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1.
Proc Natl Acad Sci U S A ; 118(3)2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33384338

RESUMO

Human adenovirus species D (HAdV-D) types are currently being explored as vaccine vectors for coronavirus disease 2019 (COVID-19) and other severe infectious diseases. The efficacy of such vector-based vaccines depends on functional interactions with receptors on host cells. Adenoviruses of different species are assumed to enter host cells mainly by interactions between the knob domain of the protruding fiber capsid protein and cellular receptors. Using a cell-based receptor-screening assay, we identified CD46 as a receptor for HAdV-D56. The function of CD46 was validated in infection experiments using cells lacking and overexpressing CD46, and by competition infection experiments using soluble CD46. Remarkably, unlike HAdV-B types that engage CD46 through interactions with the knob domain of the fiber protein, HAdV-D types infect host cells through a direct interaction between CD46 and the hexon protein. Soluble hexon proteins (but not fiber knob) inhibited HAdV-D56 infection, and surface plasmon analyses demonstrated that CD46 binds to HAdV-D hexon (but not fiber knob) proteins. Cryoelectron microscopy analysis of the HAdV-D56 virion-CD46 complex confirmed the interaction and showed that CD46 binds to the central cavity of hexon trimers. Finally, soluble CD46 inhibited infection by 16 out of 17 investigated HAdV-D types, suggesting that CD46 is an important receptor for a large group of adenoviruses. In conclusion, this study identifies a noncanonical entry mechanism used by human adenoviruses, which adds to the knowledge of adenovirus biology and can also be useful for development of adenovirus-based vaccine vectors.


Assuntos
Adenovírus Humanos , Proteínas do Capsídeo , Regulação Viral da Expressão Gênica , Internalização do Vírus , Adenovírus Humanos/genética , Adenovírus Humanos/metabolismo , /metabolismo , Proteínas do Capsídeo/biossíntese , Proteínas do Capsídeo/genética , Linhagem Celular , Humanos
2.
Arch Virol ; 165(12): 2829-2835, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33000310

RESUMO

The swine pathogen porcine circovirus type 2 (PCV2) causes significant economic damage worldwide. The PCV2 capsid (CP) residues 169-STIDYFQPNNKR-180 have been identified as a decoy epitope that diverts the host immune response away from protective epitopes. However, the decoy epitope may include important linear or conformational protective epitopes against PCV2. In this study, we used the baculovirus system to express recombinant complete CP (1-233) and mutant CP (Δ169-180), in which the decoy epitope was deleted, and evaluated the immune response to these in mice. Immunization with mutant CP (Δ169-180) protein, which formed very low level of virus-like particles (VLPs), elicited significantly lower levels of PCV2 CP-specific IgG antibodies and a slightly lower neutralizing activity than immunization with the complete CP (1-233) protein. This finding suggests that the complete CP is important for efficient VLP assembly and induction of PCV2-specific IgG antibodies and neutralizing antibodies in mice. This study may provide useful information for next-generation vaccine design for PCV2 control.


Assuntos
Proteínas do Capsídeo/imunologia , Circovirus/imunologia , Epitopos/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Animais , Anticorpos Neutralizantes/análise , Anticorpos Antivirais/análise , Proteínas do Capsídeo/biossíntese , Proteínas do Capsídeo/genética , Circovirus/genética , Epitopos/biossíntese , Epitopos/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Síndrome Definhante Multissistêmico de Suínos Desmamados/imunologia , Síndrome Definhante Multissistêmico de Suínos Desmamados/prevenção & controle , Suínos , Vacinação , Vacinas de Partículas Semelhantes a Vírus/genética
3.
Arch Virol ; 165(10): 2301-2309, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32757056

RESUMO

Porcine circovirus type 2 (PCV2) is a major pathogen associated with swine diseases. It is the smallest single-stranded DNA virus, and its genome contains four major open reading frames (ORFs). ORF2 encodes the major structural protein Cap, which can self-assemble into virus-like particles (VLPs) in vitro and contains the primary antigenic determinants. In this study, we developed a high-efficiency method for obtaining VLPs and optimized the purification conditions. In this method, we expressed the protein Cap with a 6× His tag using baculovirus-infected silkworm larvae as well as the E. coli BL21(DE3) prokaryotic expression system. The PCV2 Cap proteins produced by the silkworm larvae and E. coli BL21(DE3) were purified. Cap proteins purified from silkworm larvae self-assembled into VLPs in vitro, while the Cap proteins purified from bacteria were unable to self-assemble. Transmission electron microscopy confirmed the self-assembly of VLPs. The immunogenicity of the VLPs produced using the baculovirus system was demonstrated using an enzyme-linked immunosorbent assay (ELISA). Furthermore, the purification process was optimized. The results demonstrated that the expression system using baculovirus-infected silkworm larvae is a good choice for obtaining VLPs of PCV2 and has potential for the development of a low-cost and efficient vaccine.


