RESUMO
Aedes aegypti is a major vector for arboviruses such as dengue, chikungunya and Zika viruses. During acquisition of a viremic bloodmeal, an arbovirus infects mosquito midgut cells before disseminating to secondary tissues, including the salivary glands. Once virus is released into the salivary ducts it can be transmitted to another vertebrate host. The midgut is surrounded by a basal lamina (BL) in the extracellular matrix, consisting of a proteinaceous mesh composed of collagen IV and laminin. BL pore size exclusion limit prevents virions from passing through. Thus, the BL probably requires remodelling via enzymatic activity to enable efficient virus dissemination. Matrix metalloproteinases (MMPs) are extracellular endopeptidases that are involved in remodelling of the extracellular matrix. Here, we describe and characterize the nine Ae. aegypti encoded MMPs, AeMMPs 1-9, which share common features with other invertebrate and vertebrate MMPs. Expression profiling in Ae. aegypti revealed that Aemmp4 and Aemmp6 were upregulated during metamorphosis, whereas expression of Aemmp1 and Aemmp2 increased during bloodmeal digestion. Aemmp1 expression was also upregulated in the presence of a bloodmeal containing chikungunya virus. Using polyclonal antibodies, AeMMP1 and AeMMP2 were specifically detected in tissues associated with the mosquito midgut.
Assuntos
Aedes/enzimologia , Metaloproteinases da Matriz/metabolismo , Aedes/genética , Aedes/crescimento & desenvolvimento , Aedes/virologia , Sequência de Aminoácidos , Animais , Vírus Chikungunya/fisiologia , Feminino , Trato Gastrointestinal/enzimologia , Expressão Gênica , Genoma de Inseto , Humanos , Masculino , Metaloproteinases da Matriz/genética , Metamorfose Biológica , Dados de Sequência Molecular , Homologia de Sequência do Ácido NucleicoRESUMO
Baculovirus infection of permissive cells proceeds in a cascade fashion with the transcription of early, late and very late genes. The structure of a number of baculovirus early gene promoters has been dissected in detail and the viral factors necessary to stimulate their expression have been identified. Early baculovirus gene promoters in general have a resemblance to host promoters while late and very late gene promoters are different from early baculovirus promoters and are more defined. In this study we investigated whether two key Autographa californica M nucleopolyhedrovirus (AcMNPV) transactivators have the ability to regulate the commonly used cellular promoter from the Drosophila heat shock 70 protein gene, during transient gene expression assays in two insect cell lines permissive for AcMNPV infection, SF-21 and TN-368, or during viral infection. The AcMNPV ie-1 transactivator gene stimulated gene expression of this cellular promoter in both cell lines when the promoter was cis-linked to an enhancer element, but stimulation in the absence of enhancer elements was either undetected or lower than in the presence of enhancer elements in SF-21 and TN-368 cells, respectively. The transactivator ie-2 stimulated gene expression in the presence of cis-linked enhancer elements and ie-1 in SF-21 cells. During viral infection, the heat shock 70 promoter was maximally activated at 12 hours post infection. We discuss how these results affect the interpretation of transient gene expression assays performed in the presence of viral transcription factors.
Assuntos
Proteínas de Ligação a DNA/genética , Expressão Gênica , Proteínas de Choque Térmico HSP70/genética , Proteínas Imediatamente Precoces/genética , Regiões Promotoras Genéticas/genética , Transativadores/genética , Ativação Transcricional , Animais , Linhagem Celular , Drosophila , Elementos Facilitadores Genéticos/fisiologiaRESUMO
An Autographa californica nuclear polyhedrosis virus (AcMNPV) gene required in transient expression assays for late and very late viral gene expression was identified, sequenced, and transcriptionally mapped. This gene, designated late expression factor 1 (lef-1), was located between 7.4 and 8.7 map units of the AcMNPV physical map. It was identified by cotransfecting Spodoptera frugiperda cultured cells with a collection of overlapping cloned DNA fragments covering the entire AcMNPV genome and a reporter gene controlled by an early, late, or very late AcMNPV promoter. Omission of the DNA fragment containing lef-1 curtailed most late and very late gene expression but not early gene expression. lef-1 was found to be an early gene transcribed as a 1.8-kb RNA in the presence of the protein synthesis inhibitor cycloheximide. The C terminus of the predicted polypeptide product, LEF-1, contained a sequence motif characteristic of nucleoside triphosphate-binding sites.
