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1.
Cell ; 184(13): 3426-3437.e8, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-33991487

RESUMO

We identified an emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant by viral whole-genome sequencing of 2,172 nasal/nasopharyngeal swab samples from 44 counties in California, a state in the western United States. Named B.1.427/B.1.429 to denote its two lineages, the variant emerged in May 2020 and increased from 0% to >50% of sequenced cases from September 2020 to January 2021, showing 18.6%-24% increased transmissibility relative to wild-type circulating strains. The variant carries three mutations in the spike protein, including an L452R substitution. We found 2-fold increased B.1.427/B.1.429 viral shedding in vivo and increased L452R pseudovirus infection of cell cultures and lung organoids, albeit decreased relative to pseudoviruses carrying the N501Y mutation common to variants B.1.1.7, B.1.351, and P.1. Antibody neutralization assays revealed 4.0- to 6.7-fold and 2.0-fold decreases in neutralizing titers from convalescent patients and vaccine recipients, respectively. The increased prevalence of a more transmissible variant in California exhibiting decreased antibody neutralization warrants further investigation.


Assuntos
Anticorpos Neutralizantes/imunologia , COVID-19/imunologia , COVID-19/transmissão , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Humanos , Mutação/genética , Sequenciamento Completo do Genoma/métodos
2.
J Virol ; 94(24)2020 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-32999034

RESUMO

Although fetal death is now understood to be a severe outcome of congenital Zika syndrome, the role of viral genetics is still unclear. We sequenced Zika virus (ZIKV) from a rhesus macaque fetus that died after inoculation and identified a single intrahost substitution, M1404I, in the ZIKV polyprotein, located in nonstructural protein 2B (NS2B). Targeted sequencing flanking position 1404 in 9 additional macaque mothers and their fetuses identified M1404I at a subconsensus frequency in the majority (5 of 9, 56%) of animals and some of their fetuses. Despite its repeated presence in pregnant macaques, M1404I has occurred rarely in humans since 2015. Since the primary ZIKV transmission cycle is human-mosquito-human, mutations in one host must be retained in the alternate host to be perpetuated. We hypothesized that ZIKV I1404 increases viral fitness in nonpregnant macaques and pregnant mice but is less efficiently transmitted by vectors, explaining its low frequency in humans during outbreaks. By examining competitive fitness relative to that of ZIKV M1404, we observed that ZIKV I1404 produced lower viremias in nonpregnant macaques and was a weaker competitor in tissues. In pregnant wild-type mice, ZIKV I1404 increased the magnitude and rate of placental infection and conferred fetal infection, in contrast to ZIKV M1404, which was not detected in fetuses. Although infection and dissemination rates were not different, Aedes aegypti mosquitoes transmitted ZIKV I1404 more poorly than ZIKV M1404. Our data highlight the complexity of arbovirus mutation-fitness dynamics and suggest that intrahost ZIKV mutations capable of augmenting fitness in pregnant vertebrates may not necessarily spread efficiently via mosquitoes during epidemics.IMPORTANCE Although Zika virus infection of pregnant women can result in congenital Zika syndrome, the factors that cause the syndrome in some but not all infected mothers are still unclear. We identified a mutation that was present in some ZIKV genomes in experimentally inoculated pregnant rhesus macaques and their fetuses. Although we did not find an association between the presence of the mutation and fetal death, we performed additional studies with ZIKV with the mutation in nonpregnant macaques, pregnant mice, and mosquitoes. We observed that the mutation increased the ability of the virus to infect mouse fetuses but decreased its capacity to produce high levels of virus in the blood of nonpregnant macaques and to be transmitted by mosquitoes. This study shows that mutations in mosquito-borne viruses like ZIKV that increase fitness in pregnant vertebrates may not spread in outbreaks when they compromise transmission via mosquitoes and fitness in nonpregnant hosts.