Assuntos
Anticorpos Antivirais/biossíntese , Baculoviridae/genética , Bombyx/virologia , Proteínas do Capsídeo/imunologia , Circovirus/imunologia , Vacinas de Partículas Semelhantes a Vírus/biossíntese , Vacinas Virais/biossíntese , Animais , Antígenos Virais/química , Antígenos Virais/imunologia , Baculoviridae/imunologia , Proteínas do Capsídeo/biossíntese , Proteínas do Capsídeo/genética , Infecções por Circoviridae/imunologia , Infecções por Circoviridae/prevenção & controle , Infecções por Circoviridae/virologia , Circovirus/genética , Epitopos/química , Epitopos/imunologia , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Histidina/genética , Histidina/imunologia , Soros Imunes/química , Imunogenicidade da Vacina , Larva/virologia , Camundongos , Oligopeptídeos/genética , Oligopeptídeos/imunologia , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/prevenção & controle , Doenças dos Suínos/virologia , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/genética , Vacinas de Partículas Semelhantes a Vírus/isolamento & purificação , Vacinas Virais/administração & dosagem , Vacinas Virais/genética , Vacinas Virais/isolamento & purificação
4.
J Virol ; 94(15)2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32404528

RESUMO

Caliciviruses have a positive-strand RNA genome with a length of about 7.5 kb that contains 2, 3, or 4 functional open reading frames (ORFs). A subgenomic mRNA (sg-RNA) is transcribed in the infected cell, and both major capsid protein viral protein 1 (VP1) and minor capsid protein VP2 are translated from the sg-RNA. Translation of proteins from the genomic RNA (g-RNA) and from the sg-RNA is mediated by the RNA-linked viral protein VPg (virus protein, genome linked). Most of the calicivirus genera have translation mechanisms leading to VP1 expression from the g-RNA. VP1 is part of the polyprotein for sapoviruses, lagoviruses, and neboviruses, and a termination/reinitiation mechanism was described for noroviruses. Vesiviruses have no known mechanism for the expression of VP1 from the g-RNA, and the Vesivirus genus is the only genus of the Caliciviridae that generates VP1 via a precursor capsid leader protein (LC-VP1). Analyses of feline calicivirus (FCV) g-RNA translation showed a low level of VP1 expression with an initiation downstream of the original start codon of LC-VP1, leading to a smaller, truncated LC-VP1 (tLC-VP1) protein. Deletion and substitution analyses of the region surrounding the LC-VP1 start codon allowed the identification of sequences within the leader protein coding region of FCV that have an impact on VP1 translation frequency from the g-RNA. Introduction of such mutations into the virus showed an impact of strongly reduced tLC-VP1 levels translated from the g-RNA on viral replication.IMPORTANCE Caliciviruses are a cause of important diseases in humans and animals. It is crucial to understand the prerequisites of efficient replication of these viruses in order to develop strategies for prevention and treatment of these diseases. It was shown before that all caliciviruses except vesiviruses have established mechanisms to achieve major capsid protein (VP1) translation from the genomic RNA. Here, we show for the first time that a member of the genus Vesivirus also has a translation initiation mechanism by which a precursor protein of the VP1 protein is expressed from the genomic RNA. This finding clearly points at a functional role of the calicivirus VP1 capsid protein in early replication, and we provide experimental data supporting this hypothesis.


Assuntos
Calicivirus Felino/metabolismo , Proteínas do Capsídeo/biossíntese , Regulação Viral da Expressão Gênica , Genoma Viral , Biossíntese de Proteínas , RNA Viral/metabolismo , Animais , Calicivirus Felino/genética , Proteínas do Capsídeo/genética , Gatos , Linhagem Celular , Cricetinae , RNA Viral/genética
5.
Arch Virol ; 165(6): 1441-1444, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32239294

RESUMO

Bovine papillomavirus type 9 (BPV9) is a causative agent of severe teat papillomatosis. Considering the lack of efficient BPV culture methods, recombinant proteins such as virus-like particles developed through genetic engineering may serve as a useful tool for developing effective vaccines against BPV infection. In this study, we successfully produced immunogenic particles composed of recombinant L1 protein of BPV9 (rBPV9-L1), using a baculovirus expression system. rBPV9-L1-immunized mice produced BPV9-specific IgG, which did not cross-react with BPV type 6, which is another causative agent of teat papillomatosis. Hence, immunogenic rBPV9-L1 is potentially applicable as a vaccine candidate for teat papillomatosis.


