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1.
BMC Health Serv Res ; 21(1): 941, 2021 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-34503508

RESUMO

INTRODUCTION: Despite prioritization, routine antenatal influenza vaccine coverage is < 16% in South Africa. We aimed to describe maternal influenza vaccine coverage in 27 antenatal clinics (ANCs) in Gauteng and Western Cape (WC) Provinces, where in collaboration with the Department of Health (DoH), we augmented the annual influenza vaccination programme among pregnant women. METHODS: From 2015 through 2018, 40,230 additional doses of influenza vaccine were added to the available stock and administered as part of routine antenatal care. Educational talks were given daily and data were collected on women attending ANCs. We compared characteristics of vaccinated and unvaccinated women using multivariable logistic regression. RESULTS: We screened 62,979 pregnant women during the period when Southern Hemisphere influenza vaccines were available (27,068 in Gauteng and 35,911 in WC). Vaccine coverage at the targeted clinics was 78.7% (49,355/62682), although pregnant women in WC were more likely to be vaccinated compared to those in the Gauteng (Odds ratio (OR) =3.7 p < 0.001). Women aged 25-29 and > 35 years were less likely to be vaccinated than women aged 18-24 years (OR = 0.9 p = 0.053; OR = 0.9 p < 0.001). HIV positive status was not associated with vaccination (OR = 1.0 p = 0.266). Reasons for not vaccinating included: vaccine stock-outs where ANCs depleted available stock of vaccines and/or were awaiting delivery of vaccines (54.6%, 6949/12723), refusal/indecision (25.8%, 3285), and current illness that contraindicated vaccination (19.6%, 2489). CONCLUSION: Antenatal vaccination uptake was likely improved by the increased vaccine supply and vaccine education offered during our campaign.


Assuntos
Vacinas contra Influenza , Influenza Humana , Complicações Infecciosas na Gravidez , Feminino , Humanos , Programas de Imunização , Influenza Humana/prevenção & controle , Gravidez , Complicações Infecciosas na Gravidez/prevenção & controle , Gestantes , África do Sul , Vacinação
2.
PLoS Pathog ; 17(9): e1009484, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34543344

RESUMO

The capsid (CA) lattice of the HIV-1 core plays a key role during infection. From the moment the core is released into the cytoplasm, it interacts with a range of cellular factors that, ultimately, direct the pre-integration complex to the integration site. For integration to occur, the CA lattice must disassemble. Early uncoating or a failure to do so has detrimental effects on virus infectivity, indicating that an optimal stability of the viral core is crucial for infection. Here, we introduced cysteine residues into HIV-1 CA in order to induce disulphide bond formation and engineer hyper-stable mutants that are slower or unable to uncoat, and then followed their replication. From a panel of mutants, we identified three with increased capsid stability in cells and found that, whilst the M68C/E212C mutant had a 5-fold reduction in reverse transcription, two mutants, A14C/E45C and E180C, were able to reverse transcribe to approximately WT levels in cycling cells. Moreover, these mutants only had a 5-fold reduction in 2-LTR circle production, suggesting that not only could reverse transcription complete in hyper-stable cores, but that the nascent viral cDNA could enter the nuclear compartment. Furthermore, we observed A14C/E45C mutant capsid in nuclear and chromatin-associated fractions implying that the hyper-stable cores themselves entered the nucleus. Immunofluorescence studies revealed that although the A14C/E45C mutant capsid reached the nuclear pore with the same kinetics as wild type capsid, it was then retained at the pore in association with Nup153. Crucially, infection with the hyper-stable mutants did not promote CPSF6 re-localisation to nuclear speckles, despite the mutant capsids being competent for CPSF6 binding. These observations suggest that hyper-stable cores are not able to uncoat, or remodel, enough to pass through or dissociate from the nuclear pore and integrate successfully. This, is turn, highlights the importance of capsid lattice flexibility for nuclear entry. In conclusion, we hypothesise that during a productive infection, a capsid remodelling step takes place at the nuclear pore that releases the core complex from Nup153, and relays it to CPSF6, which then localises it to chromatin ready for integration.

