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1.
PLoS Pathog ; 20(1): e1011640, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-38215165

RESUMEN

Retroviral reverse transcription starts within the capsid and uncoating and reverse transcription are mutually dependent. There is still debate regarding the timing and cellular location of HIV's uncoating and reverse transcription and whether it occurs solely in the cytoplasm, nucleus or both. HIV can infect non-dividing cells because there is active transport of the preintegration complex (PIC) across the nuclear membrane, but Murine Leukemia Virus (MLV) is thought to depend on cell division for replication and whether MLV uncoating and reverse transcription is solely cytoplasmic has not been studied. Here, we used NIH3T3 and primary mouse dendritic cells to determine where the different stages of reverse transcription occur and whether cell division is needed for nuclear entry. Our data strongly suggest that in both NIH3T3 cells and dendritic cells (DCs), the initial step of reverse transcription occurs in the cytoplasm. However, we detected MLV RNA/DNA hybrid intermediates in the nucleus of dividing NIH3T3 cells and non-dividing DCs, suggesting that reverse transcription can continue after nuclear entry. We also confirmed that the MLV PIC requires cell division to enter the nucleus of NIH3T3 cells. In contrast, we show that MLV can infect non-dividing primary DCs, although integration of MLV DNA in DCs still required the viral p12 protein. Knockdown of several nuclear pore proteins dramatically reduced the appearance of integrated MLV DNA in DCs but not NIH3T3 cells. Additionally, MLV capsid associated with the nuclear pore proteins NUP358 and NUP62 during infection. These findings suggest that simple retroviruses, like the complex retrovirus HIV, gain nuclear entry by traversing the nuclear pore complex in non-mitotic cells.


Asunto(s)
Infecciones por VIH , Proteínas de Complejo Poro Nuclear , Animales , Ratones , Proteínas de Complejo Poro Nuclear/genética , Células 3T3 NIH , Virus de la Leucemia Murina/genética , Proteínas Virales , Proteínas de la Cápside , Retroviridae , ADN , Células Dendríticas
2.
PLoS Pathog ; 17(6): e1009662, 2021 06.
Artículo en Inglés | MEDLINE | ID: mdl-34097709

RESUMEN

Signal-regulatory protein alpha (SIRPA) is a well-known inhibitor of phagocytosis when it complexes with CD47 expressed on target cells. Here we show that SIRPA decreased in vitro infection by a number of pathogenic viruses, including New World and Old World arenaviruses, Zika virus, vesicular stomatitis virus and pseudoviruses bearing the Machupo virus, Ebola virus and SARS-CoV-2 glycoproteins, but not HSV-1, MLV or mNoV. Moreover, mice with targeted mutation of the Sirpa gene that renders it non-functional were more susceptible to infection with the New World arenaviruses Junín virus vaccine strain Candid 1 and Tacaribe virus, but not MLV or mNoV. All SIRPA-inhibited viruses have in common the requirement for trafficking to a low pH endosomal compartment. This was clearly demonstrated with SARS-CoV-2 pseudovirus, which was only inhibited by SIRPA in cells in which it required trafficking to the endosome. Similar to its role in phagocytosis inhibition, SIRPA decreased virus internalization but not binding to cell surface receptors. We also found that increasing SIRPA levels via treatment with IL-4 led to even greater anti-viral activity. These data suggest that enhancing SIRPA's activity could be a target for anti-viral therapies.


Asunto(s)
Endocitosis , Virus ARN/inmunología , Receptores Inmunológicos/fisiología , Internalización del Virus , Animales , Antivirales/farmacología , Línea Celular , Membrana Celular/virología , Chlorocebus aethiops , Sistemas de Liberación de Medicamentos , Integrinas/inmunología , Interleucina-4/farmacología , Ratones , Ratones Noqueados , Dominios Proteicos , Receptores Inmunológicos/genética , Células Vero
3.
Proc Natl Acad Sci U S A ; 117(32): 19497-19506, 2020 08 11.
Artículo en Inglés | MEDLINE | ID: mdl-32719120

RESUMEN

Understanding the genetics of susceptibility to infectious agents is of great importance to our ability to combat disease. Here, we show that voltage-gated calcium channels (VGCCs) are critical for cellular binding and entry of the New World arenaviruses Junín and Tacaribe virus, suggesting that zoonosis via these receptors could occur. Moreover, we demonstrate that α1s haploinsufficiency renders cells and mice more resistant to infection by these viruses. In addition to being more resistant to infection, haploinsufficient cells and mice required a lower dosage of VGCC antagonists to block infection. These studies underscore the importance of genetic variation in susceptibility to both viruses and pharmaceutics.


