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1.
Cell ; 184(4): 1032-1046.e18, 2021 02 18.
Artículo en Inglés | MEDLINE | ID: mdl-33571428

RESUMEN

Human immunodeficiency virus (HIV-1) remains a major health threat. Viral capsid uncoating and nuclear import of the viral genome are critical for productive infection. The size of the HIV-1 capsid is generally believed to exceed the diameter of the nuclear pore complex (NPC), indicating that capsid uncoating has to occur prior to nuclear import. Here, we combined correlative light and electron microscopy with subtomogram averaging to capture the structural status of reverse transcription-competent HIV-1 complexes in infected T cells. We demonstrated that the diameter of the NPC in cellulo is sufficient for the import of apparently intact, cone-shaped capsids. Subsequent to nuclear import, we detected disrupted and empty capsid fragments, indicating that uncoating of the replication complex occurs by breaking the capsid open, and not by disassembly into individual subunits. Our data directly visualize a key step in HIV-1 replication and enhance our mechanistic understanding of the viral life cycle.


Asunto(s)
Cápside/metabolismo , VIH-1/metabolismo , Poro Nuclear/metabolismo , Transporte Activo de Núcleo Celular , Cápside/ultraestructura , Microscopía por Crioelectrón , Células HEK293 , Infecciones por VIH/virología , VIH-1/ultraestructura , Humanos , Modelos Biológicos , Poro Nuclear/ultraestructura , Poro Nuclear/virología , Transcripción Reversa , Virión/metabolismo , Internalización del Virus , Factores de Escisión y Poliadenilación de ARNm/metabolismo
2.
Nature ; 588(7838): 498-502, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-32805734

RESUMEN

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virions are surrounded by a lipid bilayer from which spike (S) protein trimers protrude1. Heavily glycosylated S trimers bind to the angiotensin-converting enzyme 2 receptor and mediate entry of virions into target cells2-6. S exhibits extensive conformational flexibility: it modulates exposure of its receptor-binding site and subsequently undergoes complete structural rearrangement to drive fusion of viral and cellular membranes2,7,8. The structures and conformations of soluble, overexpressed, purified S proteins have been studied in detail using cryo-electron microscopy2,7,9-12, but the structure and distribution of S on the virion surface remain unknown. Here we applied cryo-electron microscopy and tomography to image intact SARS-CoV-2 virions and determine the high-resolution structure, conformational flexibility and distribution of S trimers in situ on the virion surface. These results reveal the conformations of S on the virion, and provide a basis from which to understand interactions between S and neutralizing antibodies during infection or vaccination.


Asunto(s)
Microscopía por Crioelectrón , SARS-CoV-2/metabolismo , SARS-CoV-2/ultraestructura , Glicoproteína de la Espiga del Coronavirus/análisis , Glicoproteína de la Espiga del Coronavirus/ultraestructura , Virión/química , Virión/ultraestructura , Anticuerpos Neutralizantes/inmunología , COVID-19/inmunología , Vacunas contra la COVID-19/inmunología , Línea Celular Tumoral , Humanos , Modelos Moleculares , Docilidad , Conformación Proteica , Multimerización de Proteína , SARS-CoV-2/química , SARS-CoV-2/aislamiento & purificación , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/aislamiento & purificación , Virión/aislamiento & purificación , Virión/metabolismo
4.
J Virol ; 85(7): 3046-54, 2011 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-21228238