Assuntos
Baculoviridae/genética , Genes Virais/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Células Cultivadas , Cloranfenicol O-Acetiltransferase/biossíntese , Regulação Viral da Expressão Gênica , Dados de Sequência Molecular , Mariposas , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/biossíntese , Análise de Sequência de DNA , Transcrição Gênica , Transfecção , Proteínas Virais/biossínteseRESUMO
The cg30 gene of Autographa californica nuclear polyhedrosis virus (AcMNPV) encodes two sequence motifs, a zinc finger-like motif and a leucine zipper, found in other polypeptides known to be involved in gene regulation. To gain insight into the function of the cg30 product, CG30, we constructed and characterized recombinant viruses lacking a functional cg30 gene. We found that cg30 mutants had no striking phenotype in cell lines derived from Spodoptera frugiperda or Trichoplusia ni or in T. ni larvae. Although cg30 is known to be transcribed as an early monocistronic RNA and as the second cistron of an abundant late bicistronic RNA, production of a CG30-beta-galactosidase fusion protein was observed mainly at early times postinfection. Viruses containing cg30 had a subtle growth advantage over those lacking cg30 after several viral passages in cell culture. We employed transient expression assays to determine whether cg30 and pe-38, an AcMNPV gene that encodes a polypeptide with zinc finger-like and leucine zipper motifs similar to those of cg30, have redundant functions. Although pe-38 may have a role in AcMNPV gene expression, there was no indication that cg30 and pe-38 are functionally redundant.
Assuntos
Proteínas de Ligação a DNA/genética , Genes Reguladores/genética , Genes Virais/genética , Mariposas/microbiologia , Nucleopoliedrovírus/genética , Proteínas Virais , Alelos , Animais , Bioensaio , Células Cultivadas , DNA Recombinante , Regulação Viral da Expressão Gênica , Larva/microbiologia , Mariposas/citologia , Nucleopoliedrovírus/patogenicidade , Inoculações Seriadas , Fatores de Tempo , VirulênciaRESUMO
Using a previously developed method that allows the identification of Autographa californica nuclear polyhedrosis virus (AcMNPV) genes which stimulate transient expression from a late and a very late viral promoter [Passarelli and Miller, J. Virol., 67, 2149-2158 (1993)], we have identified three genes between 56.0 and 65.4 map units of the AcMNPV genome involved in expression from a late and a very late promoter but not from an early viral promoter. One gene, p143, was previously shown to be essential for viral DNA replication and shares sequence motifs with DNA helicases [Lu and Carstens, Virology, 181, 336-347 (1991)]. The second gene, previously sequenced and originally referred to as open reading frame 6 (herein renamed late expression factor-5 [lef-5]), was located just downstream of the 6.9 kilodalton core protein gene, p6.9. The third gene, late expression factor-4 (lef-4), was defined and sequenced. The lef-4 gene was located immediately upstream of, and in opposite orientation to, the major capsid protein gene, vp39. The position and direction of lef-4 appeared to be conserved in the Orgyia pseudotsugata and Lymantria dispar nuclear polyhedrosis viruses. The gene product of lef-4, LEF-4, is predicted to be an acidic polypeptide (pl 4.91) of 464 amino acids in length with a molecular mass of 53,913 daltons.
