RESUMEN

BACKGROUND: Human immunodeficiency virus (HIV)-1 infection can activate the expression of human endogenous retroviruses (HERVs), particularly HERV-K (HML-2). HIV controllers (HICs) are rare people living with HIV (PLWHs) who naturally control HIV-1 replication and overexpress some cellular restriction factors that negatively regulate the LTR-driven transcription of HIV-1 proviruses. OBJECTIVES: To understand the ability of HICs to control the expression of endogenous retroviruses. METHODS: We measured endogenous retrovirus type K6 (ERVK-6) RNA expression in peripheral blood mononuclear cells (PBMCs) of HICs (n = 23), antiretroviral (ART)-suppressed subjects (n = 8), and HIV-1-negative (NEG) individuals (n = 10) and correlated the transcript expression of ERVK-6 with multiple HIV-1 cellular restriction factors. FINDINGS: Our study revealed that ERVK-6 RNA expression in PBMCs from HICs was significantly downregulated compared with that in both the ART and NEG control groups. Moreover, we detected that ERVK-6 RNA levels in PBMCs across all groups were negatively correlated with the expression levels of p21 and MCPIP1, two cellular restriction factors that limit the activation of macrophages and T cells by downregulating the activity of NF-kB. MAIN CONCLUSIONS: These findings support the hypothesis that HICs activate innate antiviral mechanisms that may simultaneously downregulate the transcription of both exogenous (HIV-1) and endogenous (ERVK-6) retroviruses. Future studies with larger cohorts should be performed to confirm this hypothesis and to explore the role of p21 and MCPIP1 in regulating HERV-K expression in physiological and pathological conditions.

Asunto(s)

Retrovirus Endógenos , Infecciones por VIH , VIH-1 , ARN Viral , Ribonucleasas , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios de Casos y Controles , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Retrovirus Endógenos/genética , Retrovirus Endógenos/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , Infecciones por VIH/genética , VIH-1/genética , VIH-1/inmunología , Inmunidad Innata/genética , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Ribonucleasas/genética , Ribonucleasas/metabolismo , ARN Viral/genética , Factores de Transcripción/genética , Replicación Viral/genética

RESUMEN

MicroRNAs (miRNAs) are molecules that influence messenger RNA (mRNA) expression levels by binding to the 3' untranslated region (3' UTR) of target genes. Host miRNAs can influence flavivirus replication, either by inducing changes in the host transcriptome or by directly binding to viral genomes. The 3' UTR of the flavivirus genome is a conserved region crucial for viral replication. Cells might exploit this well-preserved region by generating miRNAs that interact with it, ultimately impacting viral replication. Despite significant efforts to identify miRNAs capable of arresting viral replication, the potential of all these miRNAs to interact with the flavivirus 3' UTR is still poorly characterised. In this context, bioinformatic tools have been proposed as a fundamental part of accelerating the discovery of interactions between miRNAs and the 3' UTR of viral genomes. In this study, we performed a computational analysis to reveal potential miRNAs from human and mosquito species that bind to the 3' UTR of flaviviruses. In humans, miR-6842 and miR-661 were found, while in mosquitoes, miR-9-C, miR-2945-5p, miR-11924, miR-282-5p, and miR-79 were identified. These findings open new avenues for studying these miRNAs as antivirals against flavivirus infections.

Asunto(s)

Regiones no Traducidas 3' , Biología Computacional , Flavivirus , Genoma Viral , MicroARNs , MicroARNs/genética , MicroARNs/metabolismo , Regiones no Traducidas 3'/genética , Flavivirus/genética , Humanos , Animales , Biología Computacional/métodos , Replicación Viral/genética , Antivirales/farmacología , Infecciones por Flavivirus/virología , Infecciones por Flavivirus/genética , Culicidae/virología , Culicidae/genética