Assuntos
Mutação , Complicações Infecciosas na Gravidez/virologia , Infecção por Zika virus/virologia , Zika virus/genética , Aedes/virologia , Animais , Chlorocebus aethiops , Surtos de Doenças , Feminino , Humanos , Macaca mulatta , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mosquitos Vetores/virologia , Gravidez , Células Vero , Proteínas não Estruturais Virais , Viremia , Zika virus/crescimento & desenvolvimento
3.
J Clin Microbiol ; 57(9)2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31315955

RESUMO

We applied metagenomic next-generation sequencing (mNGS) to detect Zaire Ebola virus (EBOV) and other potential pathogens from whole-blood samples from 70 patients with suspected Ebola hemorrhagic fever during a 2014 outbreak in Boende, Democratic Republic of the Congo (DRC) and correlated these findings with clinical symptoms. Twenty of 31 patients (64.5%) tested in Kinshasa, DRC, were EBOV positive by quantitative reverse transcriptase PCR (qRT-PCR). Despite partial degradation of sample RNA during shipping and handling, mNGS followed by EBOV-specific capture probe enrichment in a U.S. genomics laboratory identified EBOV reads in 22 of 70 samples (31.4%) versus in 21 of 70 (30.0%) EBOV-positive samples by repeat qRT-PCR (overall concordance = 87.1%). Reads from Plasmodium falciparum (malaria) were detected in 21 patients, of which at least 9 (42.9%) were coinfected with EBOV. Other positive viral detections included hepatitis B virus (n = 2), human pegivirus 1 (n = 2), Epstein-Barr virus (n = 9), and Orungo virus (n = 1), a virus in the Reoviridae family. The patient with Orungo virus infection presented with an acute febrile illness and died rapidly from massive hemorrhage and dehydration. Although the patient's blood sample was negative by EBOV qRT-PCR testing, identification of viral reads by mNGS confirmed the presence of EBOV coinfection. In total, 9 new EBOV genomes (3 complete genomes, and an additional 6 ≥50% complete) were assembled. Relaxed molecular clock phylogenetic analysis demonstrated a molecular evolutionary rate for the Boende strain 4 to 10× slower than that of other Ebola lineages. These results demonstrate the utility of mNGS in broad-based pathogen detection and outbreak surveillance.


Assuntos
Coinfecção/epidemiologia , Surtos de Doenças , Ebolavirus/classificação , Doença pelo Vírus Ebola/epidemiologia , Doença pelo Vírus Ebola/virologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Metagenômica/métodos , Adulto , Coinfecção/parasitologia , Coinfecção/patologia , Coinfecção/virologia , República Democrática do Congo/epidemiologia , Ebolavirus/genética , Ebolavirus/isolamento & purificação , Feminino , Doença pelo Vírus Ebola/parasitologia , Doença pelo Vírus Ebola/patologia , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Adulto Jovem
4.
J Virol ; 88(5): 2452-60, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24335315

RESUMO

Human astroviruses (HAstV) are a frequent cause of gastroenteritis in young children and immunocompromised patients. To understand the early steps of HAstV infection in the highly permissive Caco-2 cell line, the binding and entry processes of the virus were characterized. The half-time of virus binding to the cell surface was about 10 min, while virus decapsidation took around 130 min. Drugs affecting clathrin-mediated endocytosis, endosome acidification, and actin filament polymerization, as well as those that reduce the presence of cholesterol in the cell membrane, decreased the infectivity of the virus. The infection was also reduced by silencing the expression of the clathrin heavy chain (CHC) by RNA interference or by overexpression of dominant-negative mutants of dynamin 2 and Eps15. Furthermore, the entry of HAstV apparently depends on the maturation of endosomes, since the infection was reduced by silencing the expression of Rab7, a small GTPase involved in the early- to late-endosome maturation. Altogether, our results suggest that HAstV enters Caco-2 cells using a clathrin-dependent pathway and reaches late endosomes to enter cells. Here, we have characterized the mechanism used by human astroviruses, important agents of gastroenteritis in children, to gain entry into their host cells. Using a combination of biochemical and genetic tools, we found that these viruses enter Caco-2 cells using a clathrin-dependent endocytic pathway, where they most likely need to travel to late endosomes to reach the cytoplasm and begin their replication cycle.


Assuntos
Mamastrovirus/fisiologia , Internalização do Vírus , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Antivirais/farmacologia , Infecções por Astroviridae/genética , Infecções por Astroviridae/metabolismo , Infecções por Astroviridae/virologia , Linhagem Celular , Clatrina/genética , Clatrina/metabolismo , Dinaminas/genética , Dinaminas/metabolismo , Endorribonucleases/metabolismo , Proteínas Fúngicas/metabolismo , Inativação Gênica , Humanos , Mamastrovirus/efeitos dos fármacos , Mutação , Ligação Viral , Liberação de Vírus , Replicação Viral/efeitos dos fármacos , Desenvelopamento do Vírus , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7
5.
J Virol ; 87(13): 7680-7, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23637415

