Fv1, and yet allow sufficient production of fluorescent reporter proteins for analysis in our FACS-based restriction assay. RESULTS: We demonstrated that at concentrations close to the endogenous expression level, Fv1(b) specifically restricts only N-MLV, but restriction of NB-MLV, and to a lesser extent B-MLV, could be gained by increasing the protein level of Fv1(b). By contrast, we found that even when Fv1(n) is expressed at very high levels, no significant inhibition of N-MLV or NB-MLV could be observed. Study of Fv1 mutants using this assay led to the identification of determinants for N/B tropism at an expression level close to that of endogenous Fv1(n) and Fv1(b). We also compared the recently described restriction activities of wild mice Fv1 proteins directed against non-MLV retroviruses when expressed at different levels. Fv1 from M. spretus restricted N-MLV, B-MLV and equine infectious anaemia virus equally even at low concentrations, while Fv1 from M. macedonicus showed even stronger restriction against equine infectious anaemia virus than to N-MLV. Restriction of feline foamy virus by Fv1 of M. caroli occurred at levels equivalent to MLV restriction. CONCLUSIONS: Our data indicate that for some but not all Fv1 proteins, gain of restriction activities could be achieved by increasing the expression level of Fv1. However such a concentration dependent effect is not seen with most Fv1s and cannot explain the recently reported activities against non-MLVs. It will be interesting to examine whether overexpression of other capsid binding restriction factors such as TRIM5α or Mx2 result in novel restriction specificities.
Assuntos
Vírus da Leucemia Murina/genética , Vírus da Leucemia Murina/fisiologia , Proteínas/genética , Proteínas/metabolismo , Replicação Viral , Animais , Proteínas do Capsídeo/metabolismo , Linhagem Celular , Vetores Genéticos , Humanos , Camundongos , MutaçãoRESUMO
Fv1 is the prototypic restriction factor that protects against infection by the murine leukemia virus (MLV). It was first identified in cells that were derived from laboratory mice and was found to be homologous to the gag gene of an endogenous retrovirus (ERV). To understand the evolution of the host restriction gene from its retroviral origins, Fv1s from wild mice were isolated and characterized. Most of these possess intact open reading frames but not all restricted N-, B-, NR-or NB-tropic MLVs, suggesting that other viruses could have played a role in the selection of the gene. The Fv1s from Mus spretus and Mus caroli were found to restrict equine infectious anemia virus (EIAV) and feline foamy virus (FFV) respectively, indicating that Fv1 could have a broader target range than previously thought, including activity against lentiviruses and spumaviruses. Analyses of the Fv1 sequences revealed a number of residues in the C-terminal region that had evolved under positive selection. Four of these selected residues were found to be involved in the novel restriction by mapping studies. These results strengthen the similarities between the two capsid binding restriction factors, Fv1 and TRIM5α, which support the hypothesis that Fv1 defended mice against waves of retroviral infection possibly including non-MLVs as well as MLVs.
Assuntos
Evolução Biológica , Proteínas/genética , Infecções por Retroviridae/imunologia , Animais , Sequência de Bases , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Filogenia , Retroviridae , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The Spumaretrovirinae, or foamyviruses (FVs) are complex retroviruses that infect many species of monkey and ape. Although FV infection is apparently benign, trans-species zoonosis is commonplace and has resulted in the isolation of the Prototypic Foamy Virus (PFV) from human sources and the potential for germ-line transmission. Despite little sequence homology, FV and orthoretroviral Gag proteins perform equivalent functions, including genome packaging, virion assembly, trafficking and membrane targeting. In addition, PFV Gag interacts with the FV Envelope (Env) protein to facilitate budding of infectious particles. Presently, there is a paucity of structural information with regards FVs and it is unclear how disparate FV and orthoretroviral Gag molecules share the same function. Therefore, in order to probe the functional overlap of FV and orthoretroviral Gag and learn more about FV egress and replication we have undertaken a structural, biophysical and virological study of PFV-Gag. We present the crystal structure of a dimeric amino terminal domain from PFV, Gag-NtD, both free and in complex with the leader peptide of PFV Env. The structure comprises a head domain together with a coiled coil that forms the dimer interface and despite the shared function it is entirely unrelated to either the capsid or matrix of Gag from other retroviruses. Furthermore, we present structural, biochemical and virological data that reveal the molecular details of the essential Gag-Env interaction and in addition we also examine the specificity of Trim5α restriction of PFV. These data provide the first information with regards to FV structural proteins and suggest a model for convergent evolution of gag genes where structurally unrelated molecules have become functionally equivalent.
Assuntos
Evolução Biológica , Capsídeo/metabolismo , Produtos do Gene gag/química , Produtos do Gene gag/metabolismo , Vírus Espumoso dos Símios/metabolismo , Sequência de Aminoácidos , Capsídeo/química , Linhagem Celular , Produtos do Gene gag/genética , Humanos , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Vírus Espumoso dos Símios/química , Vírus Espumoso dos Símios/genética , TransfecçãoRESUMO
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.