Assuntos
Proteínas do Capsídeo/imunologia , Doenças dos Bovinos/prevenção & controle , Papillomaviridae/imunologia , Infecções por Papillomavirus/veterinária , Vacinas de Partículas Semelhantes a Vírus/imunologia , Animais , Proteínas do Capsídeo/biossíntese , Bovinos , Doenças dos Bovinos/virologia , Feminino , Genótipo , Camundongos , Papillomaviridae/genética , Infecções por Papillomavirus/prevenção & controle , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Vacinação
6.
J Virol ; 94(2)2020 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-31666379

RESUMO

Human bocavirus 1 (HBoV1), which belongs to the genus Bocaparvovirus of the Parvoviridae family, causes acute respiratory tract infections in young children. In vitro, HBoV1 infects polarized primary human airway epithelium (HAE) cultured at an air-liquid interface (HAE-ALI). HBoV1 encodes a small nonstructural protein, nuclear protein 1 (NP1), that plays an essential role in the maturation of capsid protein (VP)-encoding mRNAs and viral DNA replication. In this study, we determined the broad interactome of NP1 using the proximity-dependent biotin identification (BioID) assay combined with mass spectrometry (MS). We confirmed that two host mRNA processing factors, DEAH-box helicase 15 (DHX15) and cleavage and polyadenylation specificity factor 6 (CPSF6; also known as CFIm68), a subunit of the cleavage factor Im complex (CFIm), interact with HBoV1 NP1 independently of any DNA or mRNAs. Knockdown of CPSF6 significantly decreased the expression of capsid protein but not that of DHX15. We further demonstrated that NP1 directly interacts with CPSF6 in vitro and colocalizes within the virus replication centers. Importantly, we revealed a novel role of CPSF6 in the nuclear import of NP1, in addition to the critical role of CPSF6 in NP1-facilitated maturation of VP-encoding mRNAs. Thus, our study suggests that CPSF6 interacts with NP1 to escort NP1 imported into the nucleus for its function in the modulation of viral mRNA processing and viral DNA replication.IMPORTANCE Human bocavirus 1 (HBoV1) is one of the significant pathogens causing acute respiratory tract infections in young children worldwide. HBoV1 encodes a small nonstructural protein (NP1) that plays an important role in the maturation of viral mRNAs encoding capsid proteins as well as in viral DNA replication. Here, we identified a critical host factor, CPSF6, that directly interacts with NP1, mediates the nuclear import of NP1, and plays a role in the maturation of capsid protein-encoding mRNAs in the nucleus. The identification of the direct interaction between viral NP1 and host CPSF6 provides new insights into the mechanism by which a viral small nonstructural protein facilitates the multiple regulation of viral gene expression and replication and reveals a novel target for potent antiviral drug development.


Assuntos
Proteínas do Capsídeo/biossíntese , Núcleo Celular , Regulação Viral da Expressão Gênica , Bocavirus Humano/metabolismo , Proteínas Nucleares/metabolismo , RNA Mensageiro , RNA Viral , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Transporte Ativo do Núcleo Celular , Proteínas do Capsídeo/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Células HEK293 , Bocavirus Humano/genética , Humanos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/genética , RNA Helicases/genética , RNA Helicases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/genética
7.
Mol Biotechnol ; 61(11): 852-859, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31473916

RESUMO

To explore virus-like particles formation of dengue virus serotype type 2 (DENV-2) structural proteins of, C, prM, E were expressed in silkworm larvae using recombinant Bombyx mori nucleopolyhedroviruses (BmNPV). Each recombinant BmNPV bacmid coding the 2C-prM-E polypeptide and E protein fused with the signal peptide of bombyxin from B. mori was injected into silkworm larvae. The expressed proteins were collected from hemolymph and fat body, and purified using affinity chromatography. E protein was observed at 55 kDa. The DENV virus-like particles (DENV-LPs) with a diameter approximately 35 nm was observed using transmission electron microscopy (TEM) and immunogold-labelling TEM analysis. The binding of each partially purified proteins to heparin, one of receptors for DENV was confirmed. DENV-LPs were secreted in silkworm larval hemolymph even still low amount, but the E protein and heparin binding function were confirmed.


Assuntos
Proteínas do Capsídeo/metabolismo , Vírus da Dengue/genética , Nucleopoliedrovírus/genética , Proteínas do Envelope Viral/metabolismo , Proteínas Estruturais Virais/metabolismo , Vírion/genética , Animais , Bombyx/crescimento & desenvolvimento , Bombyx/metabolismo , Proteínas do Capsídeo/biossíntese , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/isolamento & purificação , Vírus da Dengue/metabolismo , Corpo Adiposo/metabolismo , Expressão Gênica , Vetores Genéticos , Hemolinfa/metabolismo , Heparina/metabolismo , Larva/metabolismo , Nucleopoliedrovírus/metabolismo , Sinais Direcionadores de Proteínas/genética , Sorogrupo , Proteínas do Envelope Viral/biossíntese , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/isolamento & purificação , Proteínas Estruturais Virais/biossíntese , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/isolamento & purificação , Vírion/ultraestrutura
8.
Arch Virol ; 164(7): 1753-1760, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31025116