RESUMO

Alphaviruses are small enveloped RNA viruses that include important emerging human pathogens, such as chikungunya virus (CHIKV). These viruses infect cells via a low-pH-triggered membrane fusion reaction, making this step a potential target for antiviral therapies. The E1 fusion protein inserts into the target membrane, trimerizes, and refolds to a hairpin-like conformation in which the combination of E1 domain III (DIII) and the stem region (DIII-stem) pack against a core trimer composed of E1 domains I and II (DI/II). Addition of exogenous DIII proteins from Semliki Forest virus (SFV) has been shown to inhibit E1 hairpin formation and SFV fusion and infection. Here we produced and characterized DIII and DI/II proteins from CHIKV and SFV. Unlike SFV DIII, both core trimer binding and fusion inhibition by CHIKV DIII required the stem region. CHIKV DIII-stem and SFV DIII-stem showed efficient cross-inhibition of SFV, Sindbis virus, and CHIKV infections. We developed a fluorescence anisotropy-based assay for the binding of SFV DIII-stem to the core trimer and used it to demonstrate the relatively high affinity of this interaction (Kd [dissociation constant], ∼85 nM) and the importance of the stem region. Together, our results support the conserved nature of the key contacts of DIII-stem in the alphavirus E1 homotrimer and describe a sensitive and quantitative in vitro assay for this step in fusion protein refolding.


Assuntos
Infecções por Alphavirus/fisiopatologia , Vírus Chikungunya/metabolismo , Vírus da Floresta de Semliki/metabolismo , Proteínas do Envelope Viral/metabolismo , Proteínas Virais de Fusão/metabolismo , Ligação Viral , Animais , Linhagem Celular , Vírus Chikungunya/fisiologia , Cricetinae , Drosophila , Polarização de Fluorescência , Humanos , Lipossomos/metabolismo , Ligação Proteica , Vírus da Floresta de Semliki/fisiologia , Sindbis virus/fisiologia
6.
J Virol ; 85(13): 6334-42, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21543498

RESUMO

The alphavirus Semliki Forest virus (SFV) infects cells through a low-pH-dependent membrane fusion reaction mediated by the virus fusion protein E1. Acidic pH initiates a series of E1 conformational changes that culminate in membrane fusion and include dissociation of the E1/E2 heterodimer, insertion of the E1 fusion loop into the target membrane, and refolding of E1 to a stable trimeric hairpin conformation. A highly conserved histidine (H3) on the E1 protein was previously shown to promote low-pH-dependent E1 refolding. An SFV mutant with an alanine substitution at this position (H3A) has a lower pH threshold and reduced efficiency of virus fusion and E1 trimer formation than wild-type SFV. Here we addressed the mechanism by which H3 promotes E1 refolding and membrane fusion. We identified E1 mutations that rescue the H3A defect. These revertants implicated a network of interactions that connect the domain I-domain III (DI-DIII) linker region with the E1 core trimer, including H3. In support of the importance of these interactions, mutation of residues in the network resulted in more acidic pH thresholds and reduced efficiencies of membrane fusion. In vitro studies of truncated E1 proteins demonstrated that the DI-DIII linker was required for production of a stable E1 core trimer on target membranes. Together, our results suggest a critical and previously unidentified role for the DI-DIII linker region during the low-pH-dependent refolding of E1 that drives membrane fusion.


Assuntos
Fusão de Membrana , Glicoproteínas de Membrana/química , Vírus da Floresta de Semliki/patogenicidade , Proteínas do Envelope Viral/química , Proteínas Virais de Fusão/química , Alphavirus/patogenicidade , Alphavirus/fisiologia , Animais , Cricetinae , Histidina/química , Concentração de Íons de Hidrogênio , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Mutação , Conformação Proteica , Dobramento de Proteína , Multimerização Proteica , Estrutura Terciária de Proteína , Vírus da Floresta de Semliki/fisiologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo
7.
J Virol ; 84(11): 5730-40, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20335260

