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1.
BMC Bioinformatics ; 22(1): 45, 2021 Feb 04.
Artículo en Inglés | MEDLINE | ID: mdl-33541262

RESUMEN

BACKGROUND: Several bioinformatics pipelines have been developed to detect sequences from viruses that integrate into the human genome because of the health relevance of these integrations, such as in the persistence of viral infection and/or in generating genotoxic effects, often progressing into cancer. Recent genomics and metagenomics analyses have shown that viruses also integrate into the genome of non-model organisms (i.e., arthropods, fish, plants, vertebrates). However, rarely studies of endogenous viral elements (EVEs) in non-model organisms have gone beyond their characterization from reference genome assemblies. In non-model organisms, we lack a thorough understanding of the widespread occurrence of EVEs and their biological relevance, apart from sporadic cases which nevertheless point to significant roles of EVEs in immunity and regulation of expression. The concomitance of repetitive DNA, duplications and/or assembly fragmentations in a genome sequence and intrasample variability in whole-genome sequencing (WGS) data could determine misalignments when mapping data to a genome assembly. This phenomenon hinders our ability to properly identify integration sites. RESULTS: To fill this gap, we developed ViR, a pipeline which solves the dispersion of reads due to intrasample variability in sequencing data from both single and pooled DNA samples thus ameliorating the detection of integration sites. We tested ViR to work with both in silico and real sequencing data from a non-model organism, the arboviral vector Aedes albopictus. Potential viral integrations predicted by ViR were molecularly validated supporting the accuracy of ViR results. CONCLUSION: ViR will open new venues to explore the biology of EVEs, especially in non-model organisms. Importantly, while we generated ViR with the identification of EVEs in mind, its application can be extended to detect any lateral transfer event providing an ad-hoc sequence to interrogate.


Asunto(s)
Mosquitos Vectores , Integración Viral , Secuenciación Completa del Genoma , Animales , Biología Computacional , Genoma Viral , Genómica , Humanos , Integración Viral/genética
2.
Viruses ; 13(2)2021 01 26.
Artículo en Inglés | MEDLINE | ID: mdl-33530322

RESUMEN

Hepatitis B Virus (HBV) is a globally-distributed pathogen and is a major cause of liver disease. HBV (or closely-related animal hepadnaviruses) can integrate into the host genome, but (unlike retroviruses) this integrated form is replication-defective. The specific role(s) of the integrated HBV DNA has been a long-standing topic of debate. Novel in vitro models of HBV infection combined with sensitive molecular assays now enable researchers to investigate this under-characterised phenomenon with greater ease and precision. This review covers the contributions these systems have made to understanding how HBV DNA integration induces liver cancer and facilitates viral persistence. We summarise the current findings into a working model of chronic HBV infection and discuss the clinical implications of this hypothetical framework on the upcoming therapeutic strategies used to curb HBV-associated pathogenesis.


Asunto(s)
ADN Viral/metabolismo , Virus de la Hepatitis B/fisiología , Virus de la Hepatitis B/patogenicidad , Integración Viral , Animales , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/virología , Antígenos de Superficie de la Hepatitis B/metabolismo , Virus de la Hepatitis B/genética , Hepatitis B Crónica/patología , Hepatitis B Crónica/virología , Humanos , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/virología , Modelos Biológicos
3.
Viruses ; 13(2)2021 02 04.
Artículo en Inglés | MEDLINE | ID: mdl-33557409

RESUMEN

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality. Almost half of HCC cases are associated with hepatitis B virus (HBV) infections, which often lead to HBV sequence integrations in the human genome. Accurate identification of HBV integration sites at a single nucleotide resolution is critical for developing a better understanding of the cancer genome landscape and of the disease itself. Here, we performed further analyses and characterization of HBV integrations identified by our recently reported VIcaller platform in recurrent or known HCC genes (such as TERT, MLL4, and CCNE1) as well as non-recurrent cancer-related genes (such as CSMD2, NKD2, and RHOU). Our pathway enrichment analysis revealed multiple pathways involving the alcohol dehydrogenase 4 gene, such as the metabolism pathways of retinol, tyrosine, and fatty acid. Further analysis of the HBV integration sites revealed distinct patterns involving the integration upper breakpoints, integrated genome lengths, and integration allele fractions between tumor and normal tissues. Our analysis also implies that the VIcaller method has diagnostic potential through discovering novel clonal integrations in cancer-related genes. In conclusion, although VIcaller is a hypothesis free virome-wide approach, it can still be applied to accurately identify genome-wide integration events of a specific candidate virus and their integration allele fractions.