RESUMO

The expression of several structural proteins from a wide variety of viruses in heterologous cell culture systems results in the formation of virus-like particles (VLPs). These VLPs structurally resemble the wild-type virus particles and have been used to study viral assembly process and as antigens for diagnosis and/or vaccine development. Tomato blistering mosaic virus (ToBMV) is a tymovirus that has a 6.3-kb positive-sense ssRNA genome. We have employed the baculovirus expression vector system (BEVS) for the production of tymovirus-like particles (tVLPs) in insect cells. Two recombinant baculoviruses containing the ToBMV wild-type coat protein (CP) gene or a modified short amino-terminal deletion (Δ2-24CP) variant were constructed and used to infect insect cells. Both recombinant viruses were able to express ToBMV CP and Δ2-24CP from infected insect cells that self-assembled into tVLPs. Therefore, the N-terminal residues (2-24) of the native ToBMV CP were shown not to be essential for self-assembly of tVLPs. We also constructed a third recombinant baculovirus containing a small sequence coding for the major epitope of the chikungunya virus (CHIKV) envelope protein 2 (E2) replacing the native CP N-terminal 2-24 amino acids. This recombinant virus also produced tVLPs. In summary, ToBMV VLPs can be produced in a baculovirus/insect cell heterologous expression system, and the N-terminal residues 2-24 of the CP are not essential for this assembly, allowing its potential use as a protein carrier that facilitates antigen purification and might be used for diagnosis.


Assuntos
Baculoviridae/genética , Proteínas do Capsídeo/biossíntese , Tymovirus/crescimento & desenvolvimento , Tymovirus/genética , Proteínas do Envelope Viral/biossíntese , Montagem de Vírus/genética , Animais , Proteínas do Capsídeo/genética , Linhagem Celular , Vírus Chikungunya/genética , Expressão Gênica/genética , Lycopersicon esculentum/virologia , Mariposas/citologia , Proteínas do Envelope Viral/genética
9.
Plant Biotechnol J ; 17(9): 1751-1759, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-30791210

RESUMO

Porcine circovirus type 2 (PCV-2) is the main causative agent associated with a group of diseases collectively known as porcine circovirus-associated disease (PCAD). There is a significant economic strain on the global swine industry due to PCAD and the production of commercial PCV-2 vaccines is expensive. Plant expression systems are increasingly regarded as a viable technology to produce recombinant proteins for use as pharmaceutical agents and vaccines. However, successful production and purification of PCV-2 capsid protein (CP) from plants is an essential first step towards the goal of a plant-produced PCV-2 vaccine candidate. In this study, the PCV-2 CP was transiently expressed in Nicotiana benthamiana plants via agroinfiltration and PCV-2 CP was successfully purified using sucrose gradient ultracentrifugation. The CP self-assembled into virus-like particles (VLPs) resembling native virions and up to 6.5 mg of VLPs could be purified from 1 kg of leaf wet weight. Mice immunized with the plant-produced PCV-2 VLPs elicited specific antibody responses to PCV-2 CP. This is the first report describing the expression of PCV-2 CP in plants, the confirmation of its assembly into VLPs and the demonstration of their use to elicit a strong immune response in a mammalian model.


Assuntos
Proteínas do Capsídeo/imunologia , Circovirus , Imunogenicidade da Vacina , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/sangue , Proteínas do Capsídeo/biossíntese , Camundongos , Plantas Geneticamente Modificadas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Suínos , Tabaco/genética , Tabaco/metabolismo
10.
RNA ; 25(5): 645-655, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30803999

RESUMO

External guide sequences (EGSs) signify the short RNAs that induce ribonuclease P (RNase P), an enzyme responsible for processing the 5' termini of tRNA, to specifically cleave a target mRNA by forming a precursor tRNA-like complex. Hence, the EGS technology may serve as a potential strategy for gene-targeting therapy. Our previous studies have revealed that engineered EGS variants induced RNase P to efficiently hydrolyze target mRNAs. In the present research, an EGS variant was designed to be complementary to the mRNA coding for human cytomegalovirus (HCMV) major capsid protein (MCP), which is vital to form the viral capsid. In vitro, the EGS variant was about 80-fold more efficient in inducing human RNase P-mediated cleavage of the target mRNA than a natural tRNA-derived EGS. Moreover, the expressed variant and natural tRNA-originated EGSs led to a decrease of MCP expression by 98% and 73%-74% and a decrease of viral growth by about 10,000- and 200-fold in cells infected with HCMV, respectively. These results reveal direct evidence that the engineered EGS variant has higher efficiency in blocking the expression of HCMV genes and viral growth than the natural tRNA-originated EGS. Therefore, our findings imply that the EGS variant can be a potent candidate agent for the treatment of infections caused by HCMV.


