RESUMO
Cervical cancer represents a global concern with 604,000 new cases and 342,000 deaths reported annually, with the vast majority diagnosed in low income countries. Despite high-risk Human Papillomavirus (HR HPV)-induced cervical cancer has become highly preventable through prophylactic vaccines, screening programs are critical in the control of cervical carcinogenesis in populations with limited access to vaccination and in older generations of women who have already been exposed to HR HPV infection. The surge of HPV molecular tests has provided a more sensitive and accurate diagnostic alternative to cytology screening. Given that HPV DNA testing presents a low positive predicted value, leading to unnecessary treatment, the E6 oncoprotein from HR HPV types arises as a promising diagnostic marker for its overexpression in transformed HPV-positive cancer cells. For these reasons, this study aimed at obtaining monoclonal antibodies (mAbs) against the E6 oncoprotein of one of the most prevalent HR HPV types worldwide, HPV18, in order to develop a highly specific and sensitive indirect competitive ELISA (icELISA). The production of hybridomas secreting HPV18 E6 mAbs was carried out through a combined tolerization and immunization strategy, in order to avoid cross-reactivity with the E6 protein from low-risk HPV types 6 and 11. We selected the 7D2 hybridoma clone, which recognized HPV18 E6 and showed some cross-reactivity against the HR HPV45 E6 oncoprotein. The 7D2 mAb enabled the development of a sensitive, reliable and reproducible icELISA to detect and quantify small amounts of HPV18 E6 biomarker for cervical cancer progression. The present study establishes a valid 7D2-based icELISA that constitutes a promising bioanalytical method for the early detection and quantification of HPV18 E6 oncoprotein in cervical swab samples and cancer prevention.
Assuntos
Proteínas Oncogênicas Virais , Infecções por Papillomavirus , Neoplasias do Colo do Útero , Feminino , Humanos , Idoso , Papillomavirus Humano 18/genética , Papillomavirus Humano 18/metabolismo , Papillomavirus Humano , Proteínas Repressoras/genética , Proteínas Oncogênicas Virais/genética , Papillomaviridae/genética , Papillomaviridae/metabolismo , Ensaio de Imunoadsorção EnzimáticaRESUMO
Gumboro illness is caused by the highly contagious immunosuppressive infectious bursal disease virus (IBDV), which affects the poultry industry globally. We have previously shown that IBDV hijacks the endocytic pathway to construct viral replication complexes on endosomes linked to the Golgi complex (GC). Then, analyzing crucial proteins involved in the secretory pathway, we showed the essential requirement of Rab1b, the Rab1b downstream effector Golgi-specific BFA resistance factor 1 (GBF1), and its substrate, the small GTPase ADP-ribosylation factor 1 (ARF1), for IBDV replication. In the current work, we focused on elucidating the IBDV assembly sites. We show that viral assembly occurs within single-membrane compartments closely associated with endoplasmic reticulum (ER) membranes, though we failed to elucidate the exact nature of the virus-wrapping membranes. Additionally, we show that IBDV infection promotes the stress of the ER, characterized by an accumulation of the chaperone binding protein (BiP) and lipid droplets (LDs) in the host cells. Overall, our results represent further original data showing the interplay between IBDV and the secretory pathway, making a substantial contribution to the field of birnaviruses-host cell interactions.
Assuntos
Infecções por Birnaviridae , Vírus da Doença Infecciosa da Bursa , Doenças das Aves Domésticas , Animais , Gotículas Lipídicas , Montagem de Vírus , Endossomos , Estresse do Retículo Endoplasmático , GalinhasRESUMO
Birnaviruses are members of the Birnaviridae family, responsible for major economic losses to poultry and aquaculture. The family is composed of nonenveloped viruses with a segmented double-stranded RNA (dsRNA) genome. Infectious bursal disease virus (IBDV), the prototypic family member, is the etiological agent of Gumboro disease, a highly contagious immunosuppressive disease in the poultry industry worldwide. We previously demonstrated that IBDV hijacks the endocytic pathway for establishing the viral replication complexes on endosomes associated with the Golgi complex (GC). Here, we report that IBDV reorganizes the GC to localize the endosome-associated replication complexes without affecting its secretory functionality. By analyzing crucial proteins involved in the secretory pathway, we showed the essential requirement of Rab1b for viral replication. Rab1b comprises a key regulator of GC transport and we demonstrate that transfecting the negative mutant Rab1b N121I or knocking down Rab1b expression by RNA interference significantly reduces the yield of infectious viral progeny. Furthermore, we showed that the Rab1b downstream effector Golgi-specific BFA resistance factor 1 (GBF1), which activates the small GTPase ADP ribosylation factor 1 (ARF1), is required for IBDV replication, since inhibiting its activity by treatment with brefeldin A (BFA) or golgicide A (GCA) significantly reduces the yield of infectious viral progeny. Finally, we show that ARF1 dominant negative mutant T31N overexpression hampered IBDV infection. Taken together, these results demonstrate that IBDV requires the function of the Rab1b-GBF1-ARF1 axis to promote its replication, making a substantial contribution to the field of birnavirus-host cell interactions. IMPORTANCE Birnaviruses are unconventional members of the dsRNA viruses, with the lack of a transcriptionally active core being the main differential feature. This structural trait, among others that resemble those of the plus single-stranded (+ssRNA) viruses features, suggests that birnaviruses might follow a different replication program from that conducted by prototypical dsRNA members and the hypothesis that birnaviruses could be evolutionary links between +ssRNA and dsRNA viruses has been argued. Here, we present original data showing that IBDV-induced GC reorganization and the cross talk between IBDV and the Rab1b-GBF1-ARF1 mediate the intracellular trafficking pathway. The replication of several +ssRNA viruses depends on the cellular protein GBF1, but its role in the replication process is not clear. Thus, our findings make a substantial contribution to the field of birnavirus-host cell interactions and provide further evidence supporting the proposed evolutionary connection role of birnaviruses, an aspect which we consider especially relevant for researchers working in the virology field.