Asunto(s)
Carcinoma Hepatocelular/genética , Virus de la Hepatitis B/genética , Neoplasias Hepáticas/genética , Integración Viral , Carcinogénesis/genética , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/virología , ADN Viral/genética , Frecuencia de los Genes , Genoma Humano/genética , Genoma Viral/genética , Virus de la Hepatitis B/fisiología , Hepatitis B Crónica/genética , Hepatitis B Crónica/patología , Hepatitis B Crónica/virología , Humanos , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/virología , Programas Informáticos
4.
Viruses ; 13(2)2021 01 30.
Artículo en Inglés | MEDLINE | ID: mdl-33573130

RESUMEN

Human hepatitis B virus (HBV) can cause chronic, lifelong infection of the liver that may lead to persistent or episodic immune-mediated inflammation against virus-infected hepatocytes. This immune response results in elevated rates of killing of virus-infected hepatocytes, which may extend over many years or decades, lead to fibrosis and cirrhosis, and play a role in the high incidence of hepatocellular carcinoma (HCC) in HBV carriers. Immune-mediated inflammation appears to cause oxidative DNA damage to hepatocytes, which may also play a major role in hepatocarcinogenesis. An additional DNA damaging feature of chronic infections is random integration of HBV DNA into the chromosomal DNA of hepatocytes. While HBV DNA integration does not have a role in virus replication it may alter gene expression of the host cell. Indeed, most HCCs that arise in HBV carriers contain integrated HBV DNA and, in many, the integrant appears to have played a role in hepatocarcinogenesis. Clonal expansion of hepatocytes, which is a natural feature of liver biology, occurs because the hepatocyte population is self-renewing and therefore loses complexity due to random hepatocyte death and replacement by proliferation of surviving hepatocytes. This process may also represent a risk factor for the development of HCC. Interestingly, during chronic HBV infection, hepatocyte clones detected using integrated HBV DNA as lineage-specific markers, emerge that are larger than those expected to occur by random death and proliferation of hepatocytes. The emergence of these larger hepatocyte clones may reflect a survival advantage that could be explained by an ability to avoid the host immune response. While most of these larger hepatocyte clones are probably not preneoplastic, some may have already acquired preneoplastic changes. Thus, chronic inflammation in the HBV-infected liver may be responsible, at least in part, for both initiation of HCC via oxidative DNA damage and promotion of HCC via stimulation of hepatocyte proliferation through immune-mediated killing and compensatory division.


Asunto(s)
ADN Viral/genética , Virus de la Hepatitis B/fisiología , Hepatitis B Crónica/virología , Hepatocitos/virología , Animales , Virus de la Hepatitis B/genética , Hepatitis B Crónica/genética , Hepatitis B Crónica/inmunología , Hepatocitos/inmunología , Humanos , Hígado/inmunología , Hígado/virología , Integración Viral
5.
Viruses ; 13(2)2021 01 30.
Artículo en Inglés | MEDLINE | ID: mdl-33573241

RESUMEN

During the last two decades, progresses in bioimaging and the development of various strategies to fluorescently label the viral components opened a wide range of possibilities to visualize the early phase of Human Immunodeficiency Virus 1 (HIV-1) life cycle directly in infected cells. After fusion of the viral envelope with the cell membrane, the viral core is released into the cytoplasm and the viral RNA (vRNA) is retro-transcribed into DNA by the reverse transcriptase. During this process, the RNA-based viral complex transforms into a pre-integration complex (PIC), composed of the viral genomic DNA (vDNA) coated with viral and host cellular proteins. The protective capsid shell disassembles during a process called uncoating. The viral genome is transported into the cell nucleus and integrates into the host cell chromatin. Unlike biochemical approaches that provide global data about the whole population of viral particles, imaging techniques enable following individual viruses on a single particle level. In this context, quantitative microscopy has brought original data shedding light on the dynamics of the viral entry into the host cell, the cytoplasmic transport, the nuclear import, and the selection of the integration site. In parallel, multi-color imaging studies have elucidated the mechanism of action of host cell factors implicated in HIV-1 viral cycle progression. In this review, we describe the labeling strategies used for HIV-1 fluorescence imaging and report on the main advancements that imaging studies have brought in the understanding of the infection mechanisms from the viral entry into the host cell until the provirus integration step.


