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1.
Nat Commun ; 12(1): 1543, 2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33750839

RESUMO

Protein engineering has great potential for devising multifunctional recombinant proteins to serve as next-generation protein therapeutics, but it often requires drastic modifications of the parental protein scaffolds e.g., additional domains at the N/C-terminus or replacement of a domain by another. A discovery platform system, called RaPID (Random non-standard Peptides Integrated Discovery) system, has enabled rapid discovery of small de novo macrocyclic peptides that bind a target protein with high binding specificity and affinity. Capitalizing on the optimized binding properties of the RaPID-derived peptides, here we show that RaPID-derived pharmacophore sequences can be readily implanted into surface-exposed loops on recombinant proteins and maintain both the parental peptide binding function(s) and the host protein function. We refer to this protein engineering method as lasso-grafting and demonstrate that it can endow specific binding capacity toward various receptors into a diverse set of scaffolds that includes IgG, serum albumin, and even capsid proteins of adeno-associated virus, enabling us to rapidly formulate and produce bi-, tri-, and even tetra-specific binder molecules.


Assuntos
Peptídeos/química , Peptídeos/farmacologia , Engenharia de Proteínas/métodos , Proteínas do Capsídeo/química , Proteínas de Transporte/química , Linhagem Celular , Dependovirus , Humanos , Imunoglobulina G/química , Modelos Moleculares , Albumina Sérica/química , Bibliotecas de Moléculas Pequenas
2.
Nat Commun ; 12(1): 1642, 2021 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-33712599

RESUMO

Adeno-associated viruses (AAVs) are increasingly used as gene therapy vectors. AAVs package their genome in a non-enveloped T = 1 icosahedral capsid of ~3.8 megaDalton, consisting of 60 subunits of 3 distinct viral proteins (VPs), which vary only in their N-terminus. While all three VPs play a role in cell-entry and transduction, their precise stoichiometry and structural organization in the capsid has remained elusive. Here we investigate the composition of several AAV serotypes by high-resolution native mass spectrometry. Our data reveal that the capsids assemble stochastically, leading to a highly heterogeneous population of capsids of variable composition, whereby even the single-most abundant VP stoichiometry represents only a small percentage of the total AAV population. We estimate that virtually every AAV capsid in a particular preparation has a unique composition. The systematic scoring of the simulations against experimental native MS data offers a sensitive new method to characterize these therapeutically important heterogeneous capsids.


Assuntos
Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Capsídeo/química , Capsídeo/metabolismo , Dependovirus/metabolismo , Animais , Dependovirus/genética , Células HEK293 , Humanos , Sorogrupo , Células Sf9 , Proteínas Virais/metabolismo , Montagem de Vírus
3.
BMC Infect Dis ; 21(1): 166, 2021 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-33568111

RESUMO

BACKGROUND: An unexpected dengue outbreak occurred in Hunan Province in 2018. This was the first dengue outbreak in this area of inland China, and 172 cases were reported. METHODS: To verify the causative agent of this outbreak and characterise the viral genes, the genes encoding the structural proteins C/prM/E of viruses isolated from local residents were sequenced followed by mutation and phylogenetic analysis. Recombination, selection pressure, potential secondary structure and three-dimensional structure analyses were also performed. RESULTS: Phylogenetic analysis revealed that all epidemic strains were of the cosmopolitan DENV-2 genotype and were most closely related to the Zhejiang strain (MH010629, 2017) and then the Malaysia strain (KJ806803, 2013). Compared with the sequence of DENV-2SS, 151 base substitutions were found in the sequences of 89 isolates; these substitutions resulted in 20 non-synonymous mutations, of which 17 mutations existed in all samples (two in the capsid protein, six in the prM/M proteins, and nine in the envelope proteins). Moreover, amino acid substitutions at the 602nd (E322:Q → H) and 670th (E390: N → S) amino acids may have enhanced the virulence of the epidemic strains. One new DNA binding site and five new protein binding sites were observed. Two polynucleotide binding sites and seven protein binding sites were lost in the epidemic strains compared with DENV-2SS. Meanwhile, five changes were found in helical regions. Minor changes were observed in helical transmembrane and disordered regions. The 429th amino acid of the E protein switched from a histamine (positively charged) to an asparagine (neutral) in all 89 isolated strains. No recombination events or positive selection pressure sites were observed. To our knowledge, this study is the first to analyse the genetic characteristics of epidemic strains in the first dengue outbreak in Hunan Province in inland China. CONCLUSIONS: The causative agent is likely to come from Zhejiang Province, a neighbouring province where dengue fever broke out in 2017. This study may help clarify the intrinsic geographical relatedness of DENV-2 and contribute to further research on pathogenicity and vaccine development.