Assuntos
Fator 1 de Ribosilação do ADP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Vírus da Doença Infecciosa da Bursa/fisiologia , Via Secretória/fisiologia , Replicação Viral/fisiologia , Proteínas rab1 de Ligação ao GTP/metabolismo , Fator 1 de Ribosilação do ADP/genética , Animais , Brefeldina A/farmacologia , Linhagem Celular , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Interações Hospedeiro-Patógeno , Piridinas/farmacologia , Quinolinas/farmacologia , Via Secretória/efeitos dos fármacos , Compartimentos de Replicação Viral/metabolismo , Replicação Viral/efeitos dos fármacos , Proteínas rab1 de Ligação ao GTP/genéticaRESUMO
Zika virus (ZIKV) is a flavivirus that has emerged as a global health threat after the 2015 outbreak in the Americas, where devastating congenital defects were documented. There are currently no vaccines to prevent ZIKV infections nor commercially available clinical diagnostic tests demonstrated to identify ZIKV without cross-reactive interference of related flaviviruses. Early diagnosis is critical when treating symptomatic patients and in preventing ZIKV transmission. In this context, the development of sensitive and accurate diagnostic methods are urgently needed for the detection of ZIKV acute infection. The aim of this study consisted of obtaining monoclonal antibodies (mAbs) against denatured monomeric ZIKV Nonstructural protein 1 (ZNS1), a useful diagnostic marker for flavivirus early detection, in order to develop a highly specific and sensitive ZNS1 indirect competitive ELISA (icELISA). The production of hybridomas secreting ZNS1 mAbs was carried out through immunizations with denatured monomeric ZNS1. We selected 1F5 and 6E2 hybridoma clones, which recognized the heat-denatured ZNS1 hexameric form by indirect ELISA. Cross-reaction studies indicated that these mAbs specifically bind to a ZNS1 linear epitope, and that they do not cross-react with the NS1 protein from other related flaviviruses. The 1F5 mAb enabled the development of a sensitive and reproducible icELISA to detect and quantify small amounts of ZNS1 disease marker in heat-denatured human sera. Here, we establish a reliable 1F5 based-icELISA that constitutes a promising diagnostic tool for control strategies and the prevention of ZIKV propagation.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Antivirais/química , Antígenos Virais/genética , Ensaio de Imunoadsorção Enzimática/métodos , Proteínas não Estruturais Virais/genética , Infecção por Zika virus/diagnóstico , Zika virus/imunologia , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/isolamento & purificação , Antígenos Virais/administração & dosagem , Antígenos Virais/imunologia , Ligação Competitiva , Clonagem Molecular , Diagnóstico Precoce , Ensaio de Imunoadsorção Enzimática/normas , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Células HEK293 , Humanos , Hibridomas/química , Hibridomas/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Multimerização Proteica , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Proteínas não Estruturais Virais/administração & dosagem , Proteínas não Estruturais Virais/imunologia , Zika virus/genética , Infecção por Zika virus/imunologia , Infecção por Zika virus/virologiaRESUMO
Human Cytomegalovirus (HCMV) is a frequent opportunistic pathogen in immunosuppressed patients, which can be involved in kidney allograft dysfunction and rejection. In order to study the pathophysiology of HCMV renal diseases, we concentrated on the impact of HCMV infection on human renal tubular epithelial HK-2 cells. Our aim was to develop a model of infection of HK-2 cells by using the viral strain TB40/E, that contains the extended cell tropism of clinical isolates and the efficient viral multiplication in cell culture of laboratory-adapted strains. We observed that HK-2 cells can be infected by HCMV and expressed viral antigens, but they do not produce extracellular viral particles. We then studied the interplay of HCMV with ciliogenesis and autophagy. Primary cilium (PC) is a stress sensor important to maintain renal tissue homeostasis that projects from the apical side into the lumen of tubule cells. PC formation and length were not modified by HCMV infection. Autophagy, another stress response process critically required for normal kidney functions, was inhibited by HCMV in HK-2 cells with a reduction in the autophagic flux. HCMV classically induces an enlargement of infected cells in vivo and in vitro, and we observed that HCMV infection led to an enlargement of the HK-2 cell volume. Our results constitute therefore an excellent starting point to further explore the role of these mechanisms in renal cells dysfunction.
