RESUMO
Dengue is an important arboviral infectious disease for which there is currently no specific cure. We report gemini-like (geminoid) alkylated amphiphilic peptides containing lysines in combination with glycines or alanines (C15H31C(O)-Lys-(Gly or Ala)nLys-NHC16H33, shorthand notation C16-KXnK-C16 with X = A or G, and n = 0-2). The representatives with 1 or 2 Ala inhibit dengue protease and human furin, two serine proteases involved in dengue virus infection that have peptides with cationic amino acids as their preferred substrates, with IC50 values in the lower µM range. The geminoid C16-KAK-C16 combined inhibition of DENV2 protease (IC50 2.3 µM) with efficacy against replication of wildtype DENV2 in LLC-MK2 cells (EC50 4.1 µM) and an absence of toxicity. We conclude that the lysine-based geminoids have activity against dengue virus infection, which is based on their inhibition of the proteases involved in viral replication and are therefore promising leads to further developing antiviral therapeutics, not limited to dengue.
Assuntos
Antivirais , Vírus da Dengue , Furina , Inibidores de Proteases , Replicação Viral , Antivirais/farmacologia , Dengue/tratamento farmacológico , Vírus da Dengue/efeitos dos fármacos , Vírus da Dengue/fisiologia , Furina/antagonistas & inibidores , Humanos , Peptídeo Hidrolases , Peptídeos/farmacologia , Inibidores de Proteases/farmacologia , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
Despite the enormous disease burden associated with dengue virus infections, a licensed antiviral drug is lacking. Here, we show that the paracetamol (acetaminophen) metabolite AM404 inhibits dengue virus replication. Moreover, we find that mutations in NS4B that were previously found to confer resistance to the antiviral compounds NITD-618 and SDM25N also render dengue virus insensitive to AM404. Our work provides further support for NS4B as a direct or indirect target for antiviral drug development.
Assuntos
Antivirais/farmacologia , Ácidos Araquidônicos/farmacologia , Vírus da Dengue/efeitos dos fármacos , Farmacorresistência Viral Múltipla/genética , Mutação , Proteínas não Estruturais Virais/genética , Acetaminofen/metabolismo , Acetaminofen/farmacologia , Ácidos Araquidônicos/metabolismo , Biotransformação , Vírus da Dengue/genética , Vírus da Dengue/crescimento & desenvolvimento , Vírus da Dengue/metabolismo , Expressão Gênica , Genes Reporter , Células HeLa , Humanos , Luciferases/genética , Luciferases/metabolismo , Testes de Sensibilidade Microbiana , Naltrexona/análogos & derivados , Naltrexona/farmacologia , Ribavirina/farmacologia , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
The ongoing conflict between viruses and their hosts can drive the co-evolution between host immune genes and viral suppressors of immunity. It has been suggested that an evolutionary 'arms race' may occur between rapidly evolving components of the antiviral RNAi pathway of Drosophila and viral genes that antagonize it. We have recently shown that viral protein 1 (VP1) of Drosophila melanogaster Nora virus (DmelNV) suppresses Argonaute-2 (AGO2)-mediated target RNA cleavage (slicer activity) to antagonize antiviral RNAi. Here we show that viral AGO2 antagonists of divergent Nora-like viruses can have host specific activities. We have identified novel Nora-like viruses in wild-caught populations of D. immigrans (DimmNV) and D. subobscura (DsubNV) that are 36% and 26% divergent from DmelNV at the amino acid level. We show that DimmNV and DsubNV VP1 are unable to suppress RNAi in D. melanogaster S2 cells, whereas DmelNV VP1 potently suppresses RNAi in this host species. Moreover, we show that the RNAi suppressor activity of DimmNV VP1 is restricted to its natural host species, D. immigrans. Specifically, we find that DimmNV VP1 interacts with D. immigrans AGO2, but not with D. melanogaster AGO2, and that it suppresses slicer activity in embryo lysates from D. immigrans, but not in lysates from D. melanogaster. This species-specific interaction is reflected in the ability of DimmNV VP1 to enhance RNA production by a recombinant Sindbis virus in a host-specific manner. Our results emphasize the importance of analyzing viral RNAi suppressor activity in the relevant host species. We suggest that rapid co-evolution between RNA viruses and their hosts may result in host species-specific activities of RNAi suppressor proteins, and therefore that viral RNAi suppressors could be host-specificity factors.