Asunto(s)
Infecciones por VIH/virología , VIH-1/fisiología , Internalización del Virus , Animales , Núcleo Celular/virología , VIH-1/química , VIH-1/genética , Humanos , Microscopía Fluorescente , Integración Viral
6.
Viruses ; 13(2)2021 02 10.
Artículo en Inglés | MEDLINE | ID: mdl-33578999

RESUMEN

Since the discovery of HIV-1, the viral capsid has been recognized to have an important role as a structural protein that holds the viral genome, together with viral proteins essential for viral life cycle, such as the reverse transcriptase (RT) and the integrase (IN). The reverse transcription process takes place between the cytoplasm and the nucleus of the host cell, thus the Reverse Transcription Complexes (RTCs)/Pre-integration Complexes (PICs) are hosted in intact or partial cores. Early biochemical assays failed to identify the viral CA associated to the RTC/PIC, possibly due to the stringent detergent conditions used to fractionate the cells or to isolate the viral complexes. More recently, it has been observed that some host partners of capsid, such as Nup153 and CPSF6, can only bind multimeric CA proteins organized in hexamers. Those host factors are mainly located in the nuclear compartment, suggesting the entrance of the viral CA as multimeric structure inside the nucleus. Recent data show CA complexes within the nucleus having a different morphology from the cytoplasmic ones, clearly highlighting the remodeling of the viral cores during nuclear translocation. Thus, the multimeric CA complexes lead the viral genome into the host nuclear compartment, piloting the intranuclear journey of HIV-1 in order to successfully replicate. The aim of this review is to discuss and analyze the main discoveries to date that uncover the viral capsid as a key player in the reverse transcription and PIC maturation until the viral DNA integration into the host genome.


Asunto(s)
Cápside/metabolismo , Núcleo Celular/virología , VIH-1/fisiología , Transporte Activo de Núcleo Celular , Cápside/química , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismo , Núcleo Celular/metabolismo , VIH-1/química , VIH-1/metabolismo , Modelos Biológicos , Proteínas de Complejo Poro Nuclear/metabolismo , Transcripción Reversa , Integración Viral , Replicación Viral
7.
Viruses ; 13(1)2021 Jan 18.
Artículo en Inglés | MEDLINE | ID: mdl-33477441

RESUMEN

Integration of retroviral reverse transcripts into the chromosomes of the cells that they infect is required for efficient viral gene expression and the inheritance of viral genomes to daughter cells. Before integration can occur, retroviral reverse transcription complexes (RTCs) must access the nuclear environment where the chromosomes reside. Retroviral integration is non-random, with different types of virus-host interactions impacting where in the host chromatin integration takes place. Lentiviruses such as HIV efficiently infect interphase cells because their RTCs have evolved to usurp cellular nuclear import transport mechanisms, and research over the past decade has revealed specific interactions between the HIV capsid protein and nucleoporin (Nup) proteins such as Nup358 and Nup153. The interaction of HIV capsid with cleavage and polyadenylation specificity factor 6 (CPSF6), which is a component of the cellular cleavage and polyadenylation complex, helps to dictate nuclear import as well as post-nuclear RTC invasion. In the absence of the capsid-CPSF6 interaction, RTCs are precluded from reaching nuclear speckles and gene-rich regions of chromatin known as speckle-associated domains, and instead mis-target lamina-associated domains out at the nuclear periphery. Highlighting this area of research, small molecules that inhibit capsid-host interactions important for integration site targeting are highly potent antiviral compounds.


Asunto(s)
Proteínas de la Cápside/metabolismo , Cápside/metabolismo , Infecciones por VIH/virología , VIH-1/fisiología , Interacciones Huésped-Patógeno , Integración Viral , Transporte Activo de Núcleo Celular , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Cromatina , Infecciones por VIH/genética , Infecciones por VIH/metabolismo , Humanos , Proteínas de Complejo Poro Nuclear/metabolismo , Unión Proteica , Relación Estructura-Actividad
8.
Microbiome ; 9(1): 13, 2021 01 12.
Artículo en Inglés | MEDLINE | ID: mdl-33436089

RESUMEN

BACKGROUND: Polintons are large mobile genetic elements found in the genomes of eukaryotic organisms that are considered the ancient ancestors of most eukaryotic dsDNA viruses. Originally considered as transposons, they have been found to encode virus capsid genes, suggesting they may actually be integrated viruses; however, an extracellular form has yet to be detected. Recently, circa 25 Polinton-like viruses have been discovered in environmental metagenomes and algal genomes, which shared distantly related genes to both Polintons and virophages (Lavidaviridae). These entities could be the first members of a major class of ancient eukaryotic viruses; however, owing to the lack of available genomes for analysis, information on their global diversity, evolutionary relationships, eukaryotic hosts, and status as free virus particles is limited. RESULTS: Here, we analysed the metaviromes of an alpine lake to show that Polinton-like virus genome sequences are abundant in the water column. We identify major capsid protein genes belonging to 82 new Polinton-like viruses and use these to interrogate publicly available metagenomic datasets, identifying 543 genomes and a further 16 integrated into eukaryotic genomes. Using an analysis of shared gene content and major capsid protein phylogeny, we define large groups of Polinton-like viruses and link them to diverse eukaryotic hosts, including a new group of viruses, which possess all the core genes of virophages and infect oomycetes and Chrysophyceae. CONCLUSIONS: Our study increased the number of known Polinton-like viruses by 25-fold, identifying five major new groups of eukaryotic viruses, which until now have been hidden in metagenomic datasets. The large enrichment (> 100-fold) of Polinton-like virus sequences in the virus-sized fraction of this alpine lake and the fact that their viral major capsid proteins are found in eukaryotic host transcriptomes support the hypothesis that Polintons in unicellular eukaryotes are viruses. In summary, our data reveals a diverse assemblage of globally distributed viruses, associated with a wide range of unicellular eukaryotic hosts. We anticipate that the methods we have developed for Polinton-like virus detection and the database of over 20,000 genes we present will allow for continued discovery and analysis of these new viral groups. Video abstract.