RESUMEN

Chikungunya virus (CHIKV) is a rapidly spreading re-emergent virus transmitted from mosquitoes to humans. The emergence of epidemic variants has been associated with changes in the viral genome, such as the duplication of repeated sequences in the 3' untranslated region (UTR). Indeed, blocks of repeated sequences seemingly favor RNA recombination, providing the virus with a unique ability to continuously change the 3'UTR architecture during host switching. In this work, we provide experimental data on the molecular mechanism of RNA recombination and describe specific sequence and structural elements in the viral 3'UTR that favor template switching of the viral RNA-dependent RNA polymerase on the 3'UTR. Furthermore, we found that a 3'UTR deletion mutant that exhibits markedly delayed replication in mosquito cells and impaired transmission in vivo, recombines in reference laboratory strains of mosquitoes. Altogether, our data provide novel experimental evidence indicating that RNA recombination can act as a nucleic acid repair mechanism to add repeated sequences that are associated to high viral fitness in mosquito during chikungunya virus replication.

Asunto(s)

Regiones no Traducidas 3' , Virus Chikungunya , Genoma Viral , ARN Viral , Recombinación Genética , Replicación Viral , Virus Chikungunya/genética , Regiones no Traducidas 3'/genética , ARN Viral/genética , ARN Viral/metabolismo , Animales , Replicación Viral/genética , Fiebre Chikungunya/virología , Fiebre Chikungunya/genética , Fiebre Chikungunya/transmisión , Humanos , Aedes/virología , Aedes/genética , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismo , Línea Celular

RESUMEN

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus that causes severe outbreaks in human populations. ZIKV infection leads to the accumulation of small non-coding viral RNAs (known as sfRNAs) that are crucial for evasion of antiviral responses and for viral pathogenesis. However, the mechanistic understanding of how sfRNAs function remains incomplete. Here, we use recombinant ZIKVs and ribosome profiling of infected human cells to show that sfRNAs block translation of antiviral genes. Mechanistically, we demonstrate that specific RNA structures present in sfRNAs trigger PKR activation, which instead of limiting viral replication, enhances viral particle production. Although ZIKV infection induces mRNA expression of antiviral genes, translation efficiency of type I interferon and interferon stimulated genes were significantly downregulated by PKR activation. Our results reveal a novel viral adaptation mechanism mediated by sfRNAs, where ZIKV increases its fitness by repurposing the antiviral role of PKR into a proviral factor.

Asunto(s)

Biosíntesis de Proteínas , ARN Viral , Replicación Viral , Infección por el Virus Zika , Virus Zika , eIF-2 Quinasa , Virus Zika/genética , Humanos , eIF-2 Quinasa/metabolismo , eIF-2 Quinasa/genética , ARN Viral/metabolismo , ARN Viral/genética , Infección por el Virus Zika/virología , Infección por el Virus Zika/genética , Infección por el Virus Zika/inmunología , Replicación Viral/genética , ARN no Traducido/genética , ARN no Traducido/metabolismo , Animales , Chlorocebus aethiops , Células HEK293 , Línea Celular

RESUMEN

MicroRNAs (miRNAs), a class of small, non-coding RNAs, play a pivotal role in regulating gene expression at the post-transcriptional level. These regulatory molecules are integral to many biological processes and have been implicated in the pathogenesis of various diseases, including Human Immunodeficiency Virus (HIV) infection. This review aims to cover the current understanding of the multifaceted roles miRNAs assume in the context of HIV infection and pathogenesis. The discourse is structured around three primary focal points: (i) elucidation of the mechanisms through which miRNAs regulate HIV replication, encompassing both direct targeting of viral transcripts and indirect modulation of host factors critical for viral replication; (ii) examination of the modulation of miRNA expression by HIV, mediated through either viral proteins or the activation of cellular pathways consequent to viral infection; and (iii) assessment of the impact of miRNAs on the immune response and the progression of disease in HIV-infected individuals. Further, this review delves into the potential utility of miRNAs as biomarkers and therapeutic agents in HIV infection, underscoring the challenges and prospects inherent to this line of inquiry. The synthesis of current evidence positions miRNAs as significant modulators of the host-virus interplay, offering promising avenues for enhancing the diagnosis, treatment, and prevention of HIV infection.