RESUMO

The flavivirus dengue virus (DV) infects cells through a low-pH-triggered membrane fusion reaction mediated by the viral envelope protein E. E is an elongated transmembrane protein with three domains and is organized as a homodimer on the mature virus particle. During fusion, the E protein homodimer dissociates, inserts the hydrophobic fusion loop into target membranes, and refolds into a trimeric hairpin in which domain III (DIII) packs against the central trimer. It is clear that E refolding drives membrane fusion, but the steps in hairpin formation and their pH requirements are unclear. Here, we have used truncated forms of the DV E protein to reconstitute trimerization in vitro. Protein constructs containing domains I and II (DI/II) were monomeric and interacted with membranes to form core trimers. DI/II-membrane interaction and trimerization occurred efficiently at both neutral and low pH. The DI/II core trimer was relatively unstable and could be stabilized by binding exogenous DIII or by the formation of mixed trimers containing DI/II plus E protein with all three domains. The mixed trimer had unoccupied DIII interaction sites that could specifically bind exogenous DIII at either low or neutral pH. Truncated DV E proteins thus reconstitute hairpin formation and define properties of key domain interactions during DV fusion.


Assuntos
Vírus da Dengue/química , Multimerização Proteica , Proteínas Virais de Fusão/química , Membrana Celular/metabolismo , Concentração de Íons de Hidrogênio , Dobramento de Proteína , Proteínas do Envelope Viral , Internalização do Vírus
8.
J Virol ; 84(18): 9161-9, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20631149

RESUMO

Rotaviruses, the single most important agents of acute severe gastroenteritis in children, are nonenveloped viruses formed by a three-layered capsid that encloses a genome formed by 11 segments of double-stranded RNA. The mechanism of entry of these viruses into the host cell is not well understood. The best-studied strain, RRV, which is sensitive to neuraminidase (NA) treatment of the cells, uses integrins alpha2 beta1 and alphav beta3 and the heat shock protein hsc70 as receptors and enters MA104 cells through a non-clathrin-, non-caveolin-mediated pathway that depends on a functional dynamin and on the presence of cholesterol on the cell surface. In this work, using a combination of pharmacological, biochemical, and genetic approaches, we compared the entry characteristics of four rotavirus strains known to have different receptor requirements. We chose four rotavirus strains that represent all phenotypic combinations of NA resistance or sensitivity and integrin dependence or independence. We found that even though all the strains share their requirements for hsc70, dynamin, and cholesterol, three of them differ from the simian strain RRV in the endocytic pathway used. The human strain Wa, porcine strain TFR-41, and bovine strain UK seem to enter the cell through clathrin-mediated endocytosis, since treatments that inhibit this pathway block their infectivity; consistent with this entry route, these strains were sensitive to changes in the endosomal pH. The inhibition of other endocytic mechanisms, such as macropinocytosis or caveola-mediated uptake, had no effect on the internalization of the rotavirus strains tested here.


Assuntos
Endocitose , Células Epiteliais/virologia , Rotavirus/fisiologia , Internalização do Vírus , Animais , Bovinos , Linhagem Celular , Colesterol/metabolismo , Vesículas Revestidas por Clatrina/virologia , Dinaminas/metabolismo , Proteínas de Choque Térmico HSC70/metabolismo , Haplorrinos , Humanos , Suínos
9.
J Virol ; 82(18): 9245-53, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18632857

RESUMO

The class II fusion proteins of the alphaviruses and flaviviruses mediate virus infection by driving the fusion of the virus membrane with that of the cell. These fusion proteins are triggered by low pH, and their structures are strikingly similar in both the prefusion dimer and the postfusion homotrimer conformations. Here we have compared cholesterol interactions during membrane fusion by these two groups of viruses. Using cholesterol-depleted insect cells, we showed that fusion and infection by the alphaviruses Semliki Forest virus (SFV) and Sindbis virus were strongly promoted by cholesterol, with similar sterol dependence in laboratory and field isolates and in viruses passaged in tissue culture. The E1 fusion protein from SFV bound cholesterol, as detected by labeling with photocholesterol and by cholesterol extraction studies. In contrast, fusion and infection by numerous strains of the flavivirus dengue virus (DV) and by yellow fever virus 17D were cholesterol independent, and the DV fusion protein did not show significant cholesterol binding. SFV E1 is the first virus fusion protein demonstrated to directly bind cholesterol. Taken together, our results reveal important functional differences conferred by the cholesterol-binding properties of class II fusion proteins.