Asunto(s)
Infecciones por Arenaviridae/genética , Canales de Calcio Tipo L/genética , Resistencia a la Enfermedad/genética , Animales , Infecciones por Arenaviridae/tratamiento farmacológico , Arenavirus del Nuevo Mundo/fisiología , Agonistas de los Canales de Calcio/farmacología , Agonistas de los Canales de Calcio/uso terapéutico , Bloqueadores de los Canales de Calcio/farmacología , Bloqueadores de los Canales de Calcio/uso terapéutico , Canales de Calcio Tipo L/deficiencia , Canales de Calcio Tipo L/metabolismo , Células Cultivadas , Relación Dosis-Respuesta a Droga , Heterocigoto , Humanos , Ratones , Ratones Mutantes , Mutación , Acoplamiento Viral/efectos de los fármacos , Internalización del Virus/efectos de los fármacos
4.
J Virol ; 95(22): e0124421, 2021 10 27.
Artículo en Inglés | MEDLINE | ID: mdl-34468176

RESUMEN

Apolipoprotein B mRNA editing enzyme catalytic subunit 3 (APOBEC3) proteins are critical for the control of infection by retroviruses. These proteins deaminate cytidines in negative-strand DNA during reverse transcription, leading to G-to-A changes in coding strands. Uracil DNA glycosylase (UNG) is a host enzyme that excises uracils in genomic DNA, which the base excision repair machinery then repairs. Whether UNG removes uracils found in retroviral DNA after APOBEC3-mediated mutation is not clear, and whether this occurs in vivo has not been demonstrated. To determine if UNG plays a role in the repair of retroviral DNA, we used APOBEC3G (A3G) transgenic mice which we showed previously had extensive deamination of murine leukemia virus (MLV) proviruses. The A3G transgene was crossed onto an Ung and mouse Apobec3 knockout background (UNG-/-APO-/-), and the mice were infected with MLV. We found that virus infection levels were decreased in A3G UNG-/-APO-/- compared with A3G APO-/- mice. Deep sequencing of the proviruses showed that there were significantly higher levels of G-to-A mutations in proviral DNA from A3G transgenic UNG-/-APO-/- than A3G transgenic APO-/- mice, suggesting that UNG plays a role in the repair of uracil-containing proviruses. In in vitro studies, we found that cytoplasmic viral DNA deaminated by APOBEC3G was uracilated. In the absence of UNG, the uracil-containing proviruses integrated at higher levels into the genome than those made in the presence of UNG. Thus, UNG also functions in the nucleus prior to integration by nicking uracil-containing viral DNA, thereby blocking integration. These data show that UNG plays a critical role in the repair of the damage inflicted by APOBEC3 deamination of reverse-transcribed DNA. IMPORTANCE While APOBEC3-mediated mutation of retroviruses is well-established, what role the host base excision repair enzymes play in correcting these mutations is not clear. This question is especially difficult to address in vivo. Here, we use a transgenic mouse developed by our lab that expresses human APOBEC3G and also lacks the endogenous uracil DNA glycosylase (Ung) gene and show that UNG removes uracils introduced by this cytidine deaminase in MLV reverse transcripts, thereby reducing G-to-A mutations in proviruses. Furthermore, our data suggest that UNG removes uracils at two stages in infection-first, in unintegrated nuclear viral reverse-transcribed DNA, resulting in its degradation; and second, in integrated proviruses, resulting in their repair. These data suggest that retroviruses damaged by host cytidine deaminases take advantage of the host DNA repair system to overcome this damage.


Asunto(s)
Desaminasa APOBEC-3G/inmunología , ADN Viral/inmunología , Infecciones por Retroviridae , Retroviridae , Uracil-ADN Glicosidasa/inmunología , Animales , Reparación del ADN , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Células 3T3 NIH , Retroviridae/genética , Retroviridae/inmunología , Infecciones por Retroviridae/inmunología , Infecciones por Retroviridae/virología
5.
PLoS Biol ; 17(2): e3000137, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30726215

RESUMEN

Tripartite motif (TRIM) proteins belong to a large family with many roles in host biology, including restricting virus infection. Here, we found that TRIM2, which has been implicated in cases of Charcot-Marie-Tooth disease (CMTD) in humans, acts by blocking hemorrhagic fever New World arenavirus (NWA) entry into cells. We show that Trim2-knockout mice, as well as primary fibroblasts from a CMTD patient with mutations in TRIM2, are more highly infected by the NWAs Junín and Tacaribe virus than wild-type mice or cells are. Using mice with different Trim2 gene deletions and TRIM2 mutant constructs, we demonstrate that its antiviral activity is uniquely independent of the RING domain encoding ubiquitin ligase activity. Finally, we show that one member of the TRIM2 interactome, signal regulatory protein α (SIRPA), a known inhibitor of phagocytosis, also restricts NWA infection and conversely that TRIM2 limits phagocytosis of apoptotic cells. In addition to demonstrating a novel antiviral mechanism for TRIM proteins, these studies suggest that the NWA entry and phagocytosis pathways overlap.