Assuntos
Proteínas do Capsídeo/genética , Citomegalovirus/genética , RNA Guia/genética , RNA Mensageiro/genética , RNA de Transferência de Serina/genética , RNA Viral/genética , Ribonuclease P/metabolismo , Pareamento de Bases , Proteínas do Capsídeo/biossíntese , Linhagem Celular Transformada , Linhagem Celular Tumoral , Citomegalovirus/metabolismo , Fibroblastos/metabolismo , Fibroblastos/virologia , Regulação Viral da Expressão Gênica , Marcação de Genes/métodos , Engenharia Genética/métodos , Interações Hospedeiro-Patógeno/genética , Humanos , Terapia de Alvo Molecular , Neuroglia/metabolismo , Neuroglia/virologia , Conformação de Ácido Nucleico , Cultura Primária de Células , Clivagem do RNA , RNA Guia/química , RNA Guia/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , RNA de Transferência de Serina/química , RNA de Transferência de Serina/metabolismo , RNA Viral/química , RNA Viral/metabolismo , Ribonuclease P/química , Ribonuclease P/genética , Replicação Viral/fisiologia
11.
PLoS One ; 14(2): e0211740, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30707739

RESUMO

Macrobrachium rosenbergii nodavirus (MrNV) is the causative agent of white tail disease (WTD) which seriously impedes the production of the giant freshwater prawn and has a major economic impact. MrNV contains two segmented RNA molecules, which encode the RNA dependent RNA polymerase (RdRp) and the capsid protein (MrNV-CP) containing 371 amino acid residues. MrNV-CP comprises of the Shell (S) and the Protruding (P) domains, ranging from amino acid residues 1-252 and 253-371, respectively. The P-domain assembles into dimeric protruding spikes, and it is believed to be involved in host cell attachment and internalization. In this study, the recombinant P-domain of MrNV-CP was successfully cloned and expressed in Escherichia coli, purified with an immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC) up to ~90% purity. Characterization of the purified recombinant P-domain with SEC revealed that it formed dimers, and dynamic light scattering (DLS) analysis demonstrated that the hydrodynamic diameter of the dimers was ~6 nm. Circular dichroism (CD) analysis showed that the P-domain contained 67.9% of beta-sheets, but without alpha-helical structures. This is in good agreement with the cryo-electron microscopic analysis of MrNV which demonstrated that the P-domain contains only beta-stranded structures. Our findings of this study provide essential information for the production of the P-domain of MrNV-CP that will aid future studies particularly studies that will shed light on anti-viral drug discovery and provide an understanding of virus-host interactions and the viral pathogenicity.


Assuntos
Proteínas do Capsídeo/química , Nodaviridae/química , Palaemonidae/virologia , Multimerização Proteica , Animais , Proteínas do Capsídeo/biossíntese , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/isolamento & purificação , Escherichia coli/genética , Escherichia coli/metabolismo , Nodaviridae/genética , Nodaviridae/metabolismo , Domínios Proteicos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação
12.
J Am Chem Soc ; 141(9): 3875-3884, 2019 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-30730722

RESUMO

Site-specific protein modification is a widely used strategy to attach drugs, imaging agents, or other useful small molecules to protein carriers. N-terminal modification is particularly useful as a high-yielding, site-selective modification strategy that can be compatible with a wide array of proteins. However, this modification strategy is incompatible with proteins with buried or sterically hindered N termini, such as virus-like particles (VLPs) composed of the well-studied MS2 bacteriophage coat protein. To assess VLPs with improved compatibility with these techniques, we generated a targeted library based on the MS2-derived protein cage with N-terminal proline residues followed by three variable positions. We subjected the library to assembly, heat, and chemical selections, and we identified variants that were modified in high yield with no reduction in thermostability. Positive charge adjacent to the native N terminus is surprisingly beneficial for successful extension, and over 50% of the highest performing variants contained positive charge at this position. Taken together, these studies described nonintuitive design rules governing N-terminal extensions and identified successful extensions with high modification potential.


Assuntos
Bacteriófagos/metabolismo , Proteínas do Capsídeo/biossíntese , Nanopartículas/metabolismo , Engenharia de Proteínas , Bacteriófagos/química , Proteínas do Capsídeo/química , Estrutura Molecular , Nanopartículas/química
13.
J Microbiol Biotechnol ; 29(3): 482-488, 2019 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-30609882

RESUMO

Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome (PMWS) in pigs. Replicase (Rep) proteins are considered essential for viral replication. Capsid (Cap) protein is the primary immunogenic protein that induces protective immunity. Little is known about comparison on the immunogenicity of PCV2 Rep and Cap fusion protein and Cap protein. In the present study, recombinant baculoviruses expressing the Rep-Cap fusion protein (Bac-Rep-Cap) and the Cap protein (Bac-Cap) of PCV2 were constructed and confirmed with western blot and indirect fluorescence assay. Immunogenicities of the two recombinant proteins were tested in mice. The titers of antibodies were determined with a PCV2-specific enzyme-linked immunosorbent assay (ELISA) and a serum neutralization assay. The IFN-γ response of immunized mice was measured by ELISA. The mice immunized with the Bac-Rep-Cap and Bac-Cap successfully produced Cap-specific immunoreaction. The mice immunized with the Bac-Cap developed higher PCV2-specific neutralizing antibody titers than mice injected with the Bac-Rep-Cap. IFN-γ in the Bac-Rep-Cap group was increased compared to those in the Bac-Cap group. Vaccination of mice with the Bac-Rep-Cap showed significantly decreased protective efficacy compared to the Bac-Cap. Our findings will indubitably not only lead to a better understanding of the immunogenicity of PCV2, but also improved vaccines.