Assuntos
Alphavirus/patogenicidade , Colesterol/metabolismo , Flavivirus/patogenicidade , Fusão de Membrana/fisiologia , Proteínas Virais de Fusão/metabolismo , Alphavirus/genética , Alphavirus/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Cricetinae , Culicidae , Vírus da Dengue/metabolismo , Vírus da Dengue/patogenicidade , Flavivirus/genética , Flavivirus/metabolismo , Mutação , Vírus da Floresta de Semliki/metabolismo , Vírus da Floresta de Semliki/patogenicidade , Sindbis virus/metabolismo , Sindbis virus/patogenicidade , Proteínas Virais de Fusão/genética , Vírus da Febre Amarela/metabolismo , Vírus da Febre Amarela/patogenicidade
10.
Sci Rep ; 8(1): 14543, 2018 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-30266962

RESUMO

Zika virus (ZIKV) is an emerging, mosquito-borne pathogen associated with a widespread 2015-2016 epidemic in the Western Hemisphere and a proven cause of microcephaly and other fetal brain defects in infants born to infected mothers. ZIKV infections have been also linked to other neurological illnesses in infected adults and children, including Guillain-Barré syndrome (GBS), acute flaccid paralysis (AFP) and meningoencephalitis, but the viral pathophysiology behind those conditions remains poorly understood. Here we investigated ZIKV infectivity in neuroblastoma SH-SY5Y cells, both undifferentiated and following differentiation with retinoic acid. We found that multiple ZIKV strains, representing both the prototype African and contemporary Asian epidemic lineages, were able to replicate in SH-SY5Y cells. Differentiation with resultant expression of mature neuron markers increased infectivity in these cells, and the extent of infectivity correlated with degree of differentiation. New viral particles in infected cells were visualized by electron microscopy and found to be primarily situated inside vesicles; overt damage to the Golgi apparatus was also observed. Enhanced ZIKV infectivity in a neural cell line following differentiation may contribute to viral neuropathogenesis in the developing or mature central nervous system.


Assuntos
Neurônios/patologia , Infecção por Zika virus/patologia , Zika virus/fisiologia , Encéfalo/citologia , Encéfalo/patologia , Encéfalo/virologia , Diferenciação Celular , Linhagem Celular , Humanos , Neurônios/citologia , Neurônios/virologia
12.
Sci Rep ; 8(1): 6851, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29717225

RESUMO

During its most recent outbreak across the Americas, Zika virus (ZIKV) was surprisingly shown to cause fetal loss and congenital malformations in acutely and chronically infected pregnant women. However, understanding the underlying pathogenesis of ZIKV congenital disease has been hampered by a lack of relevant in vivo experimental models. Here we present a candidate New World monkey model of ZIKV infection in pregnant marmosets that faithfully recapitulates human disease. ZIKV inoculation at the human-equivalent of early gestation caused an asymptomatic seroconversion, induction of type I/II interferon-associated genes and proinflammatory cytokines, and persistent viremia and viruria. Spontaneous pregnancy loss was observed 16-18 days post-infection, with extensive active placental viral replication and fetal neurocellular disorganization similar to that seen in humans. These findings underscore the key role of the placenta as a conduit for fetal infection, and demonstrate the utility of marmosets as a highly relevant model for studying congenital ZIKV disease and pregnancy loss.


Assuntos
Aborto Espontâneo/virologia , Perda do Embrião/virologia , Feto/anormalidades , Malformações do Sistema Nervoso/virologia , Placenta/virologia , Complicações Infecciosas na Gravidez/virologia , Infecção por Zika virus/complicações , Zika virus , Animais , Callithrix , Citocinas/imunologia , Modelos Animais de Doenças , Feminino , Idade Gestacional , Humanos , Interferon Tipo I/imunologia , Interferon gama/imunologia , Gravidez , Complicações Infecciosas na Gravidez/imunologia , Viremia , Replicação Viral
13.
Sci Rep ; 7(1): 17126, 2017 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-29215081

RESUMO

A monkey model of Zika virus (ZIKV) infection is urgently needed to better understand transmission and pathogenesis, given its proven association with fetal brain defects in pregnant women and acute neurological illness. Here we experimentally infected 4 male marmosets with ZIKV (prototype 1947 African strain) and monitored them clinically with sampling of various body fluids and tissues for nearly 3 months. We show that the course of acute infection with ZIKV in these New World monkeys resembles the human illness in many respects, including (1) lack of apparent clinical symptoms in most cases, (2) persistence of the virus in body fluids such as semen and saliva for longer periods of time than in serum, and (3) generation of neutralizing antibodies as well as an antiviral immunological host response. Importantly, ZIKV-infected saliva samples (in addition to serum) were found to be infectious, suggesting potential capacity for viral transmission by the oral route. Re-challenge of a previously infected marmoset with a contemporary outbreak strain SPH2015 from Brazil resulted in continued protection against infection, no viral shedding, and boosting of the immune response. Given the key similarities to human infection, a marmoset model of ZIKV infection may be useful for testing of new drugs and vaccines.