Assuntos
Proteínas do Capsídeo/metabolismo , Capsídeo/metabolismo , Proteínas de Transporte/metabolismo , Vírus da Leucemia Murina/metabolismo , Animais , Fatores de Restrição Antivirais , Sequência de Bases , Sítios de Ligação , Proteínas do Capsídeo/genética , Linhagem Celular , Humanos , Vírus da Leucemia Murina/genética , Macaca mulatta , Camundongos , Mutação , Domínios e Motivos de Interação entre Proteínas , Análise de Sequência de DNA , Proteínas com Motivo Tripartido , Ubiquitina-Proteína LigasesRESUMO
Xenotropic murine leukemia virus-related virus (XMRV) is a recently discovered gammaretrovirus that has been linked to prostate cancer and chronic fatigue syndrome. This virus is therefore an important potential human pathogen and, as such, it is essential to understand its host cell tropism. Intriguingly, infectious virus has been recovered from patient-derived peripheral blood mononuclear cells. These cells express several antiviral restriction factors that are capable of inhibiting the replication of a wide range of retroviruses, including other gamma retroviruses. This raises the possibility that, similar to HIV, XMRV may have acquired resistance to restriction. We therefore investigated the susceptibility of XMRV to a panel of different restriction factors. We found that both human APOBEC3 and tetherin proteins are able to block XMRV replication. Expression of human TRIM5alpha, however, had no effect on viral infectivity. There was no evidence that XMRV expressed countermeasures to overcome restriction. In addition, the virus was inhibited by factors from nonhuman species, including mouse Apobec3, tetherin, and Fv1 proteins. These results have important implications for predicting the natural target cells for XMRV replication, for relating infection to viral pathogenicity and pathology, and for the design of model systems with which to study XMRV-related diseases.
Assuntos
Antivirais/metabolismo , Vírus da Leucemia Murina/fisiologia , Desaminases APOBEC , Animais , Antígenos CD/metabolismo , Fatores de Restrição Antivirais , Proteínas de Transporte/metabolismo , Linhagem Celular , Células Clonais , Citidina Desaminase/metabolismo , Citosina Desaminase/metabolismo , Proteínas Ligadas por GPI , Humanos , Glicoproteínas de Membrana/metabolismo , Camundongos , Primatas , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases , Internalização do Vírus , Replicação ViralRESUMO
Many TRIM proteins are up-regulated by interferons, suggesting that they might be involved in the innate immune response against viruses. Indeed, some members of the family have been shown to be either regulators of the interferon pathways or to be directly involved in the restriction of viruses. While the mechanisms of actions are varied, the modular organization of these proteins seems to be important for their activities, many of which are linked to the ubiquitination/proteasomal degradation system. The different domains enable the TRIM proteins to interact with either viral components or signaling molecules in the interferon induction pathways.
Assuntos
Motivos de Aminoácidos , Imunidade Inata/imunologia , Proteínas/química , Proteínas/metabolismo , Vírus/imunologia , Inativação Gênica , Humanos , Interferon beta/metabolismoRESUMO
Tripartite motif (TRIM)5 alpha has recently been identified as a host restriction factor that has the ability to block infection by certain retroviruses in a species-dependent manner. One interesting feature of this protein is that it is localized in distinct cytoplasmic clusters designated as cytoplasmic bodies. The potential role of these cytoplasmic bodies in TRIM5 alpha function remains to be defined. By using fluorescent fusion proteins and live cell microscopy, we studied the localization and dynamics of TRIM5 alpha cytoplasmic bodies. This analysis reveals that cytoplasmic bodies are highly mobile, exhibiting both short saltatory movements and unidirectional long-distance movements along the microtubule network. The morphology of the cytoplasmic bodies is also dynamic. Finally, photobleaching and photoactivation analysis reveals that the TRIM5 alpha protein present in the cytoplasmic bodies is very dynamic, rapidly exchanging between cytoplasmic bodies and a more diffuse cytoplasmic population. Therefore, TRIM5 alpha cytoplasmic bodies are dynamic structures more consistent with a role in function or regulation rather than protein aggregates or inclusion bodies that represent dead-end static structures.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Transporte/ultraestrutura , Corpos de Inclusão/ultraestrutura , Animais , Fatores de Restrição Antivirais , Proteínas de Transporte/genética , Linhagem Celular , Citoplasma/metabolismo , Citoesqueleto/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Humanos , Corpos de Inclusão/metabolismo , Microscopia de Fluorescência , Microtúbulos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas com Motivo Tripartido , Ubiquitina-Proteína LigasesRESUMO
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.