Asunto(s)

Infecciones por VIH , MicroARNs , Replicación Viral , Humanos , MicroARNs/genética , Infecciones por VIH/genética , Infecciones por VIH/virología , Replicación Viral/genética , VIH-1/genética , Interacciones Huésped-Patógeno/genética , Biomarcadores , Regulación de la Expresión Génica

RESUMEN

Dengue virus (DENV) is one of the most important and widespread arthropod-borne viruses, causing millions of infections over the years. Considering its epidemiological importance, efforts have been directed towards understanding various aspects of DENV biology, which have been facilitated by the development of different molecular strategies for engineering viral genomes, such as reverse genetics approaches. Reverse genetic systems are a powerful tool for investigating virus-host interaction, for vaccine development, and for high-throughput screening of antiviral compounds. However, stable manipulation of DENV genomes is a major molecular challenge, especially when using conventional cloning systems. To circumvent this issue, we describe a simple and efficient yeast-based reverse genetics system to recover infectious DENV clones.

Asunto(s)

Virus del Dengue , Dengue , Humanos , Virus del Dengue/genética , Genética Inversa , Ensayos Analíticos de Alto Rendimiento , Genoma Viral , Dengue/genética , Replicación Viral/genética

RESUMEN

IMPORTANCE: One of the fundamental features that make viruses intracellular parasites is the necessity to use cellular translational machinery. Hence, this is a crucial checkpoint for controlling infections. Here, we show that dengue and Zika viruses, responsible for nearly 400 million infections every year worldwide, explore such control for optimal replication. Using immunocompetent cells, we demonstrate that arrest of protein translations happens after sensing of dsRNA and that the information required to avoid this blocking is contained in viral 5'-UTR. Our work, therefore, suggests that the non-canonical translation described for these viruses is engaged when the intracellular stress response is activated.

Asunto(s)

Virus del Dengue , Estrés Fisiológico , Replicación Viral , Virus Zika , eIF-2 Quinasa , Animales , Humanos , Células A549 , Chlorocebus aethiops , Dengue/inmunología , Dengue/virología , Virus del Dengue/fisiología , eIF-2 Quinasa/genética , eIF-2 Quinasa/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Eliminación de Gen , Biosíntesis de Proteínas/genética , Biosíntesis de Proteínas/inmunología , Estrés Fisiológico/genética , Estrés Fisiológico/inmunología , Células Vero , Replicación Viral/genética , Replicación Viral/inmunología , Virus Zika/fisiología , Infección por el Virus Zika/inmunología , Infección por el Virus Zika/virología , ARN Bicatenario/metabolismo

RESUMEN

RNA viral genomes compact information into functional RNA structures. Here, using chikungunya virus as a model, we investigated the structural requirements of conserved RNA elements in the 3' untranslated region (3'UTR) for viral replication in mosquito and mammalian cells. Using structural predictions and co-variation analysis, we identified a highly stable and conserved Y-shaped structure (SLY) at the end of the 3'UTR that is duplicated in the Asian lineage. Functional studies with mutant viruses showed that the SLY has host-specific functions during viral replication and evolution. The SLY positively modulates viral replication in mosquito cells but has the opposite effect in mammalian cells. Additional structural/functional analyses showed that maintaining the Y-shaped fold and specific nucleotides in the loop are critical for full SLY functionality and optimal viral replication in mosquito cells. Experimental adaptation of viruses with duplicated SLYs to mammalian cells resulted in the generation of heterogeneous viral populations comprising variants with diverse 3'UTRs, contrasting with the homogeneous populations from viruses without SLY copies. Altogether, our findings constitute the first evidence of an RNA secondary structure in the 3'UTR of chikungunya virus genome that plays host-dependent functions.