Assuntos
Proteínas Argonautas , Proteínas de Drosophila , Interferência de RNA , Vírus de RNA , Proteínas Virais , Animais , Proteínas Argonautas/antagonistas & inibidores , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Sequência de Bases , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Dados de Sequência Molecular , Vírus de RNA/genética , Vírus de RNA/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Invertebrate RNA viruses are targets of the host RNA interference (RNAi) pathway, which limits virus infection by degrading viral RNA substrates. Several insect RNA viruses encode suppressor proteins to counteract this antiviral response. We recently demonstrated that the dsDNA virus Invertebrate iridescent virus 6 (IIV-6) induces an RNAi response in Drosophila. Here, we show that RNAi is suppressed in IIV-6-infected cells and we mapped RNAi suppressor activity to the viral protein 340R. Using biochemical assays, we reveal that 340R binds long dsRNA and prevents Dicer-2-mediated processing of long dsRNA into small interfering RNAs (siRNAs). We demonstrate that 340R additionally binds siRNAs and inhibits siRNA loading into the RNA-induced silencing complex. Finally, we show that 340R is able to rescue a Flock House virus replicon that lacks its viral suppressor of RNAi. Together, our findings indicate that, in analogy to RNA viruses, DNA viruses antagonize the antiviral RNAi response.
Assuntos
Iridovirus/fisiologia , Interferência de RNA , Proteínas de Ligação a RNA/metabolismo , Proteínas Virais/metabolismo , Animais , Proteínas Argonautas/metabolismo , Linhagem Celular , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/virologia , Nodaviridae/fisiologia , RNA Helicases/antagonistas & inibidores , RNA Helicases/metabolismo , Processamento Pós-Transcricional do RNA , RNA de Cadeia Dupla/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/química , Replicon , Ribonuclease III/antagonistas & inibidores , Ribonuclease III/metabolismo , Proteínas Virais/química , Replicação ViralRESUMO
RNA interference (RNAi) is a crucial antiviral defense mechanism in insects, including the major mosquito species that transmit important human viruses. To counteract the potent antiviral RNAi pathway, insect viruses encode RNAi suppressors. However, whether mosquito-specific viruses suppress RNAi remains unclear. We therefore set out to study RNAi suppression by Culex Y virus (CYV), a mosquito-specific virus of the Birnaviridae family that was recently isolated from Culex pipiens mosquitoes. We found that the Culex RNAi machinery processes CYV double-stranded RNA (dsRNA) into viral small interfering RNAs (vsiRNAs). Furthermore, we show that RNAi is suppressed in CYV-infected cells and that the viral VP3 protein is responsible for RNAi antagonism. We demonstrate that VP3 can functionally replace B2, the well-characterized RNAi suppressor of Flock House virus. VP3 was found to bind long dsRNA as well as siRNAs and interfered with Dicer-2-mediated cleavage of long dsRNA into siRNAs. Slicing of target RNAs by pre-assembled RNA-induced silencing complexes was not affected by VP3. Finally, we show that the RNAi-suppressive activity of VP3 is conserved in Drosophila X virus, a birnavirus that persistently infects Drosophila cell cultures. Together, our data indicate that mosquito-specific viruses may encode RNAi antagonists to suppress antiviral RNAi.