3.
Viruses ; 13(8)2021 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-34452291

RESUMO

HIV-1 can infect non-dividing cells. The nuclear envelope therefore represents a barrier that HIV-1 must traverse in order to gain access to the host cell chromatin for integration. Hence, nuclear entry is a critical step in the early stages of HIV-1 replication. Following membrane fusion, the viral capsid (CA) lattice, which forms the outer face of the retroviral core, makes numerous interactions with cellular proteins that orchestrate the progress of HIV-1 through the replication cycle. The ability of CA to interact with nuclear pore proteins and other host factors around the nuclear pore determines whether nuclear entry occurs. Uncoating, the process by which the CA lattice opens and/or disassembles, is another critical step that must occur prior to integration. Both early and delayed uncoating have detrimental effects on viral infectivity. How uncoating relates to nuclear entry is currently hotly debated. Recent technological advances have led to intense discussions about the timing, location, and requirements for uncoating and have prompted the field to consider alternative uncoating scenarios that presently focus on uncoating at the nuclear pore and within the nuclear compartment. This review describes recent advances in the study of HIV-1 nuclear entry, outlines the interactions of the retroviral CA protein, and discusses the challenges of investigating HIV-1 uncoating.

4.
Euro Surveill ; 26(29)2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34296675

RESUMO

BackgroundIn South Africa, COVID-19 control measures to prevent SARS-CoV-2 spread were initiated on 16 March 2020. Such measures may also impact the spread of other pathogens, including influenza virus and respiratory syncytial virus (RSV) with implications for future annual epidemics and expectations for the subsequent northern hemisphere winter.MethodsWe assessed the detection of influenza and RSV through facility-based syndromic surveillance of adults and children with mild or severe respiratory illness in South Africa from January to October 2020, and compared this with surveillance data from 2013 to 2019.ResultsFacility-based surveillance revealed a decline in influenza virus detection during the regular season compared with previous years. This was observed throughout the implementation of COVID-19 control measures. RSV detection decreased soon after the most stringent COVID-19 control measures commenced; however, an increase in RSV detection was observed after the typical season, following the re-opening of schools and the easing of measures.ConclusionCOVID-19 non-pharmaceutical interventions led to reduced circulation of influenza and RSV in South Africa. This has limited the country's ability to provide influenza virus strains for the selection of the annual influenza vaccine. Delayed increases in RSV case numbers may reflect the easing of COVID-19 control measures. An increase in influenza virus detection was not observed, suggesting that the measures may have impacted the two pathogens differently. The impact that lowered and/or delayed influenza and RSV circulation in 2020 will have on the intensity and severity of subsequent annual epidemics is unknown and warrants close monitoring.


Assuntos
COVID-19 , Vacinas contra Influenza , Influenza Humana , Infecções por Vírus Respiratório Sincicial , Vírus Sincicial Respiratório Humano , Adulto , Criança , Humanos , Influenza Humana/diagnóstico , Influenza Humana/epidemiologia , Influenza Humana/prevenção & controle , Pandemias/prevenção & controle , Infecções por Vírus Respiratório Sincicial/diagnóstico , Infecções por Vírus Respiratório Sincicial/epidemiologia , Infecções por Vírus Respiratório Sincicial/prevenção & controle , SARS-CoV-2 , África do Sul/epidemiologia
5.
J Vis Exp ; (172)2021 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-34180901

RESUMO

Sterile α-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) inhibits replication of HIV-1 in quiescent myeloid cells. U937 cells are widely used as a convenient cell system for analyzing SAMHD1 activity due to a low level of SAMHD1 RNA expression, leading to undetectable endogenous protein expression. Based on similar assays developed in the Stoye laboratory to characterize other retroviral restriction factors, the Bishop lab developed a two-color restriction assay to analyze SAMHD1 in U937 cells. Murine Leukaemia Virus-like particles expressing SAMHD1, alongside YFP expressed from an IRES, are used to transduce U937 cells. Cells are then treated with phorbol myristate acetate to induce differentiation to a quiescent phenotype. Following differentiation, cells are infected with HIV-1 virus-like particles expressing a fluorescent reporter. After 48 h, cells are harvested and analyzed by flow cytometry. The proportion of HIV-infected cells in the SAMHD1-expressing population is compared to that in internal control cells lacking SAMHD1. This comparison reveals a restriction ratio. SAMHD1 expression leads to a five-fold reduction in HIV infection, corresponding to a restriction ratio of 0.2. Our recent substitution of RFP for the original GFP as the reporter gene for HIV infection has facilitated flow cytometry analysis. This assay has been successfully used to characterize the effect of amino acid substitutions on SAMHD1 restriction by transducing with viruses encoding altered SAMHD1 proteins, derived from site-directed mutagenesis of the expression vector. For example, the catalytic site substitutions HD206-7AA show a restriction phenotype of 1, indicating a loss of restriction activity. Equally, the susceptibility of different tester viruses can be determined. The assay can be further adapted to incorporate the effect of differentiation status, metabolic status, and SAMHD1 modifiers to better understand the relationship between SAMHD1, cell metabolic state, and viral restriction.