Asunto(s)

Virus Chikungunya , Culicidae , Animales , Regiones no Traducidas 3'/genética , Virus Chikungunya/genética , ARN Viral/genética , ARN Viral/química , Culicidae/genética , Replicación Viral/genética , Genoma Viral , Mamíferos

RESUMEN

The COVID-19 pandemic initiated a race to determine the best measures to control the disease and to save as many people as possible. Efforts to implement social distancing, the use of masks, and massive vaccination programs turned out to be essential in reducing the devastating effects of the pandemic. Nevertheless, the high mutation rates of SARS-CoV-2 challenge the vaccination strategy and maintain the threat of new outbreaks due to the risk of infection surges and even lethal variations able to resist the effects of vaccines and upset the balance. Most of the new therapies tested against SARS-CoV-2 came from already available formulations developed to treat other diseases, so they were not specifically developed for SARS-CoV-2. In parallel, the knowledge produced regarding the molecular mechanisms involved in this disease was vast due to massive efforts worldwide. Taking advantage of such a vast molecular understanding of virus genomes and disease mechanisms, a targeted molecular therapy based on siRNA specifically developed to reach exclusive SARS-CoV-2 genomic sequences was tested in a non-transformed human cell model. Since coronavirus can escape from siRNA by producing siRNA inhibitors, a complex strategy to simultaneously strike both the viral infectious mechanism and the capability of evading siRNA therapy was developed. The combined administration of the chosen produced siRNA proved to be highly effective in successfully reducing viral load and keeping virus replication under control, even after many days of treatment, unlike the combinations of siRNAs lacking this anti-anti-siRNA capability. Additionally, the developed therapy did not harm the normal cells, which was demonstrated because, instead of testing the siRNA in nonhuman cells or in transformed human cells, a non-transformed human thyroid cell was specifically chosen for the experiment. The proposed siRNA combination could reduce the viral load and allow the cellular recovery, presenting a potential innovation for consideration as an additional strategy to counter or cope COVID-19.

Asunto(s)

COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , Pandemias , Replicación Viral/genética , Genoma Viral , ARN Interferente Pequeño/genética

RESUMEN

Virus replication frequently results in the accumulation, re-assortment and re-combination of mutations, which contributes to their rapid adaptation to environmental changes and often advances the emergence of new virus variants or species [...].

Asunto(s)

Virus , Mutación , Replicación Viral/genética , Virus/genética

RESUMEN

BACKGROUND: Porcine circovirus type 2 (PCV2)-associated diseases are a major problem for the swine industry worldwide. In addition to vaccines, the availability of antiviral polymers provides an efficient and safe option for reducing the impact of these diseases. By virtue of their molecular weight and repetitious structure, polymers possess properties not found in small-molecule drugs. In this perspective, we focus on chitosan, a ubiquitous biopolymer, that adjusts the molecular weight and sulfated-mediated functionality can act as an efficient antiviral polymer by mimicking PCV2-cell receptor interactions. METHODS: Sulfated chitosan (Chi-S) polymers of two molecular weights were synthesized and characterized by FTIR, SEM-EDS and elemental analysis. The Chi-S solutions were tested against PCV2 infection in PK15 cells in vitro and antiviral activity was evaluated by measuring the PCV2 DNA copy number, TCID50 and capsid protein expression, upon application of different molecular weights, sulfate functionalization, and concentrations of polymer. In addition, to explore the mode of action of the Chi-S against PCV2 infection, experiments were designed to elucidate whether the antiviral activity of the Chi-S would be influenced by when it was added to the cells, relative to the time and stage of viral infection. RESULTS: Chi-S significantly reduced genomic copies, TCID50 titers and capsid protein of PCV2, showing specific antiviral effects depending on its molecular weight, concentration, and chemical functionalization. Assays designed to explore the mode of action of the low molecular weight Chi-S revealed that it exerted antiviral activity through impeding viral attachment and penetration into cells. CONCLUSIONS: These findings help better understanding the interactions of PCV2 and porcine cells and reinforce the idea that sulfated polymers, such as Chi-S, represent a promising candidates for use in antiviral therapies against PCV2-associated diseases. Further studies in swine are warranted.