Asunto(s)
Organismos Acuáticos/genética , Organismos Acuáticos/virología , Virus ADN/genética , Eucariontes/genética , Eucariontes/virología , Genoma Viral/genética , Lagos , Virus ADN/clasificación , ADN Viral/genética , Ecosistema , Filogenia , Virófagos/genética , Integración Viral/genética
9.
Cells ; 10(2)2021 01 21.
Artículo en Inglés | MEDLINE | ID: mdl-33494237

RESUMEN

Novel coronavirus (COVID-19 or 2019-nCoV or SARS-CoV-2), which suddenly emerged in December 2019 is still haunting the entire human race and has affected not only the healthcare system but also the global socioeconomic balances. COVID-19 was quickly designated as a global pandemic by the World Health Organization as there have been about 98.0 million confirmed cases and about 2.0 million confirmed deaths, as of January 2021. Although, our understanding of COVID-19 has significantly increased since its outbreak, and multiple treatment approaches and pharmacological interventions have been tested or are currently under development to mitigate its risk-factors. Recently, some vaccine candidates showed around 95% clinical efficacy, and now receiving emergency use approvals in different countries. US FDA recently approved BNT162 and mRNA-1273 vaccines developed by Pfizer/BioNTech and Moderna Inc. for emergency use and vaccination in the USA. In this review, we present a succinct overview of the SARS-CoV-2 virus structure, molecular mechanisms of infection, COVID-19 epidemiology, diagnosis, and clinical manifestations. We also systematize different treatment strategies and clinical trials initiated after the pandemic outbreak, based on viral infection and replication mechanisms. Additionally, we reviewed the novel pharmacological intervention approaches and vaccine development strategies against COVID-19. We speculate that the current pandemic emergency will trigger detailed studies of coronaviruses, their mechanism of infection, development of systematic drug repurposing approaches, and novel drug discoveries for current and future pandemic outbreaks.


Asunto(s)
/terapia , /fisiología , Antivirales/uso terapéutico , /epidemiología , Humanos , Receptores Virales/metabolismo , Integración Viral
10.
Methods Mol Biol ; 2162: 243-260, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-32926387

RESUMEN

Clustered regularly interspaced short palindromic repeat (CRISPR) and other gene editing technologies such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) show great promises for research and therapeutic applications. One major concern is the off-target effects generated by these nucleases at unintended genomic sequences. In silico methods are usually used for off-target site prediction. However, based on currently available algorithms, the predicted cleavage activity at these potential off-target sites does not always reflect the true cleavage in vivo. Here we present an unbiased screening protocol using integration-defective lentiviral vector (IDLV) and deep sequencing to map the off-target sites generated by gene editing tools.


Asunto(s)
Sistemas CRISPR-Cas , Edición Génica/métodos , Marcación de Gen/métodos , Lentivirus/genética , Integración Viral , Vectores Genéticos/genética , Células HEK293 , Humanos
11.
Nucleic Acids Res ; 49(2): 621-635, 2021 01 25.
Artículo en Inglés | MEDLINE | ID: mdl-33337475

RESUMEN

The integration of retroviral reverse transcripts into the chromatin of the cells that they infect is required for virus replication. Retroviral integration has far-reaching consequences, from perpetuating deadly human diseases to molding metazoan evolution. The lentivirus human immunodeficiency virus 1 (HIV-1), which is the causative agent of the AIDS pandemic, efficiently infects interphase cells due to the active nuclear import of its preintegration complex (PIC). To enable integration, the PIC must navigate the densely-packed nuclear environment where the genome is organized into different chromatin states of varying accessibility in accordance with cellular needs. The HIV-1 capsid protein interacts with specific host factors to facilitate PIC nuclear import, while additional interactions of viral integrase, the enzyme responsible for viral DNA integration, with cellular nuclear proteins and nucleobases guide integration to specific chromosomal sites. HIV-1 integration favors transcriptionally active chromatin such as speckle-associated domains and disfavors heterochromatin including lamina-associated domains. In this review, we describe virus-host interactions that facilitate HIV-1 PIC nuclear import and integration site targeting, highlighting commonalities among factors that participate in both of these steps. We moreover discuss how the nuclear landscape influences HIV-1 integration site selection as well as the establishment of active versus latent virus infection.