6.
J Virol ; 95(17): e0055421, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34106747

RESUMO

The p12 region of murine leukemia virus (MLV) Gag and the p6 region of HIV-1 Gag contain late domains required for virus budding. Additionally, the accessory protein Vpr is recruited into HIV particles via p6. Mature p12 is essential for early viral replication events, but the role of mature p6 in early replication is unknown. Using a proviral vector in which the gag and pol reading frames are uncoupled, we have performed the first alanine-scanning mutagenesis screens across p6 to probe its importance for early HIV-1 replication and to further understand its interaction with Vpr. The infectivity of our mutants suggests that, unlike p12, p6 is not important for early viral replication. Consistent with this, we observed that p6 is rapidly lost upon target cell entry in time course immunoblot experiments. By analyzing Vpr incorporation into p6 mutant virions, we identified that the 15-FRFG-18 and 41-LXXLF-45 motifs previously identified as putative Vpr-binding sites are important for Vpr recruitment but that the 34-ELY-36 motif also suggested to be a Vpr-binding site is dispensable. Additionally, disrupting Vpr oligomerization together with removing either binding motif in p6 reduced Vpr incorporation ∼25- to 50-fold more than inhibiting Vpr oligomerization alone and ∼10- to 25-fold more than deleting each p6 motif alone, implying that multivalency/avidity is important for the interaction. Interestingly, using immunoblotting and immunofluorescence, we observed that most Vpr is lost concomitantly with p6 during infection but that a small fraction remains associated with the viral capsid for several hours. This has implications for the function of Vpr in early replication. IMPORTANCE The p12 protein of MLV and the p6 protein of HIV-1 are both supplementary Gag cleavage products that carry proline-rich motifs that facilitate virus budding. Importantly, p12 has also been found to be essential for early viral replication events. However, while Vpr, the only accessory protein packaged into HIV-1 virions, is recruited via the p6 region of Gag, the function of both mature p6 and Vpr in early replication is unclear. Here, we have systematically mutated the p6 region of Gag and have studied the effects on HIV infectivity and Vpr packaging. We have also investigated what happens to p6 and Vpr during early infection. We show that, unlike p12, mature p6 is not required for early replication and that most of the mature p6 and the Vpr that it recruits are lost rapidly upon target cell entry. This has implications for the role of Vpr in target cells.

7.
Proc Natl Acad Sci U S A ; 116(37): 18647-18654, 2019 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-31451672

RESUMO

The vertebrate protein SAMHD1 is highly unusual in having roles in cellular metabolic regulation, antiviral restriction, and regulation of innate immunity. Its deoxynucleoside triphosphohydrolase activity regulates cellular dNTP concentration, reducing levels below those required by lentiviruses and other viruses to replicate. To counter this threat, some primate lentiviruses encode accessory proteins that bind SAMHD1 and induce its degradation; in turn, positive diversifying selection has been observed in regions bound by these lentiviral proteins, suggesting that primate SAMHD1 has coevolved to evade these countermeasures. Moreover, deleterious polymorphisms in human SAMHD1 are associated with autoimmune disease linked to uncontrolled DNA synthesis of endogenous retroelements. Little is known about how evolutionary pressures affect these different SAMHD1 functions. Here, we examine the deeper history of these interactions by testing whether evolutionary signatures in SAMHD1 extend to other mammalian groups and exploring the molecular basis of this coevolution. Using codon-based likelihood models, we find positive selection in SAMHD1 within each mammal lineage for which sequence data are available. We observe positive selection at sites clustered around T592, a residue that is phosphorylated to regulate SAMHD1 activity. We verify experimentally that mutations within this cluster affect catalytic rate and lentiviral restriction, suggesting that virus-host coevolution has required adaptations of enzymatic function. Thus, persistent positive selection may have involved the adaptation of SAMHD1 regulation to balance antiviral, metabolic, and innate immunity functions.