RESUMEN

Murine polyomavirus middle T-antigen (MT) induces tumors by mimicking an activated growth factor receptor. An essential component of this action is a 22-amino-acid hydrophobic region close to the C terminus which locates MT to cell membranes. Here, we demonstrate that this sequence is a transmembrane domain (TMD) by showing that a hemagglutinin (HA) tag added to the MT C terminus is exposed on the outside of the cells, with the N terminus inside. To determine whether this MT TMD is inserted into the endoplasmic reticulum (ER) membrane, we added the ER retention signal KDEL to the MT C terminus (MTKDEL). This mutant protein locates only in the ER, demonstrating that MT does insert into membranes solely at this location. In addition, this ER-located MT failed to transform. Examination of the binding proteins associated with the MTKDEL protein demonstrated that it associates with PP2A and c-Src but fails to interact with ShcA, phosphatidylinositol 3-kinase (PI3K), and phospholipase C-γ1 (PLC-γ1), despite being tyrosine phosphorylated. Additional mutant and antibody studies show that MT binding to PP2A is probably required for MT to efficiently exit the ER and migrate to the plasma membrane though the TMD also plays a role in this relocation. Overall, these data, together with previous publications, illustrate that MT associates with signaling proteins at different sites in its maturation pathway. MT binds to PP2A in the cytoplasm, to c-Src at the endoplasmic reticulum, and to ShcA, PI3K, and PLC-γ1 at subsequent locations en route to the plasma membrane.


Asunto(s)
Antígenos Virales de Tumores/metabolismo , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Poliomavirus/patogenicidad , Transducción de Señal , Animales , Antígenos Virales de Tumores/genética , Proteína Tirosina Quinasa CSK , Línea Celular , Proteínas de la Membrana/genética , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfolipasa C gamma/metabolismo , Unión Proteica , Proteína Fosfatasa 2/metabolismo , Señales de Clasificación de Proteína , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas/metabolismo , Ratas , Proteínas Adaptadoras de la Señalización Shc/metabolismo , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Familia-src Quinasas
5.
Annu Rev Virol ; 9(1): 261-284, 2022 09 29.
Artículo en Inglés | MEDLINE | ID: mdl-35704745

RESUMEN

After cell entry, human immunodeficiency virus type 1 (HIV-1) replication involves reverse transcription of the RNA genome, nuclear import of the subviral complex without nuclear envelope breakdown, and integration of the viral complementary DNA into the host genome. Here, we discuss recent evidence indicating that completion of reverse transcription and viral genome uncoating occur in the nucleus rather than in the cytoplasm, as previously thought, and suggest a testable model for nuclear import and uncoating. Multiple recent studies indicated that the cone-shaped capsid, which encases the genome and replication proteins, not only serves as a reaction container for reverse transcription and as a shield from innate immune sensors but also may constitute the elusive HIV-1 nuclear import factor. Rupture of the capsid may be triggered in the nucleus by completion of reverse transcription, by yet-unknown nuclear factors, or by physical damage, and it appears to occur in close temporal and spatial association with the integration process.


Asunto(s)
Infecciones por VIH , VIH-1 , Cápside/metabolismo , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , ADN Complementario/metabolismo , ADN Viral/metabolismo , VIH-1/genética , VIH-1/metabolismo , Humanos , ARN/metabolismo , Transcripción Reversa , Replicación Viral
6.
mBio ; 13(5): e0195922, 2022 10 26.
Artículo en Inglés | MEDLINE | ID: mdl-35972146