Asunto(s)
Antígenos de Diferenciación/genética , Arenavirus del Nuevo Mundo/genética , Enfermedad de Charcot-Marie-Tooth/genética , Interacciones Huésped-Patógeno/genética , Proteínas Nucleares/genética , Receptores Inmunológicos/genética , Animales , Antígenos de Diferenciación/inmunología , Antígenos de Diferenciación/metabolismo , Apoptosis , Arenavirus del Nuevo Mundo/crecimiento & desarrollo , Arenavirus del Nuevo Mundo/patogenicidad , Encéfalo/inmunología , Encéfalo/metabolismo , Encéfalo/virología , Línea Celular Tumoral , Enfermedad de Charcot-Marie-Tooth/metabolismo , Enfermedad de Charcot-Marie-Tooth/patología , Chlorocebus aethiops , Fibroblastos/inmunología , Fibroblastos/metabolismo , Fibroblastos/virología , Regulación de la Expresión Génica , Células HEK293 , Interacciones Huésped-Patógeno/inmunología , Humanos , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/virología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/inmunología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/inmunología , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas de Neurofilamentos/genética , Proteínas de Neurofilamentos/inmunología , Proteínas de Neurofilamentos/metabolismo , Proteínas Nucleares/inmunología , Proteínas Nucleares/metabolismo , Osteoblastos/inmunología , Osteoblastos/metabolismo , Osteoblastos/virología , Cultivo Primario de Células , Receptores Inmunológicos/inmunología , Receptores Inmunológicos/metabolismo , Transducción de Señal , Células Vero , Internalización del Virus
6.
J Virol ; 94(18)2020 08 31.
Artículo en Inglés | MEDLINE | ID: mdl-32641479

RESUMEN

Apolipoprotein B editing enzyme, catalytic polypeptide 3 (APOBEC3) family members are cytidine deaminases that play important roles in intrinsic responses to retrovirus infection. Complex retroviruses like human immunodeficiency virus type 1 (HIV-1) encode the viral infectivity factor (Vif) protein to counteract APOBEC3 proteins. Vif induces degradation of APOBEC3G and other APOBEC3 proteins and thereby prevents their packaging into virions. It is not known if murine leukemia virus (MLV) encodes a Vif-like protein. Here, we show that the MLV P50 protein, produced from an alternatively spliced gag RNA, interacts with the C terminus of mouse APOBEC3 and prevents its packaging without causing its degradation. By infecting APOBEC3 knockout (KO) and wild-type (WT) mice with Friend or Moloney MLV P50-deficient viruses, we found that APOBEC3 restricts the mutant viruses more than WT viruses in vivo Replication of P50-mutant viruses in an APOBEC3-expressing stable cell line was also much slower than that of WT viruses, and overexpressing P50 in this cell line enhanced mutant virus replication. Thus, MLV encodes a protein, P50, that overcomes APOBEC3 restriction by preventing its packaging into virions.IMPORTANCE MLV has existed in mice for at least a million years, in spite of the existence of host restriction factors that block infection. Although MLV is considered a simple retrovirus compared to lentiviruses, it does encode proteins generated from alternatively spliced RNAs. Here, we show that P50, generated from an alternatively spliced RNA encoded in gag, counteracts APOBEC3 by blocking its packaging. MLV also encodes a protein, glycoGag, that increases capsid stability and limits APOBEC3 access to the reverse transcription complex (RTC). Thus, MLV has evolved multiple means of preventing APOBEC3 from blocking infection, explaining its survival as an infectious pathogen in mice.


Asunto(s)
Citidina Desaminasa/genética , Regulación Viral de la Expresión Génica , Productos del Gen gag/genética , Leucemia Experimental/genética , Virus de la Leucemia Murina de Moloney/genética , Infecciones por Retroviridae/genética , Infecciones Tumorales por Virus/genética , Empalme Alternativo , Animales , Cápside/metabolismo , Citidina Desaminasa/deficiencia , Productos del Gen gag/metabolismo , Células HEK293 , Interacciones Huésped-Patógeno/genética , Humanos , Leucemia Experimental/metabolismo , Leucemia Experimental/virología , Ratones , Ratones Noqueados , Virus de la Leucemia Murina de Moloney/metabolismo , Virus de la Leucemia Murina de Moloney/patogenicidad , Células 3T3 NIH , Infecciones por Retroviridae/metabolismo , Infecciones por Retroviridae/virología , Transducción de Señal , Infecciones Tumorales por Virus/metabolismo , Infecciones Tumorales por Virus/virología , Virión/genética , Virión/metabolismo , Virión/patogenicidad , Replicación Viral
7.
Neurobiol Dis ; 140: 104845, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32205255