Assuntos
Evolução Molecular , Interações Hospedeiro-Patógeno/genética , Imunidade Inata/genética , Proteína 1 com Domínio SAM e Domínio HD/genética , Seleção Genética , Animais , Coevolução Biológica , HIV-1/genética , HIV-1/imunologia , HIV-1/patogenicidade , Interações Hospedeiro-Patógeno/imunologia , Humanos , Modelos Genéticos , Mutação , Fosforilação , Ligação Proteica/genética , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Tirosina/genética , Tirosina/metabolismo , Proteínas Virais Reguladoras e Acessórias/genética , Replicação Viral/genética , Replicação Viral/imunologia , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/genética
8.
Microb Cell ; 5(8): 385-388, 2018 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-30175108

RESUMO

The gammaretroviral gag cleavage product, p12, is essential for replication at both early and late stages of the virus life cycle. During the early stage of infection, the viral core is released into the cytoplasm, the viral RNA genome is reversed transcribed to cDNA and this viral DNA is then integrated into the host cell chromatin to form a provirus. The p12 protein has N- and C-terminal domains (NTD and CTD) that are required for steps leading up to integration, but the molecular details of their functions remain poorly characterised. Using the prototypic gammaretrovirus, murine leukemia virus (MLV) as a model, we recently showed that the NTD of p12 directly binds to and stabilises the capsid (CA) lattice of the viral core. Alterations to the CTD of MLV p12 prevented the viral pre-integration complex (PIC) tethering to host chromatin in mitosis, and this could be partially rescued by addition of a heterologous chromatin binding motif. In this study we demonstrated that the CTD of p12 directly binds to nucleosomal histone proteins, targeting not only p12 but also CA to mitotic chromatin. Additionally, cell-cycle-dependent phosphorylation of p12 appeared to increase the affinity of p12 for chromatin in mitosis relative to interphase. Thus, we have revealed how p12 can link the CA-containing PIC to mitotic chromatin, ready for integration. Importantly, we observed that direct binding to nucleosomes is a conserved feature of p12 orthologs across the gammaretrovirus genus and that the nucleosomal docking site is potentially shared with that of spumaretroviral Gag proteins.

9.
PLoS Pathog ; 14(6): e1007117, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29906285

RESUMO

The murine leukaemia virus (MLV) Gag cleavage product, p12, is essential for both early and late steps in viral replication. The N-terminal domain of p12 binds directly to capsid (CA) and stabilises the mature viral core, whereas defects in the C-terminal domain (CTD) of p12 can be rescued by addition of heterologous chromatin binding sequences (CBSs). We and others hypothesised that p12 tethers the pre-integration complex (PIC) to host chromatin ready for integration. Using confocal microscopy, we have observed for the first time that CA localises to mitotic chromatin in infected cells in a p12-dependent manner. GST-tagged p12 alone, however, did not localise to chromatin and mass-spectrometry analysis of its interactions identified only proteins known to bind the p12 region of Gag. Surprisingly, the ability to interact with chromatin was conferred by a single amino acid change, M63I, in the p12 CTD. Interestingly, GST-p12_M63I showed increased phosphorylation in mitosis relative to interphase, which correlated with an increased interaction with mitotic chromatin. Mass-spectrometry analysis of GST-p12_M63I revealed nucleosomal histones as primary interactants. Direct binding of MLV p12_M63I peptides to histones was confirmed by biolayer-interferometry (BLI) assays using highly-avid recombinant poly-nucleosomal arrays. Excitingly, using this method, we also observed binding between MLV p12_WT and nucleosomes. Nucleosome binding was additionally detected with p12 orthologs from feline and gibbon ape leukemia viruses using both pull-down and BLI assays, indicating that this a common feature of gammaretroviral p12 proteins. Importantly, p12 peptides were able to block the binding of the prototypic foamy virus CBS to nucleosomes and vice versa, implying that their docking sites overlap and suggesting a conserved mode of chromatin tethering for different retroviral genera. We propose that p12 is acting in a similar capacity to CPSF6 in HIV-1 infection by facilitating initial chromatin targeting of CA-containing PICs prior to integration.