Asunto(s)
VIH-1/fisiología , Interacciones Huésped-Patógeno , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Integración Viral , Transporte Activo de Núcleo Celular , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/fisiología , Proteínas de la Cápside/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/virología , Cromatina/genética , Cromatina/metabolismo , Citoplasma/metabolismo , Citoplasma/virología , Proteínas del Citoesqueleto/metabolismo , Transcriptasa Inversa del VIH/fisiología , VIH-1/enzimología , VIH-1/genética , Proteínas del Virus de la Inmunodeficiencia Humana/genética , Humanos , Interfase , Modelos Moleculares , Complejos Multiproteicos/metabolismo , Poro Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Conformación Proteica , Dominios Proteicos , Factores de Transcripción/deficiencia , Factores de Transcripción/fisiología , Integración Viral/genética , Integración Viral/fisiología , Latencia del Virus , Replicación Viral , Factores de Escisión y Poliadenilación de ARNm/deficiencia , Factores de Escisión y Poliadenilación de ARNm/fisiología
12.
Zhonghua Zhong Liu Za Zhi ; 42(12): 1014-1019, 2020 Dec 23.
Artículo en Chino | MEDLINE | ID: mdl-33342157

RESUMEN

Objective: To investigate the relationship between human papillomavirus (HPV) integration and prognosis of cervical cancer patients. Methods: The data of 82 patients with cervical cancer treated in the Radiotherapy Department of Peking Union Medical College Hospital from October 2004 to June 2012 were retrospectively analyzed.The patients were divided into poor prognosis group (recurrence or metastasis after surgery and adjuvant radiotherapy) and good prognosis group based on a propensity score matching strategy.The HPV integration of the two groups were detected by whole exome sequencing to determine whether the integration sites were located in the common fragile sites (CFSs). HPV integration and integration into CFSs were compared between the two groups. Results: Among the enrolled 82 patients, 37 were divided in poor survival group and 45 in good survival group. A total of 90 integration breakpoints were identified, 30 of them occurred in poor prognosis group and 60 occurred in good prognosis group. In the poor prognosis group, HPV integration occurred in 20 patients, 13 of them were inserted in CFSs of 11 patients, and the numbers in good prognosis group were 26, 17, 11, respectively. There were no significantly statistical differences in the number of HPV integration events (P=0.289), HPV integration patients (P=0.735), CFSs integration events (P=0.427), and CFSs integration patients (P=0.591) between the two groups. In poor prognosis group, more CFSs integration events occurred in patients with metastasis than those in patients with only local recurrence (9 vs 2, P=0.003). Conclusions: No significant differences are observed in HPV integration and HPV integration into CFSs between cervical cancer patients with different prognoses. HPV integration into CFSs may be associated with distant metastasis.


Asunto(s)
Alphapapillomavirus , Neoplasias del Cuello Uterino , Integración Viral , Alphapapillomavirus/genética , Femenino , Humanos , Pronóstico , Estudios Retrospectivos , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/terapia , Neoplasias del Cuello Uterino/virología , Integración Viral/genética
13.
Proc Natl Acad Sci U S A ; 117(52): 32880-32882, 2020 12 29.
Artículo en Inglés | MEDLINE | ID: mdl-33318172

RESUMEN

In vivo clonal expansion of HIV-infected T cells is an important mechanism of viral persistence. In some cases, clonal expansion is driven by HIV proviral DNA integrated into one of a handful of genes. To investigate this phenomenon in vitro, we infected primary CD4+ T cells with an HIV construct expressing GFP and, after nearly 2 mo of culture and multiple rounds of activation, analyzed the resulting integration site distribution. In each of three replicates from each of two donors, we detected large clusters of integration sites with multiple breakpoints, implying clonal selection. These clusters all mapped to a narrow region within the STAT3 gene. The presence of hybrid transcripts splicing HIV to STAT3 sequences supports a model of LTR-driven STAT3 overexpression as a driver of preferential growth. Thus, HIV integration patterns linked to selective T cell outgrowth can be reproduced in cell culture. The single report of an HIV provirus in a case of AIDS-associated B-cell lymphoma with an HIV provirus in the same part of STAT3 also has implications for HIV-induced malignancy.