Assuntos
Proteínas do Capsídeo/imunologia , Infecções por Circoviridae/imunologia , Infecções por Circoviridae/prevenção & controle , Imunogenicidade da Vacina/imunologia , Proteínas Recombinantes de Fusão/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais/sangue , Formação de Anticorpos , Baculoviridae/genética , Western Blotting , Proteínas do Capsídeo/biossíntese , Proteínas do Capsídeo/genética , Circovirus/patogenicidade , Citocinas/sangue , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Vetores Genéticos , Injeções Intramusculares , Interferon gama/sangue , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Proteínas Recombinantes de Fusão/genética , Vacinação , Vacinas de Partículas Semelhantes a Vírus/biossíntese , Vacinas de Partículas Semelhantes a Vírus/genética , Vacinas Virais/genética , Vacinas Virais/farmacologia
14.
RNA ; 25(4): 431-452, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30659060

RESUMO

Noncanonical translation, and particularly initiation on non-AUG codons, are frequently used by viral and cellular mRNAs during virus infection and disease. The Sindbis virus (SINV) subgenomic mRNA (sgRNA) constitutes a unique model system to analyze the translation of a capped viral mRNA without the participation of several initiation factors. Moreover, sgRNA can initiate translation even when the AUG initiation codon is replaced by other codons. Using SINV replicons, we examined the efficacy of different codons in place of AUG to direct the synthesis of the SINV capsid protein. The substitution of AUG by CUG was particularly efficient in promoting the incorporation of leucine or methionine in similar percentages at the amino terminus of the capsid protein. Additionally, valine could initiate translation when the AUG is replaced by GUG. The ability of sgRNA to initiate translation on non-AUG codons was dependent on the integrity of a downstream stable hairpin (DSH) structure located in the coding region. The structural requirements of this hairpin to signal the initiation site on the sgRNA were examined in detail. Of interest, a virus bearing CUG in place of AUG in the sgRNA was able to infect cells and synthesize significant amounts of capsid protein. This virus infects the human haploid cell line HAP1 and the double knockout variant that lacks eIF2A and eIF2D. Collectively, these findings indicate that leucine-tRNA or valine-tRNA can participate in the initiation of translation of sgRNA by a mechanism dependent on the DSH. This mechanism does not involve the action of eIF2, eIF2A, or eIF2D.


Assuntos
Códon de Iniciação/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Viral/genética , Transdução de Sinais/genética , Vírus Sindbis/genética , Proteínas do Capsídeo/biossíntese , Proteínas do Capsídeo/genética , Linhagem Celular Tumoral , Códon de Iniciação/metabolismo , Fator de Iniciação 2 em Eucariotos/deficiência , Fator de Iniciação 2 em Eucariotos/genética , Fibroblastos/metabolismo , Fibroblastos/virologia , Regulação da Expressão Gênica , Haploidia , Interações Hospedeiro-Patógeno/genética , Humanos , Sequências Repetidas Invertidas , Leucina/genética , Leucina/metabolismo , Metionina/genética , Metionina/metabolismo , Conformação de Ácido Nucleico , RNA Mensageiro/metabolismo , RNA de Transferência de Leucina/genética , RNA de Transferência de Leucina/metabolismo , RNA de Transferência de Valina/genética , RNA de Transferência de Valina/metabolismo , RNA Viral/metabolismo , Replicon , Vírus Sindbis/metabolismo , Valina/genética , Valina/metabolismo
15.
J Virol ; 92(21)2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30111560

RESUMO

The capsid mRNA transcripts of human bocavirus 1 (HBoV1) can be generated by alternative splicing from the mRNA precursor transcribed from the P5 promoter. However, the alternative translation regulation mechanism of capsid mRNA transcripts is largely unknown. Here we report that the polycistronic capsid mRNA transcripts encode VP1, VP2, and VP3 in vitro and in vivo The 5' untranslated regions (UTRs) of capsid mRNA transcripts, which consist of exons, affected not only the abundance of mRNA but also the translation pattern of capsid proteins. Further study showed that exons 2 and 3 were critical for the abundance of mRNA, while exon 4 regulated capsid translation. Alternative translation of capsid mRNA involved a leaky scan mechanism. Mutating the upstream ATGs (uATGs) located in exon 4 resulted in more mRNA transcripts polyadenylated at the proximal polyadenylation [(pA)p] site, leading to increased capsid mRNA transcripts. Moreover, uATG mutations induced more VP1 expression, while VP3 expression was decreased, which resulted in less progeny virus production. Our data show that the 5' UTR of HBoV1 plays a critical role in the modulation of mRNA abundance, alternative RNA processing, alternative translation, and progeny virus production.IMPORTANCE Alternative translation of HBoV1 capsid mRNAs is vital for the viral life cycle, as capsid proteins perform essential functions in genome packaging, assembly, and antigenicity. The 5' untranslated regions (UTRs) of capsid mRNAs are generated by alternative splicing, and they contain different exons. Our study shows that the 5' UTR not only modulates mRNA abundance but also regulates capsid expression. Two upstream ATGs (uATGs) that were upstream of the capsid translation initiation site in the 5' UTR were found to affect viral capsid mRNA polyadenylation, alternative translation, and progeny virus production. The results reveal that uATGs play an important role in the viral life cycle and represent a new layer to regulate HBoV1 RNA processing, which could be a target for gene therapy.