RESUMEN

The cone-shaped mature HIV-1 capsid is the main orchestrator of early viral replication. After cytosolic entry, it transports the viral replication complex along microtubules toward the nucleus. While it was initially believed that the reverse transcribed genome is released from the capsid in the cytosol, recent observations indicate that a high amount of capsid protein (CA) remains associated with subviral complexes during import through the nuclear pore complex (NPC). Observation of postentry events via microscopic detection of HIV-1 CA is challenging, since epitope shielding limits immunodetection and the genetic fragility of CA hampers direct labeling approaches. Here, we present a minimally invasive strategy based on genetic code expansion and click chemistry that allows for site-directed fluorescent labeling of HIV-1 CA, while retaining virus morphology and infectivity. Thereby, we could directly visualize virions and subviral complexes using advanced microscopy, including nanoscopy and correlative imaging. Quantification of signal intensities of subviral complexes revealed an amount of CA associated with nuclear complexes in HeLa-derived cells and primary T cells consistent with a complete capsid and showed that treatment with the small molecule inhibitor PF74 did not result in capsid dissociation from nuclear complexes. Cone-shaped objects detected in the nucleus by electron tomography were clearly identified as capsid-derived structures by correlative microscopy. High-resolution imaging revealed dose-dependent clustering of nuclear capsids, suggesting that incoming particles may follow common entry routes. IMPORTANCE The cone-shaped capsid of HIV-1 has recently been recognized as a master organizer of events from cell entry of the virus to the integration of the viral genome into the host cell DNA. Fluorescent labeling of the capsid is essential to study its role in these dynamic events by microscopy, but viral capsid proteins are extremely challenging targets for the introduction of labels. Here we describe a minimally invasive strategy that allows us to visualize the HIV-1 capsid protein in infected cells by live-cell imaging and superresolution microscopy. Applying this strategy, we confirmed that, contrary to earlier assumptions, an equivalent of a complete capsid can enter the host cell nucleus through nuclear pores. We also observed that entering capsids cluster in the nucleus in a dose-dependent manner, suggesting that they may have followed a common entry route to a site suitable for viral genome release.


Asunto(s)
Seropositividad para VIH , VIH-1 , Humanos , VIH-1/genética , VIH-1/metabolismo , Cápside/metabolismo , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Replicación Viral/genética , Núcleo Celular/metabolismo , Seropositividad para VIH/metabolismo , Código Genético , Epítopos/metabolismo
7.
Elife ; 102021 04 27.
Artículo en Inglés | MEDLINE | ID: mdl-33904396

RESUMEN

HIV-1 replication commences inside the cone-shaped viral capsid, but timing, localization, and mechanism of uncoating are under debate. We adapted a strategy to visualize individual reverse-transcribed HIV-1 cDNA molecules and their association with viral and cellular proteins using fluorescence and correlative-light-and-electron-microscopy (CLEM). We specifically detected HIV-1 cDNA inside nuclei, but not in the cytoplasm. Nuclear cDNA initially co-localized with a fluorescent integrase fusion (IN-FP) and the viral CA (capsid) protein, but cDNA-punctae separated from IN-FP/CA over time. This phenotype was conserved in primary HIV-1 target cells, with nuclear HIV-1 complexes exhibiting strong CA-signals in all cell types. CLEM revealed cone-shaped HIV-1 capsid-like structures and apparently broken capsid-remnants at the position of IN-FP signals and elongated chromatin-like structures in the position of viral cDNA punctae lacking IN-FP. Our data argue for nuclear uncoating by physical disruption rather than cooperative disassembly of the CA-lattice, followed by physical separation from the pre-integration complex.


When viruses infect human cells, they hijack the cell's machinery to produce the proteins they need to replicate. Retroviruses like HIV-1 do this by entering the nucleus and inserting their genetic information into the genome of the infected cell. This requires HIV-1 to convert its genetic material into DNA, which is then released from the protective shell surrounding it (known as the capsid) via a process called uncoating. The nucleus is enclosed within an envelope containing pores that molecules up to a certain size can pass through. Until recently these pores were thought to be smaller than the viral capsid, which led scientists to believe that the HIV-1 genome must shed this coat before penetrating the nucleus. However, recent studies have found evidence for HIV-1 capsid proteins and capsid structures inside the nucleus of some infected cells. This suggests that the capsid may not be removed before nuclear entry or that it may even play a role in helping the virus get inside the nucleus. To investigate this further, Müller et al. attached fluorescent labels to the newly made DNA of HIV-1 and some viral and cellular proteins. Powerful microscopy tools were then used to monitor the uncoating process in various cells that had been infected with the virus. Müller et al. found large amounts of capsid protein inside the nuclei of all the infected cells studied. During the earlier stages of infection, the capsid proteins were mostly associated with viral DNA and the capsid structure appeared largely intact. At later time points, the capsid structure had been broken down and the viral DNA molecules were gradually separating themselves from these remnants. These findings suggest that the HIV-1 capsid helps the virus get inside the nucleus and may protect its genetic material during conversion into DNA until right before integration into the cell's genome. Further experiments studying this process could lead to new therapeutic approaches that target the capsid as a way to prevent or treat HIV-1.