Assuntos
Infecções por Citomegalovirus , Citomegalovirus , Autofagia , Células Cultivadas , Células Epiteliais , HumanosRESUMO
Sensitive, accurate and cost-effective diagnostic tests are urgently needed to detect Zika virus (ZIKV) infection. Nonstructural 1 (NS1) glycoprotein is an excellent diagnostic marker since it is released in a hexameric conformation from infected cells into the patient's bloodstream early in the course of the infection. We established a stable rZNS1-His-expression system in HEK293 cells through lentiviral transduction. A novel optimization approach to enhance rZNS1-His protein secretion in the mammalian expression system was accomplished through 50â¯nM rapamycin incubation followed by serum-free media incubation for 9 days, reaching protein yields of â¼10â¯mg/l of culture medium. Purified rZNS1-His hexamer was recognized by anti-NS1 antibodies in ZIKV patient's serum, and showed the ability to induce a humoral response in immunized mice. The obtained recombinant protein is a reliable biological tool that can potentially be applied in the development of diagnostic tests to detect ZIKV in infected patients during the acute phase.
RESUMO
Our perspective on nature has changed throughout history and at the same time has affected directly or indirectly our perception of biological processes. In that sense, the "fluxus" of information in a viral population arises a result of a much more complex process than the encoding of a protein by a gene, but as the consequence of the interaction between all the components of the genome and its products: DNA, RNA, and proteins and its modulation by the environment. Even modest "agents of life" like viruses display an intricate way to express their information. This conclusion can be withdrawn from the huge quantity of data furnished by new and potent technologies available now to analyze viral populations. Based on this premise, evolutive processes for viruses are now interpreted as a simultaneous and coordinated phenomenon that leads to global (i.e., not gradual or 'random') remodeling of the population. Our system of study involves the modulation of herpes simplex virus populations through the selective pressure exerted by carrageenans, natural compounds that interfere with virion attachment to cells. On this line, we demonstrated that the passaging of virus in the presence of carrageenans leads to the appearance of progeny virus phenotipically different from the parental seed, particularly, the emergence of syncytial (syn) variants. This event precedes the emergence of mutations in the population which can be readily detected five passages after from the moment of the appearance of syn virus. This observation can be explained taking into consideration that the onset of phenotypic changes may be triggered by "environmental-sensitive" glycoproteins. These "environmental-sensitive" glycoproteins may act by themselves or may transmit the stimulus to "adapter" proteins, particularly, proteins of the tegument, which eventually modulate the expression of genomic products in the "virocell." The modulation of the RNA network is a common strategy of the virocell to respond to environmental changes. This "fast" adaptive mechanism is followed eventually by the appearance of mutations in the viral genome. In this paper, we interpret these findings from a philosophical and scientific point of view interconnecting epigenetic action, exerted by carragenans from early RNA network-DNA interaction to late DNA mutation. The complexity of HSV virion structure is an adequate platform to envision new studies on this topic that may be complemented in a near future through the analysis of the genetic dynamics of HSV populations.
RESUMO
Tacaribe virus (TCRV) entry occurs by receptor-mediated endocytosis. To explore the entry mechanism used by TCRV, the inhibitory effects of drugs and dominant negative (DN) constructions affecting the main endocytic pathways were analyzed. In cells lacking the human transferrin receptor (hTfR), compounds and DN proteins that impair clathrin-mediated endocytosis were shown to reduce virus internalization without affecting virion binding. In contrast, in cells expressing the hTfR, compounds that affect clathrin-mediated endocytosis did not affect TCRV infection. Destabilization of cholesterol-rich plasma membrane microdomains by treatment with nystatin was not able to block virus entry in the presence of hTfR. However methyl-ß-cyclodextrin, which extracts cholesterol from cell membranes, reduced virus internalization in cells expressing the hTfR. Inhibition of dynamin and neutralization of the pH of intracellular vesicles reduced virus internalization in all cell lines tested. Taken together, these results demonstrate that in cells expressing the hTfR, TCRV internalization depends on the presence of cholesterol, dynamin and acidic intracellular vesicles, while in the rest of the cell lines analyzed, clathrin-mediated endocytosis is the main TCRV entry pathway and, as expected, depends on dynamin and acidic intracellular vesicles. These results represent an important contribution to the characterization of the arenavirus replication cycle.