Assuntos
Regiões 5' não Traduzidas/genética , Processamento Alternativo/genética , Bocavirus/genética , Proteínas do Capsídeo/genética , RNA Viral/biossíntese , Capsídeo/metabolismo , Proteínas do Capsídeo/biossíntese , Linhagem Celular , Células HEK293 , Humanos , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , RNA Viral/genética , Transcrição Genética/genética
16.
J Virol ; 92(22)2018 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-30135129

RESUMO

Human bocavirus 1 (HBoV1) encodes a genus-specific protein, NP1, which regulates viral alternative pre-mRNA processing. Similar to NP1 of the related bocavirus minute virus of canine (MVC), HBoV1 NP1 suppressed cleavage and polyadenylation of RNAs at the viral internal polyadenylation site (pA)p. HBoV1 (pA)p is a complex region. It contains 5 significant cleavage and polyadenylation sites, and NP1 was found to regulate only the three of these sites that are governed by canonical AAUAAA hexamer signals. HBoV1 NP1 also facilitated splicing of the upstream intron adjacent to (pA)p. Alternative polyadenylation and splicing of the upstream intron were independent of each other, functioned efficiently within an isolated transcription unit, and were responsive independent of NP1. Characterization of HBoV1 NP1 generalizes its function within the genus Bocaparvovirus, uncovers important differences, and provides important comparisons with MVC NP1 for mechanistic and evolutionary considerations.IMPORTANCE The Parvovirinae are small nonenveloped icosahedral viruses that are important pathogens in many animal species, including humans. The NP1 protein of human bocavirus 1 (HBoV1), similar to NP1 of the bocavirus minute virus of canine (MVC), regulates viral alternative RNA processing by both suppressing polyadenylation at an internal site, (pA)p, and facilitating splicing of an upstream adjacent intron. These effects allow both extension into the capsid gene and splicing of the viral pre-mRNA that correctly registers the capsid gene open reading frame. Characterization of HBoV1 NP1 generalizes this central mode of parvovirus gene regulation to another member of the bocavirus genus and uncovers both important similarities and differences in function compared to MVC NP1 that will be important for future comparative studies.


Assuntos
Processamento Alternativo/genética , Proteínas do Capsídeo/genética , Regulação Viral da Expressão Gênica/genética , Bocavirus Humano/genética , RNA Viral/genética , Proteínas não Estruturais Virais/metabolismo , Capsídeo/metabolismo , Proteínas do Capsídeo/biossíntese , Linhagem Celular , Células HEK293 , Bocavirus Humano/metabolismo , Humanos , Poliadenilação , Replicação Viral/genética
17.
Virol Sin ; 33(4): 314-322, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29959686

RESUMO

Stress granules (SGs) are intracellular granules formed when cellular translation is blocked and have been reported to be involved in a variety of viral infections. Our previous studies revealed that SGs are involved in the coxsackievirus B (CVB) infection process, but the role of SGs in CVB infection has not been fully explored. In this study, we found that CVB type 3 (CVB3) could induce SG formation in the early phase of infection. Results showed that levels of CVB3 RNA and protein were significantly inhibited during the early stage of CVB3 infection by the elevated formation of SGs, while viral RNA and protein synthesis were significantly promoted when SG formation was blocked. Our findings suggest that SG formation is one of the early antiviral mechanisms for host cells against CVB infection.


Assuntos
Antivirais/metabolismo , Infecções por Coxsackievirus/virologia , Grânulos Citoplasmáticos/metabolismo , Enterovirus Humano B/fisiologia , Proteínas do Capsídeo/biossíntese , DNA Helicases/genética , Proteína Semelhante a ELAV 1/genética , Proteína Semelhante a ELAV 1/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Técnicas de Silenciamento de Genes , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Fosforilação , Proteínas de Ligação a Poli-ADP-Ribose/genética , RNA Helicases/genética , Proteínas com Motivo de Reconhecimento de RNA/genética , RNA Viral/biossíntese , Estresse Fisiológico , Antígeno-1 Intracelular de Células T/genética , Antígeno-1 Intracelular de Células T/metabolismo , Replicação Viral
18.
Proc Natl Acad Sci U S A ; 115(28): 7344-7349, 2018 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-29941585