Asunto(s)
Núcleo Celular/virología , Replicación del ADN , ADN Viral/biosíntesis , Infecciones por VIH/virología , VIH-1/crecimiento & desarrollo , Internalización del Virus , Replicación Viral , Desencapsidación Viral , Linfocitos T CD4-Positivos/ultraestructura , Linfocitos T CD4-Positivos/virología , Proteínas de la Cápside/metabolismo , Núcleo Celular/ultraestructura , ADN Viral/genética , ADN Viral/ultraestructura , Células HEK293 , Infecciones por VIH/patología , Transcriptasa Inversa del VIH/metabolismo , VIH-1/enzimología , VIH-1/genética , VIH-1/ultraestructura , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Macrófagos/ultraestructura , Macrófagos/virología , Microscopía Electrónica , Microscopía Fluorescente , Factores de Tiempo
8.
Science ; 373(6555): 700-704, 2021 08 06.
Artículo en Inglés | MEDLINE | ID: mdl-34353956

RESUMEN

Gag, the primary structural protein of HIV-1, is recruited to the plasma membrane for virus assembly by its matrix (MA) domain. Gag is subsequently cleaved into its component domains, causing structural maturation to repurpose the virion for cell entry. We determined the structure and arrangement of MA within immature and mature HIV-1 through cryo-electron tomography. We found that MA rearranges between two different hexameric lattices upon maturation. In mature HIV-1, a lipid extends out of the membrane to bind with a pocket in MA. Our data suggest that proteolytic maturation of HIV-1 not only assembles the viral capsid surrounding the genome but also repurposes the membrane-bound MA lattice for an entry or postentry function and results in the partial removal of up to 2500 lipids from the viral membrane.


Asunto(s)
Antígenos VIH/química , Antígenos VIH/metabolismo , VIH-1/química , VIH-1/fisiología , Envoltura Viral/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/química , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Cápside/química , Cápside/fisiología , Tomografía con Microscopio Electrónico , VIH-1/ultraestructura , Membrana Dobles de Lípidos , Lípidos de la Membrana/metabolismo , Modelos Moleculares , Conformación Proteica , Dominios Proteicos , Estructura Secundaria de Proteína , Envoltura Viral/química , Envoltura Viral/ultraestructura , Virión/química , Virión/fisiología , Virión/ultraestructura , Ensamble de Virus , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismo
9.
Sci Rep ; 10(1): 9035, 2020 Jun 03.
Artículo en Inglés | MEDLINE | ID: mdl-32493963

RESUMEN

Chicxulub impact (66 Ma) event resulted in deposition of spheroids and melt glass, followed by deposition of diamectite and carbonate ejecta represented by large polished striated rounded pebbles and cobbles, henceforth, called Albion Formation1 Pook's Pebbles, name given from the first site identified in central Belize, Cayo District. Here we report that magnetic analysis of the Pook's Pebbles samples revealed unique electric discharge signatures. Sectioning of Pook's Pebbles from the Chicxulub ejecta from the Albion Formation at Belize showed that different parts of Pook's Pebbles had not only contrasting magnetization directions, but also sharply different level of magnetizations. Such behavior is indicative of electric discharge taking place sometimes during the formation of the Chicxulub ejecta blanket. In addition, some of the Pook's Pebbles' surface had recrystallized down to 0.2 mm depth. This is evidence of localized extreme pressures and temperatures during the fluidized ejecta formation which was imprinted in the outer layer of Pook's Pebbles. Recrystallization caused formation of nanophase iron along the surface, which was revealed by mapping of both natural remanent magnetization and of saturation remanence magnetization signatures. While the spheroids' magnetization orientation is consistent with reversed magnetic field at the time of impact, the study of the Pook's Pebbles provided, in addition, new evidence of electric charging during the vapor plume cloud processes.