RESUMEN

We analyzed Trim2A/A mice, generated by CRISPR-Cas9, which have a recessive, null mutation of Trim2. Trim2A/A mice develop ataxia that is associated with a severe loss of cerebellar Purkinje cells and a peripheral neuropathy. Myelinated axons in the CNS, including those in the deep cerebellar nuclei, have focal enlargements that contain mitochondria and neurofilaments. In the PNS, there is a loss of myelinated axons, particularly in the most distal nerves. The pathologically affected neuronal populations - primary sensory and motor neurons as well as cerebellar Purkinje cells - express TRIM2, suggesting that loss of TRIM2 in these neurons results in cell autonomous effects on their axons. In contrast, these pathological findings were not found in a second strain of Trim2 mutant mice (Trim2C/C), which has a partial deletion in the RING domain that is needed for ubiquitin ligase activity. Both the Trim2Aand the Trim2C alleles encode mutant TRIM2 proteins with reduced ubiquitination activity. In sum, Trim2A/A mice are a genetically authentic animal model of a recessive axonal neuropathy of humans, apparently for a function that does not depend on the ubiquitin ligase activity.


Asunto(s)
Axones/patología , Enfermedad de Charcot-Marie-Tooth/genética , Mutación , Proteínas de Motivos Tripartitos/genética , Ubiquitina-Proteína Ligasas/genética , Animales , Filamentos Intermedios/metabolismo , Ratones , Neuronas Motoras/patología
8.
J Virol ; 93(20)2019 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-31341050

RESUMEN

Endogenous retroviruses (ERV) are found throughout vertebrate genomes, and failure to silence their activation can have deleterious consequences on the host. Mutation and subsequent disruption of ERV loci is therefore an indispensable component of the cell-intrinsic defenses that maintain the integrity of the host genome. Abundant in vitro and in silico evidence have revealed that APOBEC3 cytidine-deaminases, including human APOBEC3G (hA3G), can potently restrict retrotransposition; yet, in vivo data demonstrating such activity is lacking, since no replication-competent human ERV have been identified. In mice deficient for Toll-like receptor 7 (TLR7), transcribed ERV loci can recombine and generate infectious ERV. In this study, we show that ectopic expression of hA3G can prevent the emergence of replication-competent, infectious ERV in Tlr7-/- mice. Mice encode one copy of Apobec3 in their genome. ERV reactivation in Tlr7-/- mice was comparable in the presence or absence of Apobec3 In contrast, expression of a human APOBEC3G transgene abrogated emergence of infectious ERV in the Tlr7-/- background. No ERV RNA was detected in the plasma of hA3G+Apobec3-/-Tlr7-/- mice, and infectious ERV virions could not be amplified through coculture with permissive cells. These data reveal that hA3G can potently restrict active ERV in vivo and suggest that expansion of the APOBEC3 locus in primates may have helped to provide for the continued restraint of ERV in the human genome.IMPORTANCE Although APOBEC3 proteins are known to be important antiviral restriction factors in both mice and humans, their roles in the restriction of endogenous retroviruses (ERV) have been limited to in vitro studies. Here, we report that human APOBEC3G expressed as a transgene in mice prevents the emergence of infectious ERV from endogenous loci. This study reveals that APOBEC3G can powerfully restrict active retrotransposons in vivo and demonstrates how transgenic mice can be used to investigate host mechanisms that inhibit retrotransposons and reinforce genomic integrity.


Asunto(s)
Desaminasa APOBEC-3G/metabolismo , Retrovirus Endógenos/fisiología , Infecciones por Retroviridae/metabolismo , Infecciones por Retroviridae/virología , Replicación Viral , Animales , Dosificación de Gen , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Inmunidad Innata , Ratones , Ratones Noqueados , Sistemas de Lectura Abierta , Infecciones por Retroviridae/inmunología , Receptores Toll-Like/metabolismo
9.
J Virol ; 92(11)2018 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-29593034