Assuntos
Infecção por Zika virus/patologia , Animais , Callithrix , Chlorocebus aethiops , Modelos Animais de Doenças , Masculino , Saliva/virologia , Células Vero , Zika virus/patogenicidade , Infecção por Zika virus/transmissão , Infecção por Zika virus/virologia
14.
Trends Microbiol ; 17(11): 514-21, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19796949

RESUMO

The alphaviruses and flaviviruses include many important human pathogens, such as the dengue, West Nile, and Chikungunya viruses. These enveloped viruses infect cells by a membrane fusion reaction triggered by the low pH in endosomes. Fusion is mediated by viral membrane proteins through their acid-dependent conversion from a dimer on the virus surface to a homotrimer inserted into the host cell membrane. Here we review recent studies on the regulatory mechanisms that silence these fusion proteins during virus exit and that sense low pH and mediate protein refolding during virus entry. We discuss results using truncated proteins to dissect the fusion reaction, and future research directions including the development of antiviral therapies against these medically important viruses.


Assuntos
Alphavirus/fisiologia , Flavivirus/fisiologia , Fusão de Membrana , Internalização do Vírus , Membrana Celular/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Modelos Biológicos , Deleção de Sequência , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo
15.
Cell Host Microbe ; 4(6): 600-8, 2008 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-19064260

RESUMO

Alphaviruses infect cells via a low-pH-triggered membrane fusion reaction mediated by the class II virus fusion protein E1, an elongated molecule with three extramembrane domains (DI-III). E1 drives fusion by inserting its fusion peptide loop into the target membrane and refolding to a hairpin-like trimer in which DIII moves toward the target membrane and packs against the central trimer. Three-dimensional structures provide static pictures of prefusion and postfusion E1 but do not explain this transition. Using truncated forms of E1, we reconstituted a low-pH-dependent intermediate composed of trimers of DI/II. Unexpectedly, DI/II trimers were stable in the absence of DIII. Once formed at a low pH, DI/II trimers efficiently and specifically bound recombinant DIII through a pH-independent reaction. Even in the absence of DIII, DI/II trimers interacted to form hexagonal lattices and to cause membrane deformation and tubulation. These studies identify a prefusion intermediate in class II membrane fusion.


Assuntos
Alphavirus/fisiologia , Proteínas Virais de Fusão/metabolismo , Internalização do Vírus , Concentração de Íons de Hidrogênio , Substâncias Macromoleculares , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Ligação Proteica , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/ultraestrutura
16.
J Virol ; 78(5): 2310-8, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-14963127

RESUMO

While recently we have learned much about the viral and cellular proteins involved in the initial attachment of rotaviruses to MA104 cells, the mechanism by which these viruses reach the interior of the cell is poorly understood. For this study, we observed the effects of drugs and of dominant-negative mutants, known to impair clathrin-mediated endocytosis and endocytosis mediated by caveolae, on rotavirus cell infection. Rotaviruses were able to enter cells in the presence of compounds that inhibit clathrin-mediated endocytosis as well as cells overexpressing a dominant-negative form of Eps15, a protein crucial for the assembly of clathrin coats. We also found that rotaviruses infected cells in which caveolar uptake was blocked; treatment with the cholesterol binding agents nystatin and filipin, as well as transfection of cells with dominant-negative caveolin-1 and caveolin-3 mutants, had no effect on rotavirus infection. Interestingly, cells treated with methyl-beta-cyclodextrin, a drug that sequesters cholesterol from membranes, and cells expressing a dominant-negative mutant of the large GTPase dynamin, which is known to function in several membrane scission events, were not infected by rotaviruses, indicating that cholesterol and dynamin play a role in the entry of rotaviruses.


Assuntos
Endocitose , Rotavirus/fisiologia , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Cavéolas/metabolismo , Linhagem Celular , Toxina da Cólera/metabolismo , Clatrina/antagonistas & inibidores , Clatrina/metabolismo , Dinaminas/genética , Dinaminas/metabolismo , Filipina/farmacologia , Macaca mulatta , Mutação , Nistatina/farmacologia
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