10.
mBio ; 10(6)2019 11 05.
Artículo en Inglés | MEDLINE | ID: mdl-31690677

RESUMEN

HIV-1 infects host cells by fusion at the plasma membrane, leading to cytoplasmic entry of the viral capsid encasing the genome and replication machinery. The capsid eventually needs to disassemble, but time and location of uncoating are not fully characterized and may vary depending on the host cell. To study the fate of the capsid by fluorescence and superresolution (STED) microscopy, we established an experimental system that allows discrimination of subviral structures in the cytosol from intact virions at the plasma membrane or in endosomes without genetic modification of the virus. Quantitative microscopy of infected SupT1-R5 cells revealed that the CA signal on cytosolic HIV-1 complexes corresponded to ∼50% of that found in virions at the cell surface, in agreement with dissociation of nonassembled CA molecules from entering capsids after membrane fusion. The relative amount of CA in postfusion complexes remained stable until they reached the nuclear pore complex, while subviral structures in the nucleus of infected cells lacked detectable CA. An HIV-1 variant defective in binding of the host protein cleavage and polyadenylation specificity factor 6 (CPSF6) exhibited accumulation of CA-positive subviral complexes close to the nuclear envelope without loss of infectivity; STED microscopy revealed direct association of these complexes with nuclear pores. These results support previous observations indicating capsid uncoating at the nuclear pore in infected T-cell lines. They suggest that largely intact HIV-1 capsids dock at the nuclear pore in infected SupT1-R5 cells, with CPSF6 being a facilitator of nucleoplasmic entry in this cell type, as has been observed for infected macrophages.IMPORTANCE The HIV-1 capsid performs essential functions during early viral replication and is an interesting target for novel antivirals. Thus, understanding molecular and structural details of capsid function will be important for elucidating early HIV-1 (and retroviral in general) replication in relevant target cells and may also aid antiviral development. Here, we show that HIV-1 capsids stay largely intact during transport to the nucleus of infected T cells but appear to uncoat upon entry into the nucleoplasm. These results support the hypothesis that capsids protect the HIV-1 genome from cytoplasmic defense mechanisms and target the genome toward the nucleus. A protective role of the capsid could be a paradigm that also applies to other viruses. Our findings raise the question of how reverse transcription of the HIV-1 genome is accomplished in the context of the capsid structure and whether the process is completed before the capsid is uncoated at the nuclear pore.


Asunto(s)
Infecciones por VIH/genética , VIH-1/genética , Replicación Viral/genética , Factores de Escisión y Poliadenilación de ARNm/genética , Proteínas de la Cápside/genética , Línea Celular , Membrana Celular/genética , Membrana Celular/virología , Núcleo Celular/genética , Núcleo Celular/virología , Citoplasma/genética , Citoplasma/virología , Células HEK293 , Infecciones por VIH/virología , Interacciones Huésped-Patógeno/genética , Humanos , Macrófagos/virología , Linfocitos T/virología
11.
Int J Oncol ; 28(2): 559-65, 2006 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-16391813

RESUMEN

The differentiation of colorectal cancer cells is associated with the arrest of tumor growth and tumor regression. However, the mechanism of such tumor cell differentiation has not yet been elucidated. Several adenocarcinoma cell lines, including HT29 which differentiates only upon stimulation with a differentiation agent, have been used for the study of colorectal cells. Since we had previously obtained variable results during analyses of these cells, we selected several clones of this cell line. In this study, four clones of the parental HT29 cells, H8, G9, G10 and A3, were characterized. All of them differentiated upon treatment with sodium butyrate as the differentiation agent but they appeared different in their response regarding some of the markers of differentiation. As revealed by ultrastructural analysis, H8 and G10 clones formed numerous intercellular cysts with microvilli whereas these structures were found only occasionally in G9 and A3 clones. An elevated level of the indicator of cell differentiation, CEACAM 1, was found in H8 and G10 clones and the activity of alkaline phosphatase, another important marker of colorectal cell differentiation, was up-regulated and highly increased upon butyrate treatment in the H8 clone. Phosphorylation of p38 MAPK was increased in H8 and A3 butyrate-treated clones. According to the levels of cleaved PARP and activated caspase-3, the apoptotic response to butyrate appeared similar in all four clones, while electronoptic analysis revealed that clones G9 and A3 were more perceptive to butyrate-induced apoptosis. In conclusion, our data showed considerable heterogeneity in morphology and some enzymatic activity of the cloned cells. This fact may contribute to the evidence that many HT29 cells possess multipotent information similar to that of stem cells of the normal intestinal crypt.