Asunto(s)

Quitosano , Infecciones por Circoviridae , Circovirus , Enfermedades de los Porcinos , Animales , Antivirales/metabolismo , Antivirales/farmacología , Proteínas de la Cápside/genética , Quitosano/metabolismo , Quitosano/farmacología , Infecciones por Circoviridae/prevención & control , Circovirus/genética , Peso Molecular , Sulfatos/metabolismo , Porcinos , Replicación Viral/genética

RESUMEN

The genome of Alphaviruses can be modified to produce self-replicating RNAs and virus-like particles, which are useful virological tools. In this work, we generated three plasmids for the transfection of mammalian cells: an infectious clone of Chikungunya virus (CHIKV), one that codes for the structural proteins (helper plasmid), and another one that codes nonstructural proteins (replicon plasmid). All of these plasmids contain a reporter gene (mKate2). The reporter gene in the replicon RNA and the infectious clone are synthesized from subgenomic RNA. Co-transfection with the helper and replicon plasmids has biotechnological/biomedical applications because they allow for the delivery of self-replicating RNA for the transient expression of one or more genes to the target cells.

Asunto(s)

Virus Chikungunya , Animales , Virus Chikungunya/genética , Virus Chikungunya/metabolismo , Replicación Viral/genética , Transfección , Plásmidos/genética , ARN/metabolismo , Replicón , Vectores Genéticos/genética , Mamíferos

RESUMEN

Although RNA viruses have high mutation rates, host cells and organisms work as selective environments, maintaining the viability of virus populations by eliminating deleterious genotypes. In serial passages of RNA viruses in a single cell line, most of these selective bottlenecks are absent, with no virus circulation and replication in different tissues or host alternation. In this work, Aedes aegypti Aag-2 cells were accidentally infected with Chikungunya virus (CHIKV) and Mayaro virus (MAYV). After numerous passages to achieve infection persistency, the infectivity of these viruses was evaluated in Ae. albopictus C6/36 cells, African green monkey Vero cells and primary-cultured human fibroblasts. While these CHIKV and MAYV isolates were still infectious to mosquito cells, they lost their ability to infect mammalian cells. After genome sequencing, it was observed that CHIKV accumulated many nonsynonymous mutations and a significant deletion in the coding sequence of the hypervariable domain in the nsP3 gene. Since MAYV showed very low titres, it was not sequenced successfully. Persistently infected Aag-2 cells also accumulated high loads of short and recombinant CHIKV RNAs, which seemed to have been originated from virus-derived DNAs. In conclusion, the genome of this CHIKV isolate could guide mutagenesis strategies for the production of attenuated or non-infectious (to mammals) CHIKV vaccine candidates. Our results also reinforce that a paradox is expected during passages of cells persistently infected by RNA viruses: more loosening for the development of more diverse virus genotypes and more pressure for virus specialization to this constant cellular environment.

Asunto(s)

Virus Chikungunya/crecimiento & desarrollo , Virus Chikungunya/genética , Genoma Viral/genética , Alphavirus/genética , Alphavirus/crecimiento & desarrollo , Animales , Línea Celular , Culicidae , Especificidad del Huésped , Humanos , Mamíferos , Mutación , ARN Viral/genética , Carga Viral/genética , Proteínas no Estructurales Virales/genética , Replicación Viral/genética

RESUMEN

The stress of the Golgi apparatus is an autoregulatory mechanism that is induced to compensate for greater demand in the Golgi functions. No examples of Golgi stress responses due to physiological stimuli are known. Furthermore, the impact on this organelle of viral infections that occupy the vesicular transport during replication is unknown. In this work, we evaluated if a Golgi stress response is triggered during dengue and Zika viruses replication, two flaviviruses whose replicative cycle is heavily involved with the Golgi complex, in vertebrate and mosquito cells. Using GM-130 as a Golgi marker, and treatment with monensin as a positive control for the induction of the Golgi stress response, a significant expansion of the Golgi cisternae was observed in BHK-21, Vero E6 and mosquito cells infected with either virus. Activation of the TFE3 pathway was observed in the infected cells as indicated by the translocation from the cytoplasm to the nucleus of TFE3 and increased expression of pathway targeted genes. Of note, no sign of activation of the stress response was observed in CRFK cells infected with Feline Calicivirus (FCV), a virus released by cell lysis, not requiring vesicular transport. Finally, dilatation of the Golgi complex and translocation of TFE3 was observed in vertebrate cells expressing dengue and Zika viruses NS1, but not NS3. These results indicated that infections by dengue and Zika viruses induce a Golgi stress response in vertebrate and mosquito cells due to the increased demand on the Golgi complex imposed by virion and NS1 processing and secretion.