Assuntos
Proteínas de Transporte/metabolismo , Cercopithecidae/metabolismo , Spumavirus/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Proteínas de Transporte/genética , Linhagem Celular , Cercopithecidae/genética , Produtos do Gene gag/genética , Produtos do Gene gag/metabolismo , Vetores Genéticos/genética , Humanos , Dados de Sequência Molecular , Mutação/genética , Filogenia , Alinhamento de Sequência , Spumavirus/genética , Dedos de ZincoRESUMO
Retroviral restriction factors are cellular proteins that interfere with retrovirus replication at a postpenetration, preintegration stage in the viral life cycle. The first restriction activity described was the mouse Fv1 gene. Three different alleles of Fv1, capable of restricting different murine leukaemia viruses (MLV), have been characterized at the molecular level. Two further activities, Ref1, which acts on MLV, and Lv1, which acts on lentiviruses, have been identified in other mammalian species. Recently, it has become clear that Ref1 and Lv1 are encoded by the same gene, Trim5alpha, which inhibits retrovirus replication in a species-specific manner. A series of chimeras between the human and rhesus monkey Trim5 genes were created to map and identify these specificity determinants. The Trim5alpha SPRY domain was found to be responsible for targeting HIV-1 restriction. By contrast, N-MLV restriction appears dependent on both the coiled-coil domain and the SPRY domain. A single amino acid substitution (R332P) in the human Trim5alpha can confer the ability to restrict HIV-1, suggesting that small changes during evolution may have profound effects on our susceptibility to cross-species infection.
Assuntos
Substituição de Aminoácidos/genética , Antivirais/genética , Proteínas de Transporte/genética , HIV-1 , Proteínas Recombinantes de Fusão/genética , Replicação Viral/genética , Sequência de Aminoácidos , Animais , Fatores de Restrição Antivirais , Citometria de Fluxo , Proteínas de Fluorescência Verde , Haplorrinos , Humanos , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Especificidade da Espécie , Transdução Genética , Proteínas com Motivo Tripartido , Ubiquitina-Proteína LigasesRESUMO
SAMHD1 is an intracellular enzyme that specifically degrades deoxynucleoside triphosphates into component nucleoside and inorganic triphosphate. In myeloid-derived dendritic cells and macrophages as well as resting T-cells, SAMHD1 blocks HIV-1 infection through this dNTP triphosphohydrolase activity by reducing the cellular dNTP pool to a level that cannot support productive reverse transcription. We now show that, in addition to this direct effect on virus replication, manipulating cellular SAMHD1 activity can significantly enhance or decrease the anti-HIV-1 efficacy of nucleotide analogue reverse transcription inhibitors presumably as a result of modulating dNTP pools that compete for recruitment by viral polymerases. Further, a variety of other nucleotide-based analogues, not normally considered antiretrovirals, such as the anti-herpes drugs Aciclovir and Ganciclovir and the anti-cancer drug Clofarabine are now revealed as potent anti-HIV-1 agents, under conditions of low dNTPs. This in turn suggests novel uses for nucleotide analogues to inhibit HIV-1 in differentiated cells low in dNTPs.
Assuntos
HIV-1/fisiologia , Nucleotídeos/química , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Aciclovir/farmacologia , Nucleotídeos de Adenina/farmacologia , Regulação Alostérica , Arabinonucleosídeos/farmacologia , Linhagem Celular , Clofarabina , Ganciclovir/farmacologia , Humanos , Células Mieloides/virologia , Nucleotídeos/metabolismo , Nucleotídeos/farmacologia , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/farmacologia , Replicação Viral/efeitos dos fármacosRESUMO
To test the hypothesis that rabbit endogenous lentivirus type K (RELIK) could play a role in shaping the evolution of TRIM5α, the susceptibility of viruses containing the RELIK capsid (CA) to TRIM5 restriction was evaluated. RELIK CA-containing viruses were susceptible to the TRIM5αs from Old World monkeys but were unaffected by most ape or New World monkey factors. TRIM5αs from various lagomorph species were also isolated and tested for anti-retroviral activity. The TRIM5αs from both cottontail rabbit and pika restrict a range of retroviruses, including HIV-1, HIV-2, FIV, EIAV and N-MLV. TRIM5αs from the European and cottontail rabbit, which have previously been found to contain RELIK, also restricted RELIK CA-containing viruses, whereas a weaker restriction was observed with chimeric TRIM5α containing the B30.2 domain from the pika, which lacks RELIK. Taken together, these results could suggest that the pika had not been exposed to exogenous RELIK and that endogenized RELIK might exert a selective pressure on lagomorph TRIM5α.