Asunto(s)
Proliferación Celular , VIH/fisiología , Provirus/fisiología , Linfocitos T/virología , Integración Viral , Células Cultivadas , Evolución Clonal , ADN Viral/genética , VIH/genética , Humanos , Provirus/genética , Factor de Transcripción STAT3/genética , Linfocitos T/fisiología
14.
PLoS Pathog ; 16(12): e1009147, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-33351861

RESUMEN

The central function of the retroviral integrase protein (IN) is to catalyze the integration of viral DNA into the host genome to form the provirus. The IN protein has also been reported to play a role in a number of other processes throughout the retroviral life cycle such as reverse transcription, nuclear import and particle morphogenesis. Studies have shown that HIV-1 IN is subject to multiple post-translational modifications (PTMs) including acetylation, phosphorylation and SUMOylation. However, the importance of these modifications during infection has been contentious. In this study we attempt to clarify the role of acetylation of HIV-1 IN during the retroviral life cycle. We show that conservative mutation of the known acetylated lysine residues has only a modest effect on reverse transcription and proviral integration efficiency in vivo. However, we observe a large defect in successful expression of proviral genes at early times after infection by an acetylation-deficient IN mutant that cannot be explained by delayed integration dynamics. We demonstrate that the difference between the expression of proviruses integrated by an acetylation mutant and WT IN is likely not due to altered integration site distribution but rather directly due to a lower rate of transcription. Further, the effect of the IN mutation on proviral gene expression is independent of the Tat protein or the LTR promoter. At early times after integration when the transcription defect is observed, the LTRs of proviruses integrated by the mutant IN have altered histone modifications as well as reduced IN protein occupancy. Over time as the transcription defect in the mutant virus diminishes, histone modifications on the WT and mutant proviral LTRs reach comparable levels. These results highlight an unexpected role for the IN protein in regulating proviral transcription at early times post-integration.


Asunto(s)
ADN Viral/fisiología , Integrasa de VIH/genética , VIH-1/genética , Provirus/genética , Integración Viral/genética , Acetilación , Línea Celular , Humanos , Mutación , Procesamiento Proteico-Postraduccional/genética
15.
Science ; 370(6513)2020 10 09.
Artículo en Inglés | MEDLINE | ID: mdl-33033190

RESUMEN

During the first half of the viral life cycle, HIV-1 reverse transcribes its RNA genome and integrates the double-stranded DNA copy into a host cell chromosome. Despite progress in characterizing and inhibiting these processes, in situ mechanistic and structural studies remain challenging. This is because these operations are executed by individual viral preintegration complexes deep within cells. We therefore reconstituted and imaged the early stages of HIV-1 replication in a cell-free system. HIV-1 cores released from permeabilized virions supported efficient, capsid-dependent endogenous reverse transcription to produce double-stranded DNA genomes, which sometimes looped out from ruptured capsid walls. Concerted integration of both viral DNA ends into a target plasmid then proceeded in a cell extract-dependent reaction. This reconstituted system uncovers the role of the capsid in templating replication.


Asunto(s)
Cápside/fisiología , VIH-1/fisiología , Integración Viral , Replicación Viral , Sistema Libre de Células , Humanos
16.
Nat Commun ; 11(1): 5043, 2020 10 07.
Artículo en Inglés | MEDLINE | ID: mdl-33028863

RESUMEN

Human T-cell lymphotropic virus type 1 (HTLV-1) is a deltaretrovirus and the most oncogenic pathogen. Many of the ~20 million HTLV-1 infected people will develop severe leukaemia or an ALS-like motor disease, unless a therapy becomes available. A key step in the establishment of infection is the integration of viral genetic material into the host genome, catalysed by the retroviral integrase (IN) enzyme. Here, we use X-ray crystallography and single-particle cryo-electron microscopy to determine the structure of the functional deltaretroviral IN assembled on viral DNA ends and bound to the B56γ subunit of its human host factor, protein phosphatase 2 A. The structure reveals a tetrameric IN assembly bound to two molecules of the phosphatase via a conserved short linear motif. Insight into the deltaretroviral intasome and its interaction with the host will be crucial for understanding the pattern of integration events in infected individuals and therefore bears important clinical implications.