Asunto(s)

Culicidae/virología , Infecciones por Flavivirus/virología , Flavivirus/genética , Aparato de Golgi/virología , Vertebrados/virología , Animales , Células Cultivadas , Chlorocebus aethiops , Mesocricetus , Células Vero , Proteínas no Estructurales Virales/genética , Replicación Viral/genética

RESUMEN

Over the years, viral infections have caused severe illness in humans. Zika Virus (ZIKV) is a flavivirus transmitted by mosquito vectors that leads to notable neurological impairment, whose most dramatic impact is the Congenital ZIKV Syndrome (CZS). ZIKV targets neuronal precursor cells leading to apoptosis and further impairment of neuronal development, causing microcephaly, lissencephaly, ventriculomegaly, and calcifications. Several regulators of biological processes are involved in CZS development, and in this context, microRNAs (miRNAs) seem to have a fundamental role. miRNAs are important regulators of protein translation, as they form the RISC silencing complex and interact with complementary mRNA target sequences to further post-transcriptional repression. In this context, little is known about their participation in the pathogenesis of viral infections. In this review, we discuss how miRNAs could relate to ZIKV and other flavivirus infections.

Asunto(s)

MicroARNs/genética , Infección por el Virus Zika/genética , Infección por el Virus Zika/patología , Animales , Redes Reguladoras de Genes/genética , Humanos , Inmunidad/genética , Inmunidad/inmunología , MicroARNs/metabolismo , Replicación Viral/genética , Virus Zika/genética , Virus Zika/patogenicidad , Virus Zika/fisiología , Infección por el Virus Zika/virología

RESUMEN

Mayaro virus (MAYV) hijacks the host's cell machinery to effectively replicate. The mitogen-activated protein kinases (MAPKs) p38, JNK, and ERK1/2 have emerged as crucial cellular factors implicated in different stages of the viral cycle. However, whether MAYV uses these MAPKs to competently replicate has not yet been determined. The aim of this study was to evaluate the impact of MAPK inhibition on MAYV replication using primary human dermal fibroblasts (HDFs) and HeLa cells. Viral yields in supernatants from MAYV-infected cells treated or untreated with inhibitors SB203580, SP600125, U0126, or Losmapimod were quantified using plaque assay. Additionally, viral protein expression was analyzed using immunoblot and immunofluorescence. Knockdown of p38⍺/p38ß isoforms was performed in HDFs using the PROTACs molecule NR-7h. Our data demonstrated that HDFs are highly susceptible to MAYV infection. SB203580, a p38 inhibitor, reduced MAYV replication in a dose-dependent manner in both HDFs and HeLa cells. Additionally, SB203580 significantly decreased viral E1 protein expression. Similarly, knockdown or inhibition of p38⍺/p38ß isoforms with NR-7h or Losmapimod, respectively, affected MAYV replication in a dose-dependent manner. Collectively, these findings suggest that p38 could play an important role in MAYV replication and could serve as a therapeutic target to control MAYV infection.