Assuntos
Regulação Viral da Expressão Gênica , Genes Reguladores/genética , Genes Virais/genética , Nucleopoliedrovírus/genética , Proteínas Virais Reguladoras e Acessórias/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Genes Reporter , Genoma Viral , Insetos/microbiologia , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Mapeamento por Restrição , Análise de Sequência , Fatores de Tempo , Transfecção , Proteínas Virais/genéticaRESUMO
We developed a method to identify baculovirus genes required for late and very late gene expression that is based on subtraction of clones from an Autographa californica nuclear polyhedrosis virus genomic library which is able to trans activate promoters of reporter plasmids in transient expression assays. Using this assay, we found that three genes located between 83.7 and 7.5 map units of the Autographa californica nuclear polyhedrosis virus genome were involved in expression from the late capsid protein (vp39) and very late polyhedrin (polh) gene promoters. Two of these genes, ie-1 and ie-n, trans regulate early genes in transient expression assays. Although ie-1 was necessary and sufficient for expression from the early promoter in our assay, it was necessary but not sufficient for expression from the vp39 and polh promoters. The presence of ie-n increased expression from the early, late, and very late classes of promoters tested. However, a third gene identified in this region was specifically required for expression from the vp39 and polh promoters. This gene, a previously sequenced 630-nucleotide open reading frame, was renamed lef-2 for late expression factor 2. We also found that other genes in the region between 83.7 and 7.5 map units were not required for expression from the promoters used in this assay, although we did not eliminate the possibility that they subtly modify expression. These genes include pe-38 and me53, early genes with zinc finger-like motifs, and the upstream exon of ie-0, which specifies an alternate form of IE-1.
Assuntos
Baculoviridae/genética , Regulação Viral da Expressão Gênica , Genes Reguladores , Genes Virais , Proteínas Estruturais Virais/genética , Animais , Células Cultivadas , Clonagem Molecular , Técnicas In Vitro , Mariposas , Fases de Leitura Aberta , Regiões Promotoras Genéticas , Mapeamento por Restrição , Transcrição GênicaRESUMO
We identified an Autographa californica nuclear polyhedrosis virus (AcMNPV) gene, the late expression factor 6 gene (lef-6), which is involved in expression from late and very late AcMNPV gene promoters but not from an early AcMNPV gene promoter in transient expression assays. This gene was located within the PstI I fragment of the AcMNPV genome (14.7 to 17.9 map units), immediately downstream of Ac-iap, the AcMNPV homolog of a baculovirus gene family involved in blocking apoptotic programmed cell death. The nature and temporal regulation of both Ac-iap and lef-6 transcripts was examined. Ac-iap and lef-6 were cotranscribed as bicistronic messages at both early and late times postinfection. In addition, lef-6 was transcribed as a monocistronic mRNA by initiation from an early promoter within Ac-iap.
Assuntos
Regulação Viral da Expressão Gênica , Genes Virais , Nucleopoliedrovírus/genética , Animais , Apoptose , Sequência de Bases , Northern Blotting , Células Cultivadas , DNA Viral , Dados de Sequência Molecular , Mariposas , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Viral/genética , Transcrição Gênica , Proteínas Virais/genéticaRESUMO
We report the identification of four additional genes of the Autographa californica nuclear polyhedrosis virus involved in expression from a late baculovirus promoter in transient expression assays. Three of these genes, p35, 39K, and p47, have been previously described. The role of the p35 gene product in late gene expression may be related to its ability to block apoptosis, since two other baculovirus genes also known to block apoptosis, Cp-iap and Op-iap, were able to functionally replace p35 in the transient expression assay. The requirement for p47 in this assay confirms its role in late gene expression, a role previously established by characterization of a temperature-sensitive mutant of p47, while the requirement for 39K may be related to its known association with the virogenic stroma. The fourth gene identified as a late expression factor gene, lef-11, was located immediately upstream of 39K and is predicted to encode a 13-kDa polypeptide. When plasmids containing these 4 genes were cotransfected with plasmids containing the 14 genes previously identified as late gene expression factors, the level of expression from the late capsid promoter was similar to that observed for a library of clones representing the entire viral genome. The genes provided by these 18 plasmids thus represent the viral genes necessary and sufficient to support expression from a late viral promoter in this transient expression assay.