RESUMEN

The apolipoprotein B editing complex 3 (APOBEC3) proteins are potent retroviral restriction factors that are under strong positive selection, both in terms of gene copy number and sequence diversity. A common feature of all the members of the APOBEC3 family is the presence of one or two cytidine deamination domains, essential for cytidine deamination of retroviral reverse transcripts as well as packaging into virions. Several studies have indicated that human and mouse APOBEC3 proteins restrict retrovirus infection via cytidine deaminase (CD)-dependent and -independent means. To understand the relative contribution of CD-independent restriction in vivo, we created strains of transgenic mice on an APOBEC3 knockout background that express a deaminase-dead mouse APOBEC3 due to point mutations in both CD domains (E73Q/E253Q). Here, we show that the CD-dead APOBEC3 can restrict murine retroviruses in vivo Moreover, unlike the wild-type protein, the mutant APOBEC3 is not packaged into virions but acts only as a cell-intrinsic restriction factor that blocks reverse transcription by incoming viruses. Finally, we show that wild-type and CD-dead mouse APOBEC3 can bind to murine leukemia virus (MLV) reverse transcriptase. Our findings suggest that the mouse APOBEC3 cytidine deaminase activity is not required for retrovirus restriction.IMPORTANCE APOBEC3 proteins are important host cellular restriction factors essential for restricting retrovirus infection by causing mutations in the virus genome and by blocking reverse transcription. While both methods of restriction function in vitro, little is known about their role during in vivo infection. By developing transgenic mice with mutations in the cytidine deamination domains needed for enzymatic activity and interaction with viral RNA, we show that APOBEC3 proteins can still restrict in vivo infection by interacting with reverse transcriptase and blocking its activity. These studies demonstrate that APOBEC3 proteins have evolved multiple means for blocking retrovirus infection and that all of these means function in vivo.


Asunto(s)
Citidina Desaminasa/genética , Virus de la Leucemia Murina/genética , Infecciones por Retroviridae/prevención & control , Inhibidores de la Transcriptasa Inversa/metabolismo , Transcripción Reversa/genética , Animales , Línea Celular , Citidina Desaminasa/metabolismo , Desaminación/genética , Células HEK293 , Humanos , Virus de la Leucemia Murina/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , ARN Viral/genética , ADN Polimerasa Dirigida por ARN/genética
11.
J Virol ; 90(17): 8005-12, 2016 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-27356895

RESUMEN

UNLABELLED: APOBEC3 knockout and human APOBEC3A and -3G transgenic mice were tested for their ability to be infected by the herpesviruses herpes simplex virus 1 and murine herpesvirus 68 and the parvovirus minute virus of mice (MVM). Knockout, APOBEC3A and APOBEC3G transgenic, and wild-type mice were equally infected by the herpesviruses, while APOBEC3A but not mouse APOBEC3 conferred resistance to MVM. No viruses showed evidence of cytidine deamination by mouse or human APOBEC3s. These data suggest that in vitro studies implicating APOBEC3 proteins in virus resistance may not reflect their role in vivo IMPORTANCE: It is well established that APOBEC3 proteins in different species are a critical component of the host antiretroviral defense. Whether these proteins also function to inhibit other viruses is not clear. There have been a number of in vitro studies suggesting that different APOBEC3 proteins restrict herpesviruses and parvoviruses, among others, but whether they also work in vivo has not been demonstrated. Our studies looking at the role of mouse and human APOBEC3 proteins in transgenic and knockout mouse models of viral infection suggest that these restriction factors are not broadly antiviral and demonstrate the importance of testing their activity in vivo.


Asunto(s)
Desaminasa APOBEC-3G/metabolismo , Citidina Desaminasa/metabolismo , Infecciones por Herpesviridae/inmunología , Infecciones por Parvoviridae/inmunología , Proteínas/metabolismo , Animales , Modelos Animales de Enfermedad , Resistencia a la Enfermedad , Herpesvirus Humano 1/inmunología , Ratones , Ratones Noqueados , Ratones Transgénicos , Virus Diminuto del Ratón/inmunología , Rhadinovirus/inmunología
12.
J Virol ; 90(9): 4494-4510, 2016 May.
Artículo en Inglés | MEDLINE | ID: mdl-26912630