Asunto(s)
Virus Linfotrópico T Tipo 1 Humano/patogenicidad , Integrasas/ultraestructura , Proteína Fosfatasa 2/ultraestructura , Virus Linfotrópico T Tipo 1 de los Simios/enzimología , Proteínas Virales/ultraestructura , Integración Viral , Secuencias de Aminoácidos/genética , Clonación Molecular , Microscopía por Crioelectrón , Cristalografía por Rayos X , ADN Viral/metabolismo , ADN Viral/ultraestructura , Virus Linfotrópico T Tipo 1 Humano/enzimología , Virus Linfotrópico T Tipo 1 Humano/genética , Humanos , Integrasas/genética , Integrasas/metabolismo , Leucemia-Linfoma de Células T del Adulto/patología , Leucemia-Linfoma de Células T del Adulto/virología , Simulación del Acoplamiento Molecular , Mutagénesis Sitio-Dirigida , Paraparesia Espástica Tropical/patología , Paraparesia Espástica Tropical/virología , Multimerización de Proteína , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/metabolismo , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura , Homología de Secuencia de Aminoácido , Virus Linfotrópico T Tipo 1 de los Simios/genética , Imagen Individual de Molécula , Proteínas Virales/genética , Proteínas Virales/metabolismo
17.
Viruses ; 12(10)2020 09 26.
Artículo en Inglés | MEDLINE | ID: mdl-32993136

RESUMEN

In a short time, the COVID-19 pandemic has left the world with over 25 million cases and staggering death tolls that are still rising. Treatments for SARS-CoV-2 infection are desperately needed as there are currently no approved drug therapies. With limited knowledge of viral mechanisms, a network controllability method of prioritizing existing drugs for repurposing efforts is optimal for quickly moving through the drug approval pipeline using limited, available, virus-specific data. Based on network topology and controllability, 16 proteins involved in translation, cellular transport, cellular stress, and host immune response are predicted as regulators of the SARS-CoV-2 infected cell. Of the 16, eight are prioritized as possible drug targets where two, PVR and SCARB1, are previously unexplored. Known compounds targeting these genes are suggested for viral inhibition study. Prioritized proteins in agreement with previous analysis and viral inhibition studies verify the ability of network controllability to predict biologically relevant candidates.


Asunto(s)
Betacoronavirus/efectos de los fármacos , Infecciones por Coronavirus/tratamiento farmacológico , Reposicionamiento de Medicamentos/métodos , Neumonía Viral/tratamiento farmacológico , Betacoronavirus/aislamiento & purificación , Betacoronavirus/fisiología , Infecciones por Coronavirus/metabolismo , Infecciones por Coronavirus/virología , Aprobación de Drogas , Sistemas de Liberación de Medicamentos , Interacciones Huésped-Patógeno , Humanos , Pandemias , Neumonía Viral/metabolismo , Neumonía Viral/virología , Mapas de Interacción de Proteínas/efectos de los fármacos , Receptores Virales/genética , Receptores Virales/metabolismo , Receptores Depuradores de Clase B/metabolismo , Integración Viral
18.
PLoS Comput Biol ; 16(9): e1007470, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32941445

RESUMEN

Human T-lymphotropic virus type-1 (HTLV-1) persists within hosts via infectious spread (de novo infection) and mitotic spread (infected cell proliferation), creating a population structure of multiple clones (infected cell populations with identical genomic proviral integration sites). The relative contributions of infectious and mitotic spread to HTLV-1 persistence are unknown, and will determine the efficacy of different approaches to treatment. The prevailing view is that infectious spread is negligible in HTLV-1 persistence beyond early infection. However, in light of recent high-throughput data on the abundance of HTLV-1 clones, and recent estimates of HTLV-1 clonal diversity that are substantially higher than previously thought (typically between 104 and 105 HTLV-1+ T cell clones in the body of an asymptomatic carrier or patient with HTLV-1-associated myelopathy/tropical spastic paraparesis), ongoing infectious spread during chronic infection remains possible. We estimate the ratio of infectious to mitotic spread using a hybrid model of deterministic and stochastic processes, fitted to previously published HTLV-1 clonal diversity estimates. We investigate the robustness of our estimates using three alternative estimators. We find that, contrary to previous belief, infectious spread persists during chronic infection, even after HTLV-1 proviral load has reached its set point, and we estimate that between 100 and 200 new HTLV-1 clones are created and killed every day. We find broad agreement between all estimators. The risk of HTLV-1-associated malignancy and inflammatory disease is strongly correlated with proviral load, which in turn is correlated with the number of HTLV-1-infected clones, which are created by de novo infection. Our results therefore imply that suppression of de novo infection may reduce the risk of malignant transformation.