Asunto(s)

Alphavirus/fisiología , Fibroblastos/virología , Replicación Viral/genética , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Apoptosis , Células Cultivadas , Ciclopropanos/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Células HeLa , Interacciones Microbiota-Huesped/efectos de los fármacos , Humanos , Imidazoles/farmacología , Sistema de Señalización de MAP Quinasas , Fosforilación , Piridinas/farmacología , Piel/citología , Piel/virología , Replicación Viral/efectos de los fármacos

RESUMEN

Murine leukemia virus (MLV) requires the infected cell to divide to access the nucleus to integrate into the host genome. It has been determined that MLV uses the microtubule and actin network to reach the nucleus at the early stages of infection. Several studies have shown that viruses use the dynein motor protein associated with microtubules for their displacement. We have previously reported that dynein light-chain roadblock type 2 (Dynlrb2) knockdown significantly decreases MLV infection compared to nonsilenced cells, suggesting a functional association between this dynein light chain and MLV preintegration complex (PIC). In this study, we aimed to determine if the dynein complex Dynlrb2 subunit plays an essential role in the retrograde transport of MLV. For this, an MLV mutant containing the green fluorescent protein (GFP) fused to the viral protein p12 was used to assay the PIC localization and speed in cells in which the expression of Dynlrb2 was modulated. We found a significant decrease in the arrival of MLV PIC to the nucleus and a reduced net speed of MLV PICs when Dynlrb2 was knocked down. In contrast, an increase in nuclear localization was observed when Dynlrb2 was overexpressed. Our results suggest that Dynlrb2 plays an essential role in MLV retrograde transport. IMPORTANCE Different viruses use different components of cytoplasmic dynein complex to traffic to their replication site. We have found that murine leukemia virus (MLV) depends on dynein light-chain Dynlrb2 for infection, retrograde traffic, and nuclear entry. Our study provides new information regarding the molecular requirements for retrograde transport of MLV preintegration complex and demonstrates the essential role of Dynlrb2 in MLV infection.

Asunto(s)

Transporte Activo de Núcleo Celular/fisiología , Dineínas Citoplasmáticas/genética , Dineínas/metabolismo , Virus de la Leucemia Murina/crecimiento & desarrollo , Replicación Viral/genética , Células 3T3 , Transporte Activo de Núcleo Celular/genética , Animales , Línea Celular , Núcleo Celular/virología , Dineínas/genética , Productos del Gen gag/genética , Células HEK293 , Interacciones Huésped-Patógeno/fisiología , Humanos , Ratones , Microtúbulos/metabolismo

RESUMEN

Venezuelan equine encephalitis virus (VEEV) is a reemerging arthropod-borne virus causing encephalitis in humans and domesticated animals. VEEV possesses a positive single-stranded RNA genome capped at its 5' end. The capping process is performed by the nonstructural protein nsP1, which bears methyl and guanylyltransferase activities. The capping reaction starts with the methylation of GTP. The generated m7GTP is complexed to the enzyme to form an m7GMP-nsP1 covalent intermediate. The m7GMP is then transferred onto the 5'-diphosphate end of the viral RNA. Here, we explore the specificities of the acceptor substrate in terms of length, RNA secondary structure, and/or sequence. Any diphosphate nucleosides but GDP can serve as acceptors of the m7GMP to yield m7GpppA, m7GpppC, or m7GpppU. We show that capping is more efficient on small RNA molecules, whereas RNAs longer than 130 nucleotides are barely capped by the enzyme. The structure and sequence of the short, conserved stem-loop, downstream to the cap, is an essential regulatory element for the capping process. IMPORTANCE The emergence, reemergence, and expansion of alphaviruses (genus of the family Togaviridae) are a serious public health and epizootic threat. Venezuelan equine encephalitis virus (VEEV) causes encephalitis in human and domesticated animals, with a mortality rate reaching 80% in horses. To date, no efficient vaccine or safe antivirals are available for human use. VEEV nonstructural protein 1 (nsP1) is the viral capping enzyme characteristic of the Alphavirus genus. nsP1 catalyzes methyltransferase and guanylyltransferase reactions, representing a good therapeutic target. In the present report, we provide insights into the molecular features and specificities of the cap acceptor substrate for the guanylylation reaction.