RESUMEN

UNLABELLED: Virus entry into cells is a multistep process that often requires the subversion of subcellular machineries. A more complete understanding of these steps is necessary to develop new antiviral strategies. While studying the potential role of the actin network and one of its master regulators, the small GTPase Cdc42, during Junin virus (JUNV) entry, we serendipitously uncovered the small molecule ZCL278, reported to inhibit Cdc42 function as an entry inhibitor for JUNV and for vesicular stomatitis virus, lymphocytic choriomeningitis virus, and dengue virus but not for the nonenveloped poliovirus. Although ZCL278 did not interfere with JUNV attachment to the cell surface or virus particle internalization into host cells, it prevented the release of JUNV ribonucleoprotein cores into the cytosol and decreased pH-mediated viral fusion with host membranes. We also identified SVG-A astroglial cell-derived cells to be highly permissive for JUNV infection and generated new cell lines expressing fluorescently tagged Rab5c or Rab7a or lacking Cdc42 using clustered regularly interspaced short palindromic repeat (CRISPR)-caspase 9 (Cas9) gene-editing strategies. Aided by these tools, we uncovered that perturbations in the actin cytoskeleton or Cdc42 activity minimally affect JUNV entry, suggesting that the inhibitory effect of ZCL278 is not mediated by ZCL278 interfering with the activity of Cdc42. Instead, ZCL278 appears to redistribute viral particles from endosomal to lysosomal compartments. ZCL278 also inhibited JUNV replication in a mouse model, and no toxicity was detected. Together, our data suggest the unexpected antiviral activity of ZCL278 and highlight its potential for use in the development of valuable new tools to study the intracellular trafficking of pathogens. IMPORTANCE: The Junin virus is responsible for outbreaks of Argentine hemorrhagic fever in South America, where 5 million people are at risk. Limited options are currently available to treat infections by Junin virus or other viruses of the Arenaviridae, making the identification of additional tools, including small-molecule inhibitors, of great importance. How Junin virus enters cells is not yet fully understood. Here we describe new cell culture models in which the cells are susceptible to Junin virus infection and to which we applied CRISPR-Cas9 genome engineering strategies to help characterize early steps during virus entry. We also uncovered ZCL278 to be a new antiviral small molecule that potently inhibits the cellular entry of the Junin virus and other enveloped viruses. Moreover, we show that ZCL278 also functions in vivo, thereby preventing Junin virus replication in a mouse model, opening the possibility for the discovery of ZCL278 derivatives of therapeutic potential.


Asunto(s)
Antivirales/farmacología , Benzamidas/farmacología , Descubrimiento de Drogas , Tiourea/análogos & derivados , Internalización del Virus/efectos de los fármacos , Actinas/metabolismo , Animales , Línea Celular , Células Cultivadas , Clatrina/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Endocitosis/efectos de los fármacos , Endosomas/efectos de los fármacos , Endosomas/virología , Técnicas de Inactivación de Genes , Fiebre Hemorrágica Americana/genética , Fiebre Hemorrágica Americana/metabolismo , Fiebre Hemorrágica Americana/virología , Humanos , Virus Junin/efectos de los fármacos , Virus Junin/fisiología , Ratones , Unión Proteica , Transporte de Proteínas , Proteolisis , Ribonucleoproteínas/metabolismo , Tiourea/farmacología , Carga Viral , Proteínas Virales/metabolismo , Acoplamiento Viral/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismo
13.
J Immunol ; 195(10): 4565-70, 2015 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-26546688

RESUMEN

Apolipoprotein B editing complex 3 family members are cytidine deaminases that play important roles in intrinsic responses to infection by retroviruses and have been implicated in the control of other viruses, such as parvoviruses, herpesviruses, papillomaviruses, hepatitis B virus, and retrotransposons. Although their direct effect on modification of viral DNA has been clearly demonstrated, whether they play additional roles in innate and adaptive immunity to viruses is less clear. We review the data regarding the various steps in the innate and adaptive immune response to virus infection in which apolipoprotein B editing complex 3 proteins have been implicated.


Asunto(s)
Citosina Desaminasa/inmunología , ADN Viral/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Virosis/inmunología , Desaminasas APOBEC , Inmunidad Adaptativa/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Citidina Desaminasa , Citosina Desaminasa/genética , ADN Viral/genética , Infecciones por VIH/virología , Hepatitis B/inmunología , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/virología , Humanos , Infecciones por Papillomavirus/inmunología , Infecciones por Parvoviridae/inmunología , Infecciones por Parvoviridae/virología
14.
Retrovirology ; 13(1): 45, 2016 06 30.
Artículo en Inglés | MEDLINE | ID: mdl-27363431

RESUMEN

Humans encode seven APOBEC3 proteins (A-H), with A3G, 3F and 3H as the major factors restricting HIV-1 replication. HIV-1, however, encodes Vif, which counteracts A3 proteins by chaperoning them to the proteasome where they are degraded. Vif polymorphisms found in HIV-1s isolated from infected patients have varying anti-A3G potency when assayed in vitro, but there are few studies demonstrating this in in vivo models. Here, we created Friend murine leukemia viruses encoding vif alleles that were previously shown to differentially neutralize A3G in cell culture or that were originally found in primary HIV-1 isolates. Infection of transgenic mice expressing different levels of human A3G showed that these naturally occurring Vif variants differed in their ability to counteract A3G during in vivo infection, although the effects on viral replication were not identical to those seen in cultured cells. We also found that the polymorphic Vifs that attenuated viral replication reverted to wild type only in A3G transgenic mice. Finally, we found that the level of A3G-mediated deamination was inversely correlated with the level of viral replication. This animal model should be useful for studying the functional significance of naturally occurring vif polymorphisms, as well as viral evolution in the presence of A3G.