Assuntos
Culex/genética , Drosophila melanogaster/genética , Entomobirnavirus/fisiologia , Interferência de RNA , RNA de Cadeia Dupla/metabolismo , RNA Interferente Pequeno/metabolismo , Animais , Células Cultivadas , Culex/virologia , Drosophila melanogaster/virologia , Entomobirnavirus/genética , Entomobirnavirus/metabolismo , Proteínas Virais/metabolismoRESUMO
UNLABELLED: Insects are a reservoir for many known and novel viruses. We discovered an unknown virus, tentatively named mosinovirus (MoNV), in mosquitoes from a tropical rainforest region in Côte d'Ivoire. The MoNV genome consists of two segments of positive-sense RNA of 2,972 nucleotides (nt) (RNA 1) and 1,801 nt (RNA 2). Its putative RNA-dependent RNA polymerase shares 43% amino acid identity with its closest relative, that of the Pariacoto virus (family Nodaviridae). Unexpectedly, for the putative capsid protein, maximal pairwise identity of 16% to Lake Sinai virus 2, an unclassified virus with a nonsegmented RNA genome, was found. Moreover, MoNV virions are nonenveloped and about 50 nm in diameter, larger than any of the known nodaviruses. Mature MoNV virions contain capsid proteins of â¼ 56 kDa, which do not seem to be cleaved from a longer precursor. Northern blot analyses revealed that MoNV expresses two subgenomic RNAs of 580 nt (RNA 3) and 292 nt (RNA 4). RNA 4 encodes a viral suppressor of RNA interference (RNAi) that shares its mechanism with the B2 RNAi suppressor protein of other nodaviruses despite lacking recognizable similarity to these proteins. MoNV B2 binds long double-stranded RNA (dsRNA) and, accordingly, inhibits Dicer-2-mediated processing of dsRNA into small interfering RNAs (siRNAs). Phylogenetic analyses indicate that MoNV is a novel member of the family Nodaviridae that acquired its capsid gene via reassortment from an unknown, distantly related virus beyond the family level. IMPORTANCE: The identification of novel viruses provides important information about virus evolution and diversity. Here, we describe an unknown unique nodavirus in mosquitoes, named mosinovirus (MoNV). MoNV was classified as a nodavirus based on its genome organization and on phylogenetic analyses of the RNA-dependent RNA polymerase. Notably, its capsid gene was acquired from an unknown virus with a distant relationship to nodaviruses. Another remarkable feature of MoNV is that, unlike other nodaviruses, it expresses two subgenomic RNAs (sgRNAs). One of the sgRNAs expresses a protein that counteracts antiviral defense of its mosquito host, whereas the function of the other sgRNA remains unknown. Our results show that complete genome segments can be exchanged beyond the species level and suggest that insects harbor a large repertoire of exceptional viruses.
Assuntos
Regulação Viral da Expressão Gênica , Nodaviridae/genética , Nodaviridae/fisiologia , RNA Viral/biossíntese , Replicação Viral , Animais , Proteínas do Capsídeo/análise , Proteínas do Capsídeo/genética , Análise por Conglomerados , Côte d'Ivoire , Culicidae/virologia , Dados de Sequência Molecular , Filogenia , RNA de Cadeia Dupla/metabolismo , RNA Viral/genética , Proteínas de Ligação a RNA/metabolismo , Recombinação Genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Vírion/químicaRESUMO
RNA viruses in insects are targets of an RNA interference (RNAi)-based antiviral immune response, in which viral replication intermediates or viral dsRNA genomes are processed by Dicer-2 (Dcr-2) into viral small interfering RNAs (vsiRNAs). Whether dsDNA virus infections are controlled by the RNAi pathway remains to be determined. Here, we analyzed the role of RNAi in DNA virus infection using Drosophila melanogaster infected with Invertebrate iridescent virus 6 (IIV-6) as a model. We show that Dcr-2 and Argonaute-2 mutant flies are more sensitive to virus infection, suggesting that vsiRNAs contribute to the control of DNA virus infection. Indeed, small RNA sequencing of IIV-6-infected WT and RNAi mutant flies identified abundant vsiRNAs that were produced in a Dcr-2-dependent manner. We observed a highly uneven distribution with strong clustering of vsiRNAs to small defined regions (hotspots) and modest coverage at other regions (coldspots). vsiRNAs mapped in similar proportions to both strands of the viral genome, suggesting that long dsRNA derived from convergent overlapping transcripts serves as a substrate for Dcr-2. In agreement, strand-specific RT-PCR and Northern blot analyses indicated that antisense transcripts are produced during infection. Moreover, we show that vsiRNAs are functional in silencing reporter constructs carrying fragments of the IIV-6 genome. Together, our data indicate that RNAi provides antiviral defense against dsDNA viruses in animals. Thus, RNAi is the predominant antiviral defense mechanism in insects that provides protection against all major classes of viruses.