Assuntos
Regulação Viral da Expressão Gênica , Genes Virais , Nucleopoliedrovírus/genética , Animais , Linhagem Celular , Replicação do DNA , Fases de Leitura AbertaRESUMO
A trans-acting gene required for late viral gene expression in transient expression assays was identified in the genome of Autographa californica nuclear polyhedrosis virus. A genomic library of A. californica nuclear polyhedrosis virus DNA lacking a clone spanning the region from 43 to 48 map units (mu) was unable to activate gene expression from a reporter plasmid when the reporter gene was driven by a baculovirus late or very late promoter in transient expression assays. The genomic region responsible for activating reporter gene expression was further mapped to 43.4 to 45.2 mu of the viral genome. The nucleotide sequence of this region was determined and shown to contain several small open reading frames (ORFs) and one major ORF, named lef-3, for late expression factor 3. The lef-3 ORF was predicted to encode a polypeptide of 385 amino acids and with a molecular mass of 44,529 daltons. No homolog to the lef-3 ORF was found in existing data bases. Further analysis showed that only the lef-3 ORF in the region from 43.4 to 45.2 mu was necessary for late gene activation in the assays used. The temporal regulation of lef-3 transcription was studied by Northern (RNA) blot hybridization; lef-3 was found to be an early gene that was transcribed primarily as a 2.0-kb mRNA. Primer extension analysis and S1 nuclease protection assays revealed that lef-3 transcription initiated about 280 bp upstream of the first ATG codon and terminated near a polyadenylation signal, 130 bp downstream of the last codon of the lef-3 ORF.
Assuntos
Baculoviridae/genética , Regulação Viral da Expressão Gênica , Genes Virais , Transcrição Gênica , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Códon , DNA Viral/genética , DNA Viral/isolamento & purificação , Biblioteca Genômica , Dados de Sequência Molecular , Mariposas , Oligodesoxirribonucleotídeos , RNA Viral/genética , RNA Viral/isolamento & purificação , Mapeamento por Restrição , Ativação TranscricionalRESUMO
We have identified and sequenced a novel baculovirus gene, late expression factor eight gene (lef-8), of Autographa californica nuclear polyhedrosis virus that is necessary for efficient expression from late and very late virus gene promoters in a transient expression assay. The predicted gene product, LEF-8, has a molecular mass of 102 kDa and contains a conserved sequence motif, GXKX4HGQ/NKG, found in DNA-directed RNA polymerases throughout the animal, plant, and microbial kingdoms.
Assuntos
Baculoviridae/genética , RNA Polimerases Dirigidas por DNA/genética , Genes Virais , Nucleopoliedrovírus/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Animais , Baculoviridae/enzimologia , Sequência de Bases , DNA Viral , Expressão Gênica , Humanos , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
Evidence is presented that a 26-kDa protein encoded by the vaccinia virus A2L open reading frame, originally shown to be one of three intermediate-stage genes that together can transactivate late-stage gene expression in transfection assays (J. G. Keck, C. J. Baldick, and B. Moss, Cell 61:801-809, 1990), is required for in vitro transcription of a template with a late promoter. The critical step in this analysis was the preparation of an extract containing all the required factors except for the A2L protein. This extract was prepared from cells infected with a recombinant vaccinia virus expressing the bacteriophage T7 RNA polymerase in the presence of the DNA synthesis inhibitor cytosine arabinoside and transfected with plasmids containing the two other known transactivator genes, A1L and G8R, under T7 promoter control. Reaction mixtures made with extracts of these cells had background levels of late transcription activity, unless they were supplemented with extracts of cells transfected with the A2L gene. Active transcription mixtures were also made by mixing extracts from three sets of cells, each transfected with a gene (A1L, A2L, or G8R) encoding a separate factor, indicating the absence of any requirement for their coexpression. To minimize the possibility that the A2L protein functions indirectly by activating another viral or cellular protein, this gene was expressed in insect cells by using a baculovirus vector. The partially purified recombinant protein complemented the activity of A2L-deficient cell extracts. Recombinant A1L, A2L, and G8R proteins, all produced in insect cells, together complemented extracts from mammalian cells containing only viral early proteins, concordant with previous in vivo transfection data.