Asunto(s)
Desaminasa APOBEC-3G/metabolismo , Infecciones por VIH/virología , VIH-1/genética , Polimorfismo Genético , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/genética , Desaminasa APOBEC-3G/genética , Alelos , Animales , Modelos Animales de Enfermedad , Virus de la Leucemia Murina de Friend/genética , Virus de la Leucemia Murina de Friend/fisiología , Humanos , Ratones , Ratones Transgénicos , Mutación , Replicación Viral
15.
PLoS Pathog ; 10(5): e1004145, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24851906

RESUMEN

The apolipoprotein B editing complex 3 (A3) cytidine deaminases are among the most highly evolutionarily selected retroviral restriction factors, both in terms of gene copy number and sequence diversity. Primate genomes encode seven A3 genes, and while A3F and 3G are widely recognized as important in the restriction of HIV, the role of the other genes, particularly A3A, is not as clear. Indeed, since human cells can express multiple A3 genes, and because of the lack of an experimentally tractable model, it is difficult to dissect the individual contribution of each gene to virus restriction in vivo. To overcome this problem, we generated human A3A and A3G transgenic mice on a mouse A3 knockout background. Using these mice, we demonstrate that both A3A and A3G restrict infection by murine retroviruses but by different mechanisms: A3G was packaged into virions and caused extensive deamination of the retrovirus genomes while A3A was not packaged and instead restricted infection when expressed in target cells. Additionally, we show that a murine leukemia virus engineered to express HIV Vif overcame the A3G-mediated restriction, thereby creating a novel model for studying the interaction between these proteins. We have thus developed an in vivo system for understanding how human A3 proteins use different modes of restriction, as well as a means for testing therapies that disrupt HIV Vif-A3G interactions.


Asunto(s)
Citidina Desaminasa/fisiología , Proteínas/fisiología , Infecciones por Retroviridae/genética , Infecciones por Retroviridae/virología , Carga Viral/genética , Desaminasa APOBEC-3G , Animales , Células Cultivadas , VIH-1/fisiología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células 3T3 NIH , Retroviridae/fisiología , Ensamble de Virus/genética , Internalización del Virus , Replicación Viral/genética , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/genética
16.
Proc Natl Acad Sci U S A ; 110(22): 9078-83, 2013 May 28.
Artículo en Inglés | MEDLINE | ID: mdl-23671100

RESUMEN

Pathogenic retroviruses have evolved multiple means for evading host restriction factors such as apolipoprotein B editing complex (APOBEC3) proteins. Here, we show that murine leukemia virus (MLV) has a unique means of counteracting APOBEC3 and other cytosolic sensors of viral nucleic acid. Using virus isolated from infected WT and APOBEC3 KO mice, we demonstrate that the MLV glycosylated Gag protein (glyco-Gag) enhances viral core stability. Moreover, in vitro endogenous reverse transcription reactions of the glyco-Gag mutant virus were substantially inhibited compared with WT virus, but only in the presence of APOBEC3. Thus, glyco-Gag rendered the reverse transcription complex in the viral core resistant to APOBEC3. Glyco-Gag in the virion also rendered MLV resistant to other cytosolic sensors of viral reverse transcription products in newly infected cells. Strikingly, glyco-Gag mutant virus reverted to glyco-Gag-containing virus only in WT and not APOBEC3 KO mice, indicating that counteracting APOBEC3 is the major function of glyco-Gag. Thus, in contrast to the HIV viral infectivity factor protein, which prevents APOBEC3 packaging in the virion, the MLV glyco-Gag protein uses a unique mechanism to counteract the antiviral action of APOBEC3 in vivo--namely, protecting the reverse transcription complex in viral cores from APOBEC3. These data suggest that capsid integrity may play a critical role in virus resistance to intrinsic cellular antiviral resistance factors that act at the early stages of infection.


Asunto(s)
Citidina Desaminasa/antagonistas & inhibidores , Productos del Gen gag/metabolismo , Interacciones Huésped-Patógeno/fisiología , Virus de la Leucemia Murina/fisiología , Transcripción Reversa/fisiología , Animales , Western Blotting , Citidina Desaminasa/genética , Citidina Desaminasa/metabolismo , Cartilla de ADN/genética , Productos del Gen gag/farmacología , Glicosilación , Virus de la Leucemia Murina/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Células 3T3 NIH , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
17.
18.
J Virol ; 88(14): 7703-14, 2014 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-24760892