Assuntos
Vírus de DNA/genética , Regulação Viral da Expressão Gênica , Interferência de RNA , Animais , Antivirais/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Inativação Gênica , Genoma Viral , Cinética , Modelos Genéticos , Mutação , Reação em Cadeia da Polimerase , RNA Interferente Pequeno/metabolismo , Análise de Sequência de DNA , Wolbachia/metabolismoRESUMO
After decades of being considered non-pathogenic, Zika virus (ZIKV) emerged as an important threat to human health during the epidemic of 2015-2016. ZIKV infections are usually asymptomatic, but can cause Guillain-Barré syndrome in adults and microcephaly in newborns. As there are currently no approved antiviral drugs against ZIKV, we tested anti-ZIKV activity of compounds from the NIH Clinical Collection for which we previously showed antiviral activity against the related dengue virus. One of the top hits from the screen was lacidipine, a 1,4-dihydropyridine calcium antagonist that is approved as an antihypertensive drug. Our data show that lacidipine is antiviral against ZIKV (strain H/PF/2013) in both Vero cells and induced pluripotent stem cell (iPSC)-derived human neural progenitor cells with IC50 values of 3.0 µM and <50 nM, respectively. The antiviral effect was also observed against four other ZIKV strains from the African and Asian lineages. Time-of-addition and replicon assays indicated that lacidipine acts at the post-entry stage of the viral replication cycle, inhibiting viral genome replication. Lacidipine altered the subcellular distribution of free cholesterol and neutral lipids, suggesting that the antiviral effect of lacidipine is mediated by altered trafficking of lipids. Together, these results identify lacidipine as a novel inhibitor of ZIKV replication that likely disturbs trafficking of lipids needed for replication organelle formation.
Assuntos
Bloqueadores dos Canais de Cálcio , Di-Hidropiridinas , Células-Tronco Neurais , Infecção por Zika virus , Animais , Antivirais/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio , Chlorocebus aethiops , Di-Hidropiridinas/farmacologia , Humanos , Recém-Nascido , Lipídeos , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/virologia , Células-Tronco , Células Vero , Replicação Viral , Zika virus , Infecção por Zika virus/tratamento farmacológicoRESUMO
Dengue virus (DENV) is associated with an estimated 390 million infections per year, occurring across approximately 100 countries in tropical and sub-tropical regions. To date, there are no antiviral drugs or specific therapies to treat DENV infection. Posaconazole and itraconazole are potent antifungal drugs that inhibit ergosterol biosynthesis in fungal cells, but also target a number of human proteins. Here, we show that itraconazole and posaconazole have antiviral activity against DENV. Posaconazole inhibited replication of multiple serotypes of DENV and the related flavivirus Zika virus, and reduced viral RNA replication, but not translation of the viral genome. We used a combination of knockdown and drug sensitization assays to define the molecular target of posaconazole that mediates its antiviral activity. We found that knockdown of oxysterol-binding protein (OSBP) inhibited DENV replication. Moreover, knockdown of OSBP, but not other known targets of posaconazole, enhanced the inhibitory effect of posaconazole. Our findings imply OSBP as a potential target for the development of antiviral compounds against DENV.