Asunto(s)
Butiratos/farmacología , Células HT29/patología , Adenocarcinoma , Fosfatasa Alcalina/metabolismo , Antígenos CD/metabolismo , Apoptosis , Moléculas de Adhesión Celular/metabolismo , Diferenciación Celular/efectos de los fármacos , Células Clonales , Neoplasias Colorrectales , Células HT29/efectos de los fármacos , Células HT29/ultraestructura , Humanos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo
12.
FEBS Lett ; 590(13): 1858-76, 2016 07.
Artículo en Inglés | MEDLINE | ID: mdl-27117435

RESUMEN

The recent development of fluorescence microscopy approaches overcoming the diffraction limit of light microscopy opened possibilities for studying small-scale cellular processes. The spatial resolution achieved by these novel techniques, together with the possibility to perform live-cell and multicolor imaging, make them ideally suited for visualization of native viruses and subviral structures within the complex environment of a host cell or organ, thus providing fundamentally new possibilities for investigating virus-cell interactions. Here, we review the use of super-resolution microscopy approaches to study virus-cell interactions, and discuss recent insights into human immunodeficiency virus biology obtained by exploiting these novel techniques.


Asunto(s)
VIH/fisiología , Microscopía Fluorescente/métodos , Humanos , Imagenología Tridimensional , Replicación Viral
13.
Curr Protoc Microbiol ; 38: 14F.1.1-26, 2015 Aug 03.
Artículo en Inglés | MEDLINE | ID: mdl-26237106

RESUMEN

Mouse polyomavirus (MPyV) is a member of the Polyomaviridae family, which comprises non-enveloped tumorigenic viruses infecting various vertebrates including humans and causing different pathogenic responses in the infected organisms. Despite the variations in host tropism and pathogenicity, the structure of the virions of these viruses is similar. The capsid, with icosahedral symmetry (ø, 45 nm, T = 7d), is composed of a shell of 72 capsomeres of structural proteins, arranged around the nucleocore containing approximately 5-kbp-long circular dsDNA in complex with cellular histones. MPyV has been one of the most studied polyomaviruses and serves as a model virus for studies of the mechanisms of cell transformation and virus trafficking, and for use in nanotechnology. It can be propagated in primary mouse cells (e.g., in whole mouse embryo cells) or in mouse epithelial or fibroblast cell lines. In this unit, propagation, purification, quantification, and storage of MPyV virions are presented.


Asunto(s)
Poliomavirus/crecimiento & desarrollo , Poliomavirus/aislamiento & purificación , Preservación Biológica/métodos , Carga Viral/métodos , Cultivo de Virus/métodos , Animales , Células Cultivadas , Ratones
14.
Rejuvenation Res ; 17(2): 226-8, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-24713060

RESUMEN

Pulses up to 11 Tesla magnetic fields may generate pockets of currents along the walls of cellular material and may interfere with the overall ability of cell division. We used prokaryotic cells (Escherichia coli) and eukaryotic cells (murine fibroblasts) and exposed them to magnetic pulses of intensities ranging from 1 millitesla (mT) to 11,000 mT. We found prokaryotic cells to be more sensitive to magnetic field pulses than eukaryotic cells.