Asunto(s)

Virus de la Encefalitis Equina Venezolana/genética , Caperuzas de ARN/genética , ARN Viral/genética , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/genética , Animales , Encefalomielitis Equina Venezolana/patología , Encefalomielitis Equina Venezolana/virología , Caballos , Humanos , Metiltransferasas/metabolismo , Conformación de Ácido Nucleico , Nucleotidiltransferasas/metabolismo , Proteínas no Estructurales Virales/genética

RESUMEN

Geminiviruses have genomes composed of single-stranded DNA molecules and encode a rolling-circle replication (RCR) initiation protein ("Rep"), which has multiple functions. Rep binds to specific repeated DNA motifs ("iterons"), which are major determinants of virus-specific replication. The particular amino acid (aa) residues that determine the preference of a geminivirus Rep for specific iterons (i.e., the trans-acting replication "specificity determinants", or SPDs) are largely unknown, but diverse lines of evidence indicate that most of them are closely associated with the so-called RCR motif I (FLTYP), located in the first 12-19 aa residues of the protein. In this work, we characterized two strains of a novel begomovirus, rhynchosia golden mosaic Sinaloa virus (RhGMSV), that were incompatible in replication in pseudorecombination experiments. Systematic comparisons of the Rep proteins of both RhGMSV strains in the DNA-binding domain allowed the aa residues at positions 71 and 74 to be identified as the residues most likely to be responsible for differences in replication specificity. Residue 71 is part of the ß-5 strand structural element, which was predicted in previous studies to contain Rep SPDs. Since the Rep proteins encoded by both RhGMSV strains are identical in their first 24 aa residues, where other studies have mapped potential SPDs, this is the first study lending direct support to the notion that geminivirus Rep proteins contain separate SPDs in their N-terminal domain.

Asunto(s)

Begomovirus/clasificación , Begomovirus/metabolismo , Proteínas Virales/metabolismo , Replicación Viral/fisiología , Secuencia de Aminoácidos , Begomovirus/genética , Clonación Molecular , Fabaceae/virología , Genoma Viral , Filogenia , Hojas de la Planta/virología , Conformación Proteica , Virus Reordenados , Nicotiana/virología , Proteínas Virales/genética , Replicación Viral/genética

RESUMEN

Here, we present for the first time, a site-specific N-glycosylation analysis of proteins from a Brazilian Zika virus (ZIKV) strain. The virus was propagated with high yield in an embryo-derived stem cell line (EB66, Valneva SE), and concentrated by g-force step-gradient centrifugation. Subsequently, the sample was proteolytically digested with different enzymes, measured via a LC-MS/MS-based workflow, and analyzed in a semi-automated way using the in-house developed glyXtoolMS software. The viral non-structural protein 1 (NS1) was glycosylated exclusively with high-mannose structures on both potential N-glycosylation sites. In case of the viral envelope (E) protein, no specific N-glycans could be identified with this method. Nevertheless, N-glycosylation could be proved by enzymatic de-N-glycosylation with PNGase F, resulting in a strong MS-signal of the former glycopeptide with deamidated asparagine at the potential N-glycosylation site N444. This confirmed that this site of the ZIKV E protein is highly N-glycosylated but with very high micro-heterogeneity. Our study clearly demonstrates the progress made towards site-specific N-glycosylation analysis of viral proteins, i.e. for Brazilian ZIKV. It allows to better characterize viral isolates, and to monitor glycosylation of major antigens. The method established can be applied for detailed studies regarding the impact of protein glycosylation on antigenicity and human pathogenicity of many viruses including influenza virus, HIV and corona virus.

Asunto(s)

Glicosilación , Proteínas no Estructurales Virales/genética , Infección por el Virus Zika/genética , Virus Zika/genética , Animales , Asparagina/genética , Brasil , Línea Celular , Chlorocebus aethiops , Cromatografía Liquida , Humanos , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/genética , Espectrometría de Masas en Tándem , Células Vero , Proteínas del Envoltorio Viral/genética , Replicación Viral/genética , Virus Zika/patogenicidad , Infección por el Virus Zika/virología