Assuntos
Antivirais/farmacologia , Vírus da Dengue/efeitos dos fármacos , Reposicionamento de Medicamentos , Receptores de Esteroides/antagonistas & inibidores , Triazóis/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Antifúngicos/farmacologia , Chlorocebus aethiops , Vírus da Dengue/crescimento & desenvolvimento , Células HeLa , Humanos , Itraconazol/farmacologia , Células Vero , Zika virus/efeitos dos fármacos , Zika virus/crescimento & desenvolvimentoRESUMO
BACKGROUND: Cytomegaloviruses (CMVs) have developed various sophisticated strategies to manipulate and evade the defense mechanisms of their hosts. Among the CMV genes that are predicted to be involved in these strategies are genes that encode mimics of cellular proteins, such as G protein-coupled receptors (GPCRs) and chemokines (CKs). These genes may have been pirated from the host genome during the long co-evolution of virus and host. OBJECTIVES: In this report, the putative functions of the CMV-encoded homologs of GPCRs and CKs in the pathogenesis of infection will be discussed. STUDY DESIGN: In order to present an overview of the current state of knowledge, the literature on the CMV-encoded homologs of GPCRs and CKs was reviewed. RESULTS: The GPCR and CK homologs that are encoded by the CMVs represent immunomodulatory proteins with crucial roles in the pathogenesis of infection. CONCLUSIONS: In light of their function as well as accessibility on the cell surface, the CMV-encoded GPCR homologs are attractive targets for the development of new anti-viral therapies.
Assuntos
Quimiocinas/fisiologia , Infecções por Citomegalovirus/virologia , Citomegalovirus/patogenicidade , Mimetismo Molecular , Receptores Acoplados a Proteínas G/fisiologia , Proteínas Virais/fisiologia , Quimiocinas/genética , Citomegalovirus/genética , Genes Virais , Humanos , Receptores Acoplados a Proteínas G/genética , Homologia de Sequência de Aminoácidos , Proteínas Virais/genéticaRESUMO
Dengue virus (DENV) is an important human arthropod-borne virus with a major impact on public health. Nevertheless, a licensed vaccine or specific treatment is still lacking. We therefore screened the NIH Clinical Collection (NCC), a library of drug-like small molecules, for inhibitors of DENV replication using a cell line that contains a stably replicating DENV serotype 2 (DENV2) subgenomic replicon. The most potent DENV inhibitor in the NCC was δ opioid receptor antagonist SDM25N. This compound showed antiviral activity against wild-type DENV2 in both Hela and BHK-21 cells, but not in the C6/36 cell line derived from the mosquito Aedes albopictus. The structurally related compound naltrindole also inhibited DENV replication, albeit less potently. Using a transient subgenomic replicon, we demonstrate that SDM25N restricts genomic RNA replication rather than translation of the viral genome. We identified a single amino acid substitution (F164L) in the NS4B protein that confers resistance to SDM25N. Remarkably, an NS4B amino acid substitution (P104L), which was previously shown to confer resistance to the DENV inhibitor NITD-618, also provided resistance to SDM25N. In conclusion, we have identified a new DENV inhibitor, SDM25N, which restricts genomic RNA replication by - directly or indirectly - targeting the viral NS4B protein.
Assuntos
Antivirais/farmacologia , Vírus da Dengue/efeitos dos fármacos , Naltrexona/análogos & derivados , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Aedes , Substituição de Aminoácidos , Animais , Linhagem Celular , Cricetinae , Dengue/tratamento farmacológico , Vírus da Dengue/genética , Vírus da Dengue/fisiologia , Farmacorresistência Viral/genética , Células HeLa , Humanos , Mutação , Naltrexona/farmacologia , Inibidores da Síntese de Ácido Nucleico/farmacologia , RNA Viral/biossíntese , RNA Viral/genética , Replicon/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas , Proteínas não Estruturais Virais/química , Replicação Viral/efeitos dos fármacosRESUMO
The siRNA pathway is an essential antiviral mechanism in insects. Whether other RNA interference pathways are involved in antiviral defense remains unclear. Here, we report in cells derived from the two main vectors for arboviruses, Aedes albopictus and Aedes aegypti, the production of viral small RNAs that exhibit the hallmarks of ping-pong derived piwi-associated RNAs (piRNAs) after infection with positive or negative sense RNA viruses. Furthermore, these cells produce endogenous piRNAs that mapped to transposable elements. Our results show that these mosquito cells can initiate de novo piRNA production and recapitulate the ping-pong dependent piRNA pathway upon viral infection. The mechanism of viral-piRNA production is discussed.