Asunto(s)
Electricidad , Campos Electromagnéticos , Escherichia coli/citología , Fibroblastos/citología , Animales , Línea Celular , Proliferación Celular , Ratones
15.
PLoS One ; 9(5): e96922, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24810588

RESUMEN

Infection of non-enveloped polyomaviruses depends on an intact microtubular network. Here we focus on mouse polyomavirus (MPyV). We show that the dynamics of MPyV cytoplasmic transport reflects the characteristics of microtubular motor-driven transport with bi-directional saltatory movements. In cells treated with microtubule-disrupting agents, localization of MPyV was significantly perturbed, the virus was retained at the cell periphery, mostly within membrane structures resembling multicaveolar complexes, and at later times post-infection, only a fraction of the virus was found in Rab7-positive endosomes and multivesicular bodies. Inhibition of cytoplasmic dynein-based motility by overexpression of dynamitin affected perinuclear translocation of the virus, delivery of virions to the ER and substantially reduced the numbers of infected cells, while overexpression of dominant-negative form of kinesin-1 or kinesin-2 had no significant impact on virus localization and infectivity. We also found that transport along microtubules was important for MPyV-containing endosome sequential acquisition of Rab5, Rab7 and Rab11 GTPases. However, in contrast to dominant-negative mutant of Rab7 (T22N), overexpression of dominant-negative mutant Rab11 (S25N) did not affect the virus infectivity. Altogether, our study revealed that MPyV cytoplasmic trafficking leading to productive infection bypasses recycling endosomes, does not require the function of kinesin-1 and kinesin-2, but depends on functional dynein-mediated transport along microtubules for translocation of the virions from peripheral, often caveolin-positive compartments to late endosomes and ER - a prerequisite for efficient delivery of the viral genome to the nucleus.


Asunto(s)
Endocitosis , Proteínas de Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas Motoras Moleculares/metabolismo , Poliomavirus/metabolismo , Animales , Línea Celular , Supervivencia Celular , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/virología , Endosomas/metabolismo , Endosomas/virología , Ratones
16.
FEBS J ; 277(5): 1270-83, 2010 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-20121946

RESUMEN

Minor structural proteins of mouse polyomavirus (MPyV) are essential for virus infection. To study their properties and possible contributions to cell death induction, fusion variants of these proteins, created by linking enhanced green fluorescent protein (EGFP) to their C- or N-termini, were prepared and tested in the absence of other MPyV gene products, namely the tumor antigens and the major capsid protein, VP1. The minor proteins linked to EGFP at their C-terminus (VP2-EGFP, VP3-EGFP) were found to display properties similar to their nonfused, wild-type versions: they killed mouse 3T3 cells quickly when expressed individually. Carrying nuclear localization signals at their common C-terminus, the minor capsid proteins were detected in the nucleus. However, a substantial subpopulation of both VP2 and VP3 proteins, as well as of the fusion proteins VP2-EGFP and VP3-EGFP, was detected in the cytoplasm, co-localizing with intracellular membranes. Truncated VP3 protein, composed of 103 C-terminal amino acids, exhibited reduced affinity for intracellular membranes and cytotoxicity. Biochemical studies proved each of the minor proteins to be a very potent inducer of apoptosis, which was dependent on caspase activation. Immuno-electron microscopy showed the minor proteins to be associated with damaged membranes of the endoplasmic reticulum, nuclear envelope and mitochondria as soon as 5 h post-transfection. Analysis of apoptotic markers and cell death kinetics in cells transfected with the wild-type MPyV genome and the genome mutated in both VP2 and VP3 translation start codons revealed that the minor proteins contribute moderately to apoptotic processes in the late phase of infection and both are dispensable for cell destruction at the end of the virus replication cycle.


Asunto(s)
Apoptosis/efectos de los fármacos , Proteínas de la Cápside/farmacología , Fibroblastos/virología , Infecciones por Polyomavirus/patología , Poliomavirus , Animales , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Línea Celular , Ratones , Plásmidos/genética , Factores de Tiempo
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