Assuntos
Capsídeo/metabolismo , Cromatina/metabolismo , Produtos do Gene gag/genética , Mitose , Nucleossomos/metabolismo , Vírion/genética , Integração Viral/fisiologia , Animais , Cromatina/química , Cromatina/virologia , Regulação da Expressão Gênica , Produtos do Gene gag/química , Produtos do Gene gag/metabolismo , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Camundongos , Mutação , Ligação Proteica , Vírion/crescimento & desenvolvimento , Vírion/metabolismo , Montagem de Vírus , Replicação Viral
10.
Sci Rep ; 7: 42824, 2017 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-28220857

RESUMO

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 fármacos
11.
Retrovirology ; 13(1): 58, 2016 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-27549239

RESUMO

BACKGROUND: Correct disassembly of the HIV-1 capsid shell, called uncoating, is increasingly recognised as central for multiple steps during retroviral replication. However, the timing, localisation and mechanism of uncoating are poorly understood and progress in this area is hampered by difficulties in measuring the process. Previous work suggested that uncoating occurs soon after entry of the viral core into the cell, but recent studies report later uncoating, at or in the nucleus. Furthermore, inhibiting reverse transcription delays uncoating, linking these processes. RESULTS: Here, we have used a combined approach of experimental interrogation of viral mutants and mathematical modelling to investigate the timing of uncoating with respect to reverse transcription. By developing a minimal, testable, model and employing multiple uncoating assays to overcome the disadvantages of each single assay, we find that uncoating is not concomitant with the initiation of reverse transcription. Instead, uncoating appears to be triggered once reverse transcription reaches a certain stage, namely shortly after first strand transfer. CONCLUSIONS: Using multiple approaches, we have identified a point during reverse transcription that induces uncoating of the HIV-1 CA shell. We propose that uncoating initiates after the first strand transfer of reverse transcription.


Assuntos
Capsídeo/fisiologia , HIV-1/fisiologia , Transcrição Reversa , Desenvelopamento do Vírus , Proteínas do Capsídeo , Linhagem Celular , HIV-1/genética , Interações Hospedeiro-Patógeno , Humanos , Modelos Biológicos , Mutação
12.
PLoS Pathog ; 11(10): e1005194, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26431200

RESUMO

SAMHD1 restricts HIV-1 infection of myeloid-lineage and resting CD4+ T-cells. Most likely this occurs through deoxynucleoside triphosphate triphosphohydrolase activity that reduces cellular dNTP to a level where reverse transcriptase cannot function, although alternative mechanisms have been proposed recently. Here, we present combined structural and virological data demonstrating that in addition to allosteric activation and triphosphohydrolase activity, restriction correlates with the capacity of SAMHD1 to form "long-lived" enzymatically competent tetramers. Tetramer disruption invariably abolishes restriction but has varied effects on in vitro triphosphohydrolase activity. SAMHD1 phosphorylation also ablates restriction and tetramer formation but without affecting triphosphohydrolase steady-state kinetics. However phospho-SAMHD1 is unable to catalyse dNTP turnover under conditions of nucleotide depletion. Based on our findings we propose a model for phosphorylation-dependent regulation of SAMHD1 activity where dephosphorylation switches housekeeping SAMHD1 found in cycling cells to a high-activity stable tetrameric form that depletes and maintains low levels of dNTPs in differentiated cells.


Assuntos
Biocatálise , HIV-1/patogenicidade , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Linhagem Celular , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Cristalografia por Raios X , Citometria de Fluxo , Humanos , Proteínas Monoméricas de Ligação ao GTP/química , Fosforilação , Conformação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína 1 com Domínio SAM e Domínio HD , Espectrofotometria Atômica
13.
Cell Host Microbe ; 17(4): 489-99, 2015 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-25856754