Assuntos
Arbovírus/genética , Arbovírus/fisiologia , Culicidae/genética , Culicidae/virologia , Amplificação de Genes , Integração Viral/genética , Aedes/citologia , Aedes/genética , Aedes/virologia , Animais , Infecções por Arbovirus/genética , Infecções por Arbovirus/virologia , Sequência de Bases , Células Cultivadas , Elementos de DNA Transponíveis/fisiologia , Amplificação de Genes/genética , Amplificação de Genes/fisiologia , Perfilação da Expressão Gênica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
RNA interference (RNAi) is an important pathway to combat virus infections in insects and plants. Hallmarks of antiviral RNAi in these organisms are: (1) an increase in virus replication after inactivation of major actors in the RNAi pathway, (2) production of virus-derived small interfering RNAs (v-siRNAs), and (3) suppression of RNAi by dedicated viral proteins. In this chapter, we will review the mechanism of RNAi in insects, its function as an antiviral immune system, viral small RNA profiles, and viral counterdefense strategies. We will also consider alternative, inducible antiviral immune responses.
Assuntos
Drosophila melanogaster/genética , Drosophila melanogaster/imunologia , Sistema Imunitário/imunologia , Interferência de RNA , Vírus/patogenicidade , Animais , Drosophila melanogaster/virologia , Humanos , RNA Interferente Pequeno/genética , Vírus/genéticaRESUMO
The RNA interference (RNAi) pathway plays an important role in antiviral immunity in insects. To -counteract the RNAi-mediated immune response of their hosts, several insect viruses, such as Flock house virus, Drosophila C virus, and Cricket paralysis virus, encode potent viral suppressors of RNAi (VSRs). Because of the importance of RNAi in antiviral defense in insects, other insect viruses are likely to encode VSRs as well. In this chapter, we describe a detailed protocol for an RNAi reporter assay in Drosophila S2 cells for the identification of VSR activity.
Assuntos
Drosophila melanogaster/citologia , Drosophila melanogaster/virologia , Genes Reporter/genética , Genes Supressores , Genes Virais/genética , Interferência de RNA , Animais , Linhagem Celular , Drosophila melanogaster/genética , Luciferases de Vaga-Lume/genética , RNA de Cadeia Dupla/biossíntese , RNA de Cadeia Dupla/genética , Transcrição Gênica , TransfecçãoRESUMO
Virus-derived small interfering RNAs (siRNAs) are the hallmark of RNAi-based antiviral immunity. Wu and colleagues demonstrate how viral genomes can be assembled from these small RNA sequences. Their results provide an approach for virus discovery as well as important insights into how these siRNAs mediate antiviral defense.
RESUMO
BACKGROUND: Müller cell gliosis occurs in various retinal pathologies regardless of the underlying cellular defect. Because activated Müller glial cells span the entire retina and align areas of injury, they are ideal targets for therapeutic strategies, including gene therapy. METHODOLOGY/PRINCIPAL FINDINGS: We used adeno-associated viral AAV2/6 vectors to transduce mouse retinas. The transduction pattern of AAV2/6 was investigated by studying expression of the green fluorescent protein (GFP) transgene using scanning-laser ophthalmoscopy and immuno-histochemistry. AAV2/6 vectors transduced mouse Müller glial cells aligning the retinal blood vessels. However, the transduction capacity was hindered by the inner limiting membrane (ILM) and besides Müller glial cells, several other inner retinal cell types were transduced. To obtain Müller glial cell-specific transgene expression, the cytomegalovirus (CMV) promoter was replaced by the glial fibrillary acidic protein (GFAP) promoter. Specificity and activation of the GFAP promoter was tested in a mouse model for retinal gliosis. Mice deficient for Crumbs homologue 1 (CRB1) develop gliosis after light exposure. Light exposure of Crb1(-/-) retinas transduced with AAV2/6-GFAP-GFP induced GFP expression restricted to activated Müller glial cells aligning retinal blood vessels. CONCLUSIONS/SIGNIFICANCE: Our experiments indicate that AAV2 vectors carrying the GFAP promoter are a promising tool for specific expression of transgenes in activated glial cells.