Asunto(s)
Infecciones por HTLV-I , Interacciones Huésped-Patógeno , Virus Linfotrópico T Tipo 1 Humano , Linfocitos T CD4-Positivos/virología , Infecciones por HTLV-I/fisiopatología , Infecciones por HTLV-I/virología , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/fisiología , Virus Linfotrópico T Tipo 1 Humano/clasificación , Virus Linfotrópico T Tipo 1 Humano/genética , Virus Linfotrópico T Tipo 1 Humano/patogenicidad , Humanos , Mitosis/genética , Mitosis/fisiología , Modelos Biológicos , Provirus/genética , Provirus/patogenicidad , Carga Viral/genética , Integración Viral/genética
19.
PLoS Genet ; 16(8): e1008915, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32776928

RESUMEN

Sequences homologous to human herpesvirus 6 (HHV-6) are integrated within the nuclear genome of about 1% of humans, but it is not clear how this came about. It is also uncertain whether integrated HHV-6 can reactivate into an infectious virus. HHV-6 integrates into telomeres, and this has recently been associated with polymorphisms affecting MOV10L1. MOV10L1 is located on the subtelomere of chromosome 22q (chr22q) and is required to make PIWI-interacting RNAs (piRNAs). As piRNAs block germline integration of transposons, piRNA-mediated repression of HHV-6 integration has been proposed to explain this association. In vitro, recombination of the HHV-6 genome along its terminal direct repeats (DRs) leads to excision from the telomere and viral reactivation, but the expected "solo-DR scar" has not been described in vivo. Here we screened for integrated HHV-6 in 7,485 Japanese subjects using whole-genome sequencing (WGS). Integrated HHV-6 was associated with polymorphisms on chr22q. However, in contrast to prior work, we find that the reported MOV10L1 polymorphism is physically linked to an ancient endogenous HHV-6A variant integrated into the telomere of chr22q in East Asians. Unexpectedly, an HHV-6B variant has also endogenized in chr22q; two endogenous HHV-6 variants at this locus thus account for 72% of all integrated HHV-6 in Japan. We also report human genomes carrying only one portion of the HHV-6B genome, a solo-DR, supporting in vivo excision and possible viral reactivation. Together these results explain the recently-reported association between integrated HHV-6 and MOV10L1/piRNAs, suggest potential exaptation of HHV-6 in its coevolution with human chr22q, and clarify the evolution and risk of reactivation of the only intact (non-retro)viral genome known to be present in human germlines.


Asunto(s)
Genoma Humano , Herpesvirus Humano 6/genética , Integración Viral , Grupo de Ascendencia Continental Asiática/genética , Cromosomas Humanos Par 22/genética , Evolución Molecular , Mutación de Línea Germinal , Humanos , Polimorfismo de Nucleótido Simple , ARN Interferente Pequeño/genética
20.
PLoS Pathog ; 16(8): e1008562, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32833988

RESUMEN

Merkel Cell Polyomavirus (MCPyV) is the etiological agent of the majority of Merkel Cell Carcinomas (MCC). MCPyV positive MCCs harbor integrated, defective viral genomes that constitutively express viral oncogenes. Which molecular mechanisms promote viral integration, if distinct integration patterns exist, and if integration occurs preferentially at loci with specific chromatin states is unknown. We here combined short and long-read (nanopore) next-generation sequencing and present the first high-resolution analysis of integration site structure in MCC cell lines as well as primary tumor material. We find two main types of integration site structure: Linear patterns with chromosomal breakpoints that map closely together, and complex integration loci that exhibit local amplification of genomic sequences flanking the viral DNA. Sequence analysis suggests that linear patterns are produced during viral replication by integration of defective/linear genomes into host DNA double strand breaks via non-homologous end joining, NHEJ. In contrast, our data strongly suggest that complex integration patterns are mediated by microhomology-mediated break-induced replication, MMBIR. Furthermore, we show by ChIP-Seq and RNA-Seq analysis that MCPyV preferably integrates in open chromatin and provide evidence that viral oncogene expression is driven by the viral promoter region, rather than transcription from juxtaposed host promoters. Taken together, our data explain the characteristics of MCPyV integration and may also provide a model for integration of other oncogenic DNA viruses such as papillomaviruses.


Asunto(s)
Carcinoma de Células de Merkel/patología , Reparación del ADN por Unión de Extremidades , Poliomavirus de Células de Merkel/genética , Infecciones por Polyomavirus/complicaciones , Infecciones Tumorales por Virus/complicaciones , Integración Viral , Replicación Viral , Antígenos Virales de Tumores , Neoplasias Óseas/genética , Neoplasias Óseas/secundario , Neoplasias Óseas/virología , Carcinoma de Células de Merkel/genética , Carcinoma de Células de Merkel/virología , Humanos , Infecciones por Polyomavirus/genética , Infecciones por Polyomavirus/virología , Recombinación Genética , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/virología , Infecciones Tumorales por Virus/genética , Infecciones Tumorales por Virus/virología , Proteínas Virales/genética
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