RESUMEN

Successful adaptive immunity to virus infection often depends on the initial innate response. Previously, we demonstrated that Junín virus, the etiological agent responsible for Argentine hemorrhagic fever (AHF), activates an early innate immune response via an interaction between the viral glycoprotein and Toll-like receptor 2 (TLR2). Here we show that TLR2/6 but not TLR1/2 heterodimers sense Junín virus glycoprotein and induce a cytokine response, which in turn upregulates the expression of the RNA helicases RIG-I and MDA5. NF-κB and Erk1/2 were important in the cytokine response, since both proteins were phosphorylated as a result of the interaction of virus with TLR2, and treatment with an Erk1/2-specific inhibitor blocked cytokine production. We show that the Junín virus glycoprotein activates cytokine production in a human macrophage cell line as well. Moreover, we show that TLR2-mediated immune response plays a role in viral clearance because wild-type mice cleared Candid 1 (JUNV C1), the vaccine strain of Junín virus, more rapidly than did TLR2 knockout mice. This clearance correlated with the generation of Junín virus-specific CD8(+) T cells. However, infected wild-type and TLR2 knockout mice developed TLR2-independent blocking antibody responses with similar kinetics. We also show that microglia and astrocytes but not neurons are susceptible to infection with JUNV C1. Although JUNV C1 infection of the brain also triggered a TLR2-dependent cytokine response, virus levels were equivalent in wild-type and TLR2 knockout mice. Importance: Junín virus is transmitted by rodents native to Argentina and is associated with both systemic disease and, in some patients, neurological symptoms. Humans become infected when they inhale aerosolized Junín virus. AHF has a 15 to 30% mortality rate, and patients who clear the infection develop a strong antibody response to Junín virus. Here we investigated what factors determine the immune response to Junín virus. We show that a strong initial innate immune response to JUNV C1 determines how quickly mice can clear systemic infection and that this depended on the cellular immune response. In contrast, induction of an innate immune response in the brain had no effect on virus infection levels. These findings may explain how the initial immune response to Junín virus infection could determine different outcomes in humans.


Asunto(s)
Inmunidad Adaptativa , Sangre/inmunología , Encéfalo/inmunología , Inmunidad Innata , Virus Junin/inmunología , Receptor Toll-Like 2/inmunología , Replicación Viral , Animales , Sangre/virología , Encéfalo/virología , Línea Celular , Citocinas/metabolismo , Humanos , Virus Junin/fisiología , Macrófagos/inmunología , Macrófagos/virología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados
20.
J Virol ; 87(9): 4808-17, 2013 May.
Artículo en Inglés | MEDLINE | ID: mdl-23449789

RESUMEN

The apolipoprotein B editing complex 3 (APOBEC3) family of proteins is a group of intrinsic antiviral factors active against a number of retroviral pathogens, including HIV in humans and mouse mammary tumor virus (MMTV) in mice. APOBEC3 restricts its viral targets through cytidine deamination of viral DNA during reverse transcription or via deaminase-independent means. Here, we used virions from the mammary tissue of MMTV-infected inbred wild-type mice with different allelic APOBEC3 variants (APOBEC3(BALB) and APOBEC3(BL/6)) and knockout mice to determine whether cytidine deamination was important for APOBEC3's anti-MMTV activity. First, using anti-murine APOBEC3 antiserum, we showed that both APOBEC3 allelic variants are packaged into the cores of milk-borne virions produced in vivo. Next, using an in vitro deamination assay, we determined that virion-packaged APOBEC3 retains its deamination activity and that allelic differences in APOBEC3 affect the sequence specificity. In spite of this in vitro activity, cytidine deamination by virion-packaged APOBEC3 of MMTV early reverse transcription DNA occurred only at low levels. Instead, the major means by which in vivo virion-packaged APOBEC3 restricted virus was through inhibition of early reverse transcription in both cell-free virions and in vitro infection assays. Moreover, the different wild-type alleles varied in their ability to inhibit this step. Our data suggest that while APOBEC3-mediated cytidine deamination of MMTV may occur, it is not the major means by which APOBEC3 restricts MMTV infection in vivo. This may reflect the long-term coexistence of MMTV and APOBEC3 in mice.


Asunto(s)
Citidina Desaminasa/metabolismo , Citidina/metabolismo , Virus del Tumor Mamario del Ratón/genética , Virus del Tumor Mamario del Ratón/metabolismo , Infecciones por Retroviridae/enzimología , Infecciones por Retroviridae/virología , Transcripción Reversa , Infecciones Tumorales por Virus/virología , Animales , Citidina Desaminasa/genética , Desaminación , Regulación hacia Abajo , Femenino , Regulación Viral de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infecciones por Retroviridae/genética , Infecciones por Retroviridae/metabolismo , Infecciones Tumorales por Virus/enzimología , Infecciones Tumorales por Virus/genética , Infecciones Tumorales por Virus/metabolismo , Ensamble de Virus
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