RESUMO

The SAMHD1 triphosphohydrolase inhibits HIV-1 infection of myeloid and resting T cells by depleting dNTPs. To overcome SAMHD1, HIV-2 and some SIVs encode either of two lineages of the accessory protein Vpx that bind the SAMHD1 N or C terminus and redirect the host cullin-4 ubiquitin ligase to target SAMHD1 for proteasomal degradation. We present the ternary complex of Vpx from SIV that infects mandrills (SIVmnd-2) with the cullin-4 substrate receptor, DCAF1, and N-terminal and SAM domains from mandrill SAMHD1. The structure reveals details of Vpx lineage-specific targeting of SAMHD1 N-terminal "degron" sequences. Comparison with Vpx from SIV that infects sooty mangabeys (SIVsmm) complexed with SAMHD1-DCAF1 identifies molecular determinants directing Vpx lineages to N- or C-terminal SAMHD1 sequences. Inspection of the Vpx-DCAF1 interface also reveals conservation of Vpx with the evolutionally related HIV-1/SIV accessory protein Vpr. These data suggest a unified model for how Vpx and Vpr exploit DCAF1 to promote viral replication.


Assuntos
Proteínas de Transporte/química , Interações Hospedeiro-Patógeno , Proteínas Monoméricas de Ligação ao GTP/química , Multimerização Proteica , Proteínas Virais Reguladoras e Acessórias/química , Animais , Proteínas de Transporte/metabolismo , Cristalografia por Raios X , Mandrillus , Modelos Moleculares , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Conformação Proteica , Proteínas Virais Reguladoras e Acessórias/metabolismo , Replicação Viral
14.
PLoS Pathog ; 10(10): e1004474, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25356837

RESUMO

The murine leukaemia virus (MLV) gag gene encodes a small protein called p12 that is essential for the early steps of viral replication. The N- and C-terminal regions of p12 are sequentially acting domains, both required for p12 function. Defects in the C-terminal domain can be overcome by introducing a chromatin binding motif into the protein. However, the function of the N-terminal domain remains unknown. Here, we undertook a detailed analysis of the effects of p12 mutation on incoming viral cores. We found that both reverse transcription complexes and isolated mature cores from N-terminal p12 mutants have altered capsid complexes compared to wild type virions. Electron microscopy revealed that mature N-terminal p12 mutant cores have different morphologies, although immature cores appear normal. Moreover, in immunofluorescent studies, both p12 and capsid proteins were lost rapidly from N-terminal p12 mutant viral cores after entry into target cells. Importantly, we determined that p12 binds directly to the MLV capsid lattice. However, we could not detect binding of an N-terminally altered p12 to capsid. Altogether, our data imply that p12 stabilises the mature MLV core, preventing premature loss of capsid, and that this is mediated by direct binding of p12 to the capsid shell. In this manner, p12 is also retained in the pre-integration complex where it facilitates tethering to mitotic chromosomes. These data also explain our previous observations that modifications to the N-terminus of p12 alter the ability of particles to abrogate restriction by TRIM5alpha and Fv1, factors that recognise viral capsid lattices.


Assuntos
Capsídeo/metabolismo , Produtos do Gene gag/metabolismo , Vírus da Leucemia Murina/genética , Infecções por Retroviridae/virologia , Replicação Viral , Sequência de Aminoácidos , Animais , Capsídeo/ultraestrutura , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Linhagem Celular , Cromossomos , Produtos do Gene gag/genética , Humanos , Vírus da Leucemia Murina/fisiologia , Vírus da Leucemia Murina/ultraestrutura , Camundongos , Dados de Sequência Molecular , Mutação , Estrutura Terciária de Proteína , Proteínas Recombinantes , Transcrição Reversa , Alinhamento de Sequência , Vírion
15.
Nature ; 505(7482): 234-8, 2014 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-24336198