Assuntos
Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Fluorescência Verde/genética , Neuroglia/metabolismo , Vasos Retinianos/citologia , Transdução Genética/métodos , Adenoviridae/genética , Animais , Expressão Gênica , Humanos , Injeções , Camundongos , Regiões Promotoras Genéticas/genética , Transgenes/genéticaRESUMO
Rat cytomegalovirus (RCMV) is a beta-herpesvirus with a 230-kbp genome containing over 167 open reading frames (ORFs). RCMV gene expression is tightly regulated in cultured cells, occurring in three distinct kinetic classes (immediate early, early, and late). However, the extent of viral-gene expression in vivo and its relationship to the in vitro expression are unknown. In this study, we used RCMV-specific DNA microarrays to investigate the viral transcriptional profiles in cultured, RCMV-infected endothelial cells, fibroblasts, and aortic smooth muscle cells and to compare these profiles to those found in tissues from RCMV-infected rat heart transplant recipients. In cultured cells, RCMV expresses approximately 95% of the known viral ORFs with few differences between cell types. By contrast, in vivo viral-gene expression in tissues from rat heart allograft recipients is highly restricted. In the tissues studied, a total of 80 viral genes expressing levels twice above background (5,000 to 10,000 copies per mug total RNA) were detected. In each tissue type, there were a number of genes expressed exclusively in that tissue. Although viral mRNA and genomic DNA levels were lower in the spleen than in submandibular glands, the number of individual viral genes expressed was higher in the spleen (60 versus 41). This finding suggests that the number of viral genes expressed is specific to a given tissue and is not dependent upon the viral load or viral mRNA levels. Our results demonstrate that the profiles, as well as the amplitude, of viral-gene expression are tissue specific and are dramatically different from those in infected cultured cells, indicating that RCMV gene expression in vitro does not reflect viral-gene expression in vivo.
Assuntos
Expressão Gênica , Transplante de Coração , Infecções por Herpesviridae/virologia , Muromegalovirus/genética , RNA Viral/biossíntese , Animais , Aorta/virologia , Células Cultivadas , DNA Viral/análise , Células Endoteliais/virologia , Fibroblastos/virologia , Perfilação da Expressão Gênica , Muromegalovirus/crescimento & desenvolvimento , Músculo Liso Vascular/citologia , Músculo Liso Vascular/virologia , Miócitos de Músculo Liso/virologia , Análise de Sequência com Séries de Oligonucleotídeos , Especificidade de Órgãos , RNA Mensageiro/biossíntese , Ratos , Ratos Endogâmicos F344 , Baço/virologia , Glândula Submandibular/virologia , Transplante HomólogoRESUMO
The rat cytomegalovirus (RCMV) R33 and R78 genes are conserved within members of the subfamily Betaherpesvirinae and encode proteins (pR33 and pR78, respectively) that show sequence similarity with G protein-coupled receptors. Previously, the biological relevance of these genes was demonstrated by the finding that R33- and R78-deleted RCMV strains are severely attenuated in vivo. In addition, R78-deleted strains were found to replicate less efficiently in cell culture. To monitor of the expression of R33- and R78-encoded proteins, recombinant RCMV strains, designated RCMV33G and RCMV78G, were generated. These recombinants expressed enhanced green fluorescent protein (EGFP)-tagged versions of pR33 and pR78 instead of native pR33 and pR78, respectively. Here it is reported that, although RCMV33G replicates as efficiently as wt virus in rat embryo fibroblast cultures, strain RCMV78G produces virus titres that are 3- to 4-fold lower than wt RCMV in the culture medium. This result indicates that the pR78-EGFP protein, as expressed by RCMV78G, does not completely functionally replace its native counterpart (pR78) in vitro. Interestingly, in infected rats, infectious RCMV33G was produced in significantly lower amounts than infectious wt RCMV, as well as RCMV78G, in the salivary glands. Conversely, although RCMV33G replicated to similar levels as wt virus in the spleen, both RCMV78G and an R78 knock-out strain (RCMV Delta R78a) replicated poorly in this organ. Together, these data indicate that R78 is crucial for the production of infectious RCMV in the spleen of infected rats.