RESUMO

Most double-stranded RNA (dsRNA) viruses transcribe RNA plus strands within a common innermost capsid shell. This process requires coordinated efforts by RNA-dependent RNA polymerase (RdRp) together with other capsid proteins and genomic RNA. Here we report the near-atomic resolution structure of the RdRp protein VP2 in complex with its cofactor protein VP4 and genomic RNA within an aquareovirus capsid using 200-kV cryoelectron microscopy and symmetry-mismatch reconstruction. The structure of these capsid proteins enabled us to observe the elaborate nonicosahedral structure within the double-layered icosahedral capsid. Our structure shows that the RdRp complex is anchored at the inner surface of the capsid shell and interacts with genomic dsRNA and four of the five asymmetrically arranged N termini of the capsid shell proteins under the fivefold axis, implying roles for these N termini in virus assembly. The binding site of the RNA end at VP2 is different from the RNA cap binding site identified in the crystal structure of orthoreovirus RdRp λ3, although the structures of VP2 and λ3 are almost identical. A loop, which was thought to separate the RNA template and transcript, interacts with an apical domain of the capsid shell protein, suggesting a mechanism for regulating RdRp replication and transcription. A conserved nucleoside triphosphate binding site was localized in our RdRp cofactor protein VP4 structure, and interactions between the VP4 and the genomic RNA were identified.


Assuntos
Proteínas do Capsídeo/biossíntese , RNA Polimerases Dirigidas por DNA/metabolismo , Genoma Viral , RNA Viral/biossíntese , Reoviridae/fisiologia , Transcrição Genética/fisiologia , Montagem de Vírus/fisiologia , Animais , Proteínas do Capsídeo/genética , Carpas , Linhagem Celular , RNA Viral/genética
19.
Yeast ; 35(9): 519-529, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29709079

RESUMO

Cervical cancer is ranked the fourth most common cancer in women worldwide. Despite two prophylactic vaccines being commercially available, they are unaffordable for most women in developing countries. We compared the optimized expression of monomers of the unique HPV type 16 L1-L2 chimeric protein (SAF) in two yeast strains of Pichia pastoris, KM71 (Muts ) and GS115 (Mut+ ), with Hansenula polymorpha NCYC 495 to determine the preferred host in bioreactors. SAF was uniquely created by replacing the h4 helix of the HPV-16 capsid L1 protein with an L2 peptide. Two different feeding strategies in fed-batch cultures of P. pastoris Muts were evaluated: a predetermined feed rate vs. feeding based on the oxygen consumption by maintaining constant dissolved oxygen levels (DO stat). All cultures showed a significant increase in biomass when methanol was fed using the DO stat method. In P. pastoris the SAF concentrations were higher in the Muts strains than in the Mut+ strains. However, H. polymorpha produced the highest level of SAF at 132.10 mg L-1 culture while P. pastoris Muts only produced 23.61 mg L-1 . H. polymorpha showed greater potential for the expression of HPV-16 L1/L2 chimeric proteins despite the track record of P. pastoris as a high-level producer of heterologous proteins.


Assuntos
Proteínas do Capsídeo/biossíntese , Expressão Gênica , Proteínas Oncogênicas Virais/biossíntese , Pichia/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Biomassa , Reatores Biológicos , Proteínas do Capsídeo/genética , Meios de Cultura/química , Humanos , Metanol/metabolismo , Proteínas Oncogênicas Virais/genética , Oxigênio/análise , Oxigênio/metabolismo , Pichia/genética , Pichia/crescimento & desenvolvimento , Proteínas Recombinantes de Fusão/genética
20.
Sheng Wu Gong Cheng Xue Bao ; 34(4): 586-593, 2018 Apr 25.
Artigo em Chinês | MEDLINE | ID: mdl-29701032

RESUMO

We constructed the CAP2NC prokaryotic expression vector of HIV-1 NL4-3 strain and obtained relatively pure CAP2NC protein by optimizing its purification conditions to explore its in vitro self-assembly conditions. Primers were designed according to the CAP2NC DNA sequence of HIV-1 NL4-3 strain. The target gene was amplified by PCR and cloned into prokaryotic expression vector pTO-T7. Then the recombinant strain was transformed into Escherichia coli BL21 (DE3). IPTG induced protein expression, then the protein was purified by hydrophobic chromatography. SDS-PAGE and Western blotting were performed to analyze the target protein, and the biological activity of the antigen was identified through ELISA. The self-assembly of CAP2NC protein was analyzed by transmission electron microscopy and gel filtration chromatography. The protein had good reaction with the specific antibodies of p24 and formed different structures in various conditions. When 10% yeast RNA was added to the protein complex, the recombinant protein only formed into a tubular structure, which was similar to the self-assembled structure of the HIV-1 virus capsid. The results showed that the HIV-1 CAP2NC protein had in vitro self-assembly activity, and the RNA affected the structure of CAP2NC protein assembly. The protein can be used as a simple and effective molecular model to study its structure, and then it can provide a reference for the study of HIV immature virus particles.


Assuntos
Proteínas do Capsídeo/biossíntese , HIV-1/química , Proteínas do Vírus da Imunodeficiência Humana/biossíntese , Proteínas Recombinantes/biossíntese , Clonagem Molecular , Escherichia coli , Expressão Gênica
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