Assuntos
Proteínas de Transporte/genética , Retrovirus Endógenos/genética , Evolução Molecular , Lagomorpha/virologia , Lentivirus/genética , Animais , Sequência de Bases , Proteínas de Transporte/metabolismo , Linhagem Celular , DNA Viral/genética , Retrovirus Endógenos/metabolismo , Lagomorpha/metabolismo , Lentivirus/metabolismo , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , Análise de Sequência de DNARESUMO
Lentiviruses are widespread in a variety of vertebrates, often associated with chronic disease states. However, until the recent discovery of the prehistoric endogenous lentiviruses in rabbits (RELIK) and lemurs (PSIV), it was thought that lentiviruses had no capacity for germline integration and were only spread horizontally in an exogenous fashion. The existence of RELIK and PSIV refuted these ideas, revealing lentiviruses to be present in a range of mammals, capable of germline integration, and far more ancient than previously thought. Using Gag sequences reconstructed from the remnants of these prehistoric lentiviruses, we have produced chimeric lentiviruses capable of infecting nondividing cells and determined structures of capsid domains from PSIV and RELIK. We show that the structures from these diverse viruses are highly similar, containing features found in modern-day lentiviruses, including a functional cyclophilin-binding loop. Together, these data provide evidence for an ancient capsid-cyclophilin interaction preserved throughout lentiviral evolution.
Assuntos
Proteínas do Capsídeo/química , Ciclofilina A/metabolismo , Retrovirus Endógenos/química , Retrovirus Endógenos/genética , Evolução Molecular , Lentivirus/química , Lentivirus/genética , Animais , Sequência de Bases , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Cristalografia por Raios X , Ciclofilina A/química , Metilação de DNA , Retrovirus Endógenos/fisiologia , Produtos do Gene gag/química , Produtos do Gene gag/metabolismo , Genes Virais , Genes gag , Lemur/virologia , Lentivirus/fisiologia , Lentivirus de Primatas/química , Lentivirus de Primatas/genética , Lentivirus de Primatas/fisiologia , Modelos Moleculares , Estrutura Terciária de Proteína , Coelhos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Vírion/metabolismoRESUMO
In addition to the ability to bind the retroviral capsid protein, the retroviral restriction factors Fv1, Trim5alpha and Trim5-CypA share the common property of containing sequences that promote self-association. Otherwise Fv1 and Trim5alpha appear unrelated. Mutational analyses showed that restriction was invariably lost when changes designed to disrupt the sequences responsible for multimerization were introduced. A novel restriction protein could be obtained by substituting sequences from the self-associating domain of Fv1 for the Trim5 sequences in Trim5-CypA. Similarly, a fusion protein containing cyclophilin A joined to arfaptin2, a protein known to form extended dimers, was also shown to restrict HIV-1. Hence, multimerization of a capsid-binding domain could be the common minimum design feature for capsid-dependent retroviral restriction factors. However, not all domains that promote multimerization can substitute for the N-terminal domains of Fv1 and Trim5alpha. Moreover, only CypA can provide a capsid-binding site with different N-terminal domains. It is suggested that the spatial relationship between the multiple target binding sites may be important for restriction.
Assuntos
Antirretrovirais/farmacologia , Proteínas de Transporte/genética , Ciclofilina A/genética , HIV-1/efeitos dos fármacos , Proteínas/genética , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Fatores de Restrição Antivirais , Proteínas de Transporte/fisiologia , Linhagem Celular , Ciclofilina A/fisiologia , HIV-1/crescimento & desenvolvimento , Proteínas/fisiologia , Proteínas Recombinantes de Fusão/genética , Proteínas com Motivo Tripartido , Ubiquitina-Proteína LigasesRESUMO
The Trim5alpha protein from several primates restricts retroviruses in a capsid (CA)-dependent manner. In owl monkeys, the B30.2 domain of Trim5 has been replaced by cyclophilin A (CypA) following a retrotransposition. Restriction of human immunodeficiency virus type 1 (HIV-1) by the resulting Trim5-CypA fusion protein depends on CA binding to CypA, suggesting both that the B30.2 domain might be involved in CA binding and that the tripartite RING motif, B-BOX, and coiled coil (RBCC) motif domain can function independently of the B30.2 domain in restriction. To investigate the potential of RBCCs from other Trims to participate in restricting HIV-1, CypA was fused to the RBCC of Trim1, Trim18, and Trim19 and tested for restriction. Despite low identity within the RBCC domain, all fusion proteins were found to restrict HIV-1 but not the nonbinding G89V mutant, indicating that the overall structure of RBCC and not its primary sequence was important for the restriction function. The critical interaction between CA and Trim-CypA appears to take place soon after viral entry. Quantitative PCR analysis of viral reverse transcriptase products revealed that the different fusion proteins block HIV-1 at two distinct stages of its life cycle, either prior to reverse transcription or just before integration. With Trim1 and Trim18, this timing is dependent on the length of the Trim component of the fusion protein. These observations suggest that restriction factor binding can have different mechanistic consequences.