RESUMO

Lentiviruses contain accessory genes that have evolved to counteract the effects of host cellular defence proteins that inhibit productive infection. One such restriction factor, SAMHD1, inhibits human immunodeficiency virus (HIV)-1 infection of myeloid-lineage cells as well as resting CD4(+) T cells by reducing the cellular deoxynucleoside 5'-triphosphate (dNTP) concentration to a level at which the viral reverse transcriptase cannot function. In other lentiviruses, including HIV-2 and related simian immunodeficiency viruses (SIVs), SAMHD1 restriction is overcome by the action of viral accessory protein x (Vpx) or the related viral protein r (Vpr) that target and recruit SAMHD1 for proteasomal degradation. The molecular mechanism by which these viral proteins are able to usurp the host cell's ubiquitination machinery to destroy the cell's protection against these viruses has not been defined. Here we present the crystal structure of a ternary complex of Vpx with the human E3 ligase substrate adaptor DCAF1 and the carboxy-terminal region of human SAMHD1. Vpx is made up of a three-helical bundle stabilized by a zinc finger motif, and wraps tightly around the disc-shaped DCAF1 molecule to present a new molecular surface. This adapted surface is then able to recruit SAMHD1 via its C terminus, making it a competent substrate for the E3 ligase to mark for proteasomal degradation. The structure reported here provides a molecular description of how a lentiviral accessory protein is able to subvert the cell's normal protein degradation pathway to inactivate the cellular viral defence system.


Assuntos
Proteínas de Transporte/metabolismo , HIV/química , HIV/fisiologia , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteólise , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Cercocebus atys/virologia , Cristalografia por Raios X , Interações Hospedeiro-Patógeno , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Monoméricas de Ligação ao GTP/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Serina-Treonina Quinases , Proteína 1 com Domínio SAM e Domínio HD , Vírus da Imunodeficiência Símia/química , Vírus da Imunodeficiência Símia/fisiologia , Ubiquitina-Proteína Ligases , Ubiquitinação , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/química , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/metabolismo
16.
Retrovirology ; 9: 83, 2012 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-23035841

RESUMO

BACKGROUND: The Moloney murine leukaemia virus (Mo-MLV) gag gene encodes three main structural proteins, matrix, capsid and nucleocapsid and a protein called p12. In addition to its role during the late stages of infection, p12 has an essential, but undefined, function during early post-entry events. As these stages of retroviral infection remain poorly understood, we set out to investigate the function of p12. RESULTS: Examination of the infectivity of Mo-MLV virus-like particles containing a mixture of wild type and mutant p12 revealed that the N- and C-terminal regions of p12 are sequentially acting domains, both required for p12 function, and that the N-terminal activity precedes the C-terminal activity in the viral life cycle. By creating a panel of p12 mutants in other gammaretroviruses, we showed that these domains are conserved in this retroviral genus. We also undertook a detailed mutational analysis of each domain, identifying residues essential for function. These data show that different regions of the N-terminal domain are necessary for infectivity in different gammaretroviruses, in stark contrast to the C-terminal domain where the same region is essential for all viruses. Moreover, chimeras between the p12 proteins of Mo-MLV and gibbon ape leukaemia virus revealed that the C-terminal domains are interchangeable whereas the N-terminal domains are not. Finally, we identified potential functions for each domain. We observed that particles with defects in the N-terminus of p12 were unable to abrogate restriction factors, implying that their cores were impaired. We further showed that defects in the C-terminal domain of p12 could be overcome by introducing a chromatin binding motif into the protein. CONCLUSIONS: Based on these data, we propose a model for p12 function where the N-terminus of p12 interacts with, and stabilizes, the viral core, allowing the C-terminus of p12 to tether the preintegration complex to host chromatin during mitosis, facilitating integration.


Assuntos
Produtos do Gene gag/genética , Produtos do Gene gag/metabolismo , Vírus da Leucemia Murina de Moloney/fisiologia , Replicação Viral , Análise Mutacional de DNA , Vírus da Leucemia do Macaco Gibão/genética , Vírus da Leucemia do Macaco Gibão/fisiologia , Vírus da Leucemia Murina de Moloney/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo
17.
PLoS One ; 7(3): e34221, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22470540

RESUMO

The prevalence of specific infections in UK prostate cancer patients was investigated. Serum from 84 patients and 62 controls was tested for neutralisation of xenotropic murine leukaemia virus-related virus (XMRV) Envelope. No reactivity was found in the patient samples. In addition, a further 100 prostate DNA samples were tested for XMRV, BK virus, Trichomonas vaginalis and human papilloma viruses by nucleic acid detection techniques. Despite demonstrating DNA integrity and assay sensitivity, we failed to detect the presence of any of these agents in DNA samples, bar one sample that was weakly positive for HPV16. Therefore we conclude that these infections are absent in this typical cohort of men with prostate cancer.