Assuntos
Regiões Promotoras Genéticas , Transativadores/fisiologia , Vaccinia virus/genética , Proteínas Virais/fisiologia , Cromatografia DEAE-Celulose , Clonagem Molecular , Regulação Viral da Expressão Gênica , Nucleopoliedrovírus/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Moldes Genéticos , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Transfecção , Vaccinia virus/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
A region of the baculovirus Autographa californica nuclear polyhedrosis virus genome that is frequently found to be altered after serial passage of the virus in cell culture was characterized. Sequence analysis of this region of the genome in wild-type and mutant viruses revealed that some of the mutations affected a 675 bp open reading frame, designated DA26. The DA26 gene was disrupted both by deletion and by insertion of sequences that resembled transposable elements. Northern blot analysis of DA26 showed that it was expressed very early after infection. DA26-specific transcripts could be detected after the 1 h viral adsorption period upon infection of cultured Trichoplusia ni cells. These transcripts were mapped by nuclease protection assays. A recombinant virus was constructed in which DA26 was disrupted by insertion of the Escherichia coli lacZ gene. This virus was viable in both T. ni and Spodoptera frugiperda cells and analysis of the kinetics of protein synthesis revealed no differences between wild-type and recombinant viruses. The disruption of DA26 also did not interfere with the ability of the virus to infect T. ni or S. frugiperda larvae.
Assuntos
Genes Virais , Vírus de Insetos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Células Cultivadas , Expressão Gênica , Vírus de Insetos/crescimento & desenvolvimento , Vírus de Insetos/metabolismo , Cinética , Lepidópteros/microbiologia , Dados de Sequência Molecular , Mutação , Fenótipo , Mapeamento por Restrição , Transcrição Gênica , Proteínas Virais/biossíntese , Proteínas Virais/genéticaRESUMO
Eighteen genes of Autographa californica nuclear polyhedrosis virus are necessary and sufficient to transactivate expression from the late vp39 promoter in transient-expression assays in SF-21 cells. These 18 genes, known as late expression factor genes (lefs), are also required to transactivate the very late promoter of the polyhedrin gene, polh, but expression from this promoter is relatively weak compared with expression from the vp39 promoter. To further define the factors required for late and very late promoter expression, we first determined that the eighteen lefs were also required for expression from two other major baculovirus promoters: the late basic 6.9-kDa protein gene, p6.9, and the very late 10-kDa protein gene, p10. We next examined the effect of the very late expression factor 1 gene (vlf-1), a gene previously identified by analysis of a temperature-sensitive mutant, in the transient expression assay and found that vlf-1 specifically transactivated the two very late promoters but not the two late promoters. We then surveyed the Autographa californica nuclear polyhedrosis virus genome for additional genes which might specifically regulate very late gene expression; no additional vlf genes were detected, suggesting that VLF-1 is the primary regulator of very late gene expression. Finally, we found that the relative contribution of the antiapoptosis gene p35, which behaves as a lef in these transient-expression assays, depended on the nature of the other viral genes provided in the cotransfection mixtures, suggesting that other viral genes also contribute to the ability of the virus to block apoptosis.
Assuntos
Regulação Viral da Expressão Gênica , Nucleopoliedrovírus/genética , Animais , Linhagem Celular , Genes Virais , Regiões Promotoras Genéticas , Spodoptera , Fatores de Transcrição/genética , Proteínas Virais/genéticaRESUMO
The tat gene of the human immunodeficiency virus, tat-III, when introduced into T-lymphoblastoid Jurkat cells by a Moloney retroviral recombinant DNA vector expressed high levels of the functional tat protein as measured by the chloramphenicol acetyltransferase assay. Immunofluorescence analysis with CD4-specific monoclonal antibodies demonstrated that the cell surface levels of the CD4 antigen were increased by 5- to 10-fold in the tat-III-infected Jurkat cells. Cellular cytoplasmic RNA analysis indicated that the enhanced CD4 expression was mediated at the mRNA level. Our findings suggest that the single expression of the human immunodeficiency virus tat protein in the absence of the other viral proteins causes an upregulation of CD4 gene expression on helper T cells, although infection of these cells by the virus, thus expressing all the viral gene products including tat, is known to downregulate CD4 antigen expression.