Assuntos
Infecções por Herpesviridae/virologia , Muromegalovirus/fisiologia , Receptores Acoplados a Proteínas G , Proteínas Virais/fisiologia , Animais , Modelos Animais de Doenças , Deleção de Genes , Proteínas de Fluorescência Verde , Infecções por Herpesviridae/metabolismo , Proteínas Luminescentes , Masculino , Muromegalovirus/genética , Muromegalovirus/metabolismo , RNA Mensageiro/análise , RNA Viral/análise , Ratos , Ratos Endogâmicos Lew , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Baço/virologia , Fatores de Tempo , Transfecção , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação ViralRESUMO
Rat cytomegalovirus (RCMV) possesses two adjacent genes, r131 and r129, which have the potential to encode CC chemokine homologs. Interestingly, the amino acid sequences encoded by both genes show similarity to the sequence of the murine CMV (MCMV) MCK-2 protein, which is encoded by the m131/129 gene. In order to study the significance of the r131 gene in the pathogenesis of RCMV infection, we generated two different virus strains in which the r131 open reading frame is disrupted. Replication of these null mutant strains, designated RCMVdeltar131a and RCMVdeltar131b, was evaluated in vitro and in vivo. Both strains were found to replicate with a similar efficiency as wild-type (WT) RCMV in vitro. However, in contrast to WT virus, neither RCMVdeltar131a nor RCMVdeltar131b established a high-titer infection in the salivary glands of immunocompromised rats. Furthermore, in a local, rat footpad infection model, both recombinant viruses induced a significantly lower amount of paw swelling than did WT RCMV. Also, a higher number of infiltrating macrophages was observed in paws infected with WT RCMV than in those infected with the recombinants. Taken together, these results suggest that r131 (i) promotes inflammation at initial sites of inoculation and, subsequently, efficient virus dissemination to or infection of the salivary glands and (ii) might be involved in the persistence of virus infection, at least in the spleen. In addition, our data indicate that r131 represents the functional homolog of the MCMV m131/129 gene.
Assuntos
Quimiocinas CC/genética , Genes Essenciais , Muromegalovirus/patogenicidade , Glândulas Salivares/virologia , Proteínas Virais/genética , Replicação Viral , Sequência de Aminoácidos , Animais , Linhagem Celular , Quimiocinas CC/metabolismo , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/fisiopatologia , Humanos , Camundongos , Dados de Sequência Molecular , Muromegalovirus/genética , Muromegalovirus/fisiologia , Mutação , Ratos , Homologia de Sequência de Aminoácidos , Proteínas Virais/metabolismoRESUMO
An intriguing feature of the rat cytomegalovirus (RCMV) genome is open reading frame (ORF) r127, which shows similarity to the rep genes of parvoviruses as well as the U94 genes of human herpesvirus type 6A (HHV-6A) and 6B (HHV-6B). Counterparts of these genes have not been found in other herpesviruses. Here, it is shown that the r127 gene is transcribed during the early and late phases of virus replication in vitro as an unspliced 1.1 kb transcript containing the complete r127 ORF. Transcripts of r127 were also detected in various organs of RCMV-infected rats at 1 week post-infection (p.i.), but only in the salivary gland at 4 months p.i. Using rabbit polyclonal antibodies raised against the r127-encoded protein (pr127), pr127 was found to be expressed as early as 12 h p.i. within the nuclei of RCMV-infected cells in vitro. Expression of pr127 was also observed within the nuclei of cells in various organs of RCMV-infected rats at 3 weeks p.i. Moreover, pr127 was demonstrated to bind single- as well as double-stranded DNA. Finally, an RCMV r127 deletion mutant (RCMVDeltar127) was generated, in which the r127 ORF was disrupted. This deletion mutant, however, was shown to replicate with a similar efficiency as wild-type RCMV (wt RCMV), both in vitro and in vivo. Taken together, it is concluded that the RCMV r127 gene encodes a nuclear protein with single- and double-stranded DNA-binding activity that is dispensable for virus replication, not only in vitro, but also during the acute phase of infection in vivo.