Assuntos
Vírus BK , Papillomaviridae , Neoplasias da Próstata/virologia , Trichomonas vaginalis , Vírus Relacionado ao Vírus Xenotrópico da Leucemia Murina , Anticorpos Neutralizantes/imunologia , Vírus BK/genética , Estudos de Coortes , DNA/análise , Infecções por Vírus de DNA/complicações , Humanos , Masculino , Papillomaviridae/genética , Reação em Cadeia da Polimerase , Infecções por Polyomavirus/complicações , Neoplasias da Próstata/sangue , Neoplasias da Próstata/complicações , Tricomoníase/complicações , Trichomonas vaginalis/genética , Infecções Tumorais por Vírus/complicações , Reino Unido , Vírus Relacionado ao Vírus Xenotrópico da Leucemia Murina/genética
18.
J Gen Virol ; 93(Pt 5): 915-924, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22357751

RESUMO

In 2006, a new retrovirus was isolated from prostate cancer patient tissue. Named xenotropic murine leukemia virus-related virus (XMRV), this was potentially the third class of retrovirus to be pathogenic in humans. XMRV made a more dramatic impact on the wider scientific community, and indeed the media, in 2009 when it was reported to be present in a remarkably high proportion of patients with chronic fatigue syndrome as well as a significant, albeit smaller, proportion of healthy controls. The apparent strong link to disease and the fear of a previously unknown retrovirus circulating in the general population lead to a surge in XMRV research. Subsequent studies failed to find an association of XMRV with disease and, in most cases, failed to find the virus in human samples. In 2011, the case against XMRV and human disease strengthened, ending with several decisive publications revealing the origin of the virus and demonstrating contamination of samples. In this review, we outline the passage of research on XMRV and its potential association with disease from its isolation to the present day, where we find ourselves at the end of a turbulent story.


Assuntos
Síndrome de Fadiga Crônica/virologia , Neoplasias da Próstata/virologia , Virologia/história , Vírus Relacionado ao Vírus Xenotrópico da Leucemia Murina/isolamento & purificação , Vírus Relacionado ao Vírus Xenotrópico da Leucemia Murina/patogenicidade , História do Século XXI , Humanos , Masculino
19.
Proc Natl Acad Sci U S A ; 107(11): 5166-71, 2010 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-20194752

RESUMO

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 , Animais , Antígenos CD/metabolismo , 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 Viral
20.
Retrovirology ; 7: 10, 2010 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-20156349

RESUMO

BACKGROUND: Detection of a retrovirus, xenotropic murine leukaemia virus-related virus (XMRV), has recently been reported in 67% of patients with chronic fatigue syndrome. We have studied a total of 170 samples from chronic fatigue syndrome patients from two UK cohorts and 395 controls for evidence of XMRV infection by looking either for the presence of viral nucleic acids using quantitative PCR (limit of detection <16 viral copies) or for the presence of serological responses using a virus neutralisation assay. RESULTS: We have not identified XMRV DNA in any samples by PCR (0/299). Some serum samples showed XMRV neutralising activity (26/565) but only one of these positive sera came from a CFS patient. Most of the positive sera were also able to neutralise MLV particles pseudotyped with envelope proteins from other viruses, including vesicular stomatitis virus, indicating significant cross-reactivity in serological responses. Four positive samples were specific for XMRV. CONCLUSIONS: No association between XMRV infection and CFS was observed in the samples tested, either by PCR or serological methodologies. The non-specific neutralisation observed in multiple serum samples suggests that it is unlikely that these responses were elicited by XMRV and highlights the danger of over-estimating XMRV frequency based on serological assays. In spite of this, we believe that the detection of neutralising activity that did not inhibit VSV-G pseudotyped MLV in at least four human serum samples indicates that XMRV infection may occur in the general population, although with currently uncertain outcomes.


Assuntos
Síndrome de Fadiga Crônica/virologia , Infecções por Retroviridae/diagnóstico , Retroviridae/isolamento & purificação , Adolescente , Adulto , Idoso , Anticorpos Antivirais/sangue , Estudos de Coortes , DNA Viral/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Testes de Neutralização/métodos , Reação em Cadeia da Polimerase/métodos , Reino Unido/epidemiologia , Adulto Jovem
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