Assuntos
Vírus da Dengue/genética , Dengue/diagnóstico , Proteínas do Envelope Viral/genética , Sítios de Ligação , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , China/epidemiologia , Dengue/epidemiologia , Dengue/virologia , Vírus da Dengue/classificação , Vírus da Dengue/isolamento & purificação , Surtos de Doenças , Genótipo , Humanos , Mutação , Filogenia , Estrutura Terciária de Proteína , RNA Viral/química , RNA Viral/metabolismo , Análise de Sequência de RNA , Proteínas do Envelope Viral/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
4.
Viruses ; 13(2)2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33557422

RESUMO

Understanding of the construction and function of the HIV capsid has advanced considerably in the last decade. This is due in large part to the development of more sophisticated structural techniques, particularly cryo-electron microscopy (cryoEM) and cryo-electron tomography (cryoET). The capsid is known to be a pleomorphic fullerene cone comprised of capsid protein monomers arranged into 200-250 hexamers and 12 pentamers. The latter of these induce high curvature necessary to close the cone at both ends. CryoEM/cryoET, NMR, and X-ray crystallography have collectively described these interactions to atomic or near-atomic resolutions. Further, these techniques have helped to clarify the role the HIV capsid plays in several parts of the viral life cycle, from reverse transcription to nuclear entry and integration into the host chromosome. This includes visualizing the capsid bound to host factors. Multiple proteins have been shown to interact with the capsid. Cyclophilin A, nucleoporins, and CPSF6 promote viral infectivity, while MxB and Trim5α diminish the viral infectivity. Finally, structural insights into the intra- and intermolecular interactions that govern capsid function have enabled development of small molecules, peptides, and truncated proteins to disrupt or stabilize the capsid to inhibit HIV replication. The most promising of these, GS6207, is now in clinical trial.


Assuntos
Antivirais/metabolismo , Capsídeo/metabolismo , Infecções por HIV/metabolismo , HIV-1/metabolismo , Animais , Antivirais/química , Antivirais/farmacologia , Capsídeo/química , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Infecções por HIV/virologia , HIV-1/química , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Interações Hospedeiro-Patógeno , Humanos , Ligação Proteica , Replicação Viral/efeitos dos fármacos
5.
Viruses ; 13(2)2021 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-33578999

RESUMO

Since the discovery of HIV-1, the viral capsid has been recognized to have an important role as a structural protein that holds the viral genome, together with viral proteins essential for viral life cycle, such as the reverse transcriptase (RT) and the integrase (IN). The reverse transcription process takes place between the cytoplasm and the nucleus of the host cell, thus the Reverse Transcription Complexes (RTCs)/Pre-integration Complexes (PICs) are hosted in intact or partial cores. Early biochemical assays failed to identify the viral CA associated to the RTC/PIC, possibly due to the stringent detergent conditions used to fractionate the cells or to isolate the viral complexes. More recently, it has been observed that some host partners of capsid, such as Nup153 and CPSF6, can only bind multimeric CA proteins organized in hexamers. Those host factors are mainly located in the nuclear compartment, suggesting the entrance of the viral CA as multimeric structure inside the nucleus. Recent data show CA complexes within the nucleus having a different morphology from the cytoplasmic ones, clearly highlighting the remodeling of the viral cores during nuclear translocation. Thus, the multimeric CA complexes lead the viral genome into the host nuclear compartment, piloting the intranuclear journey of HIV-1 in order to successfully replicate. The aim of this review is to discuss and analyze the main discoveries to date that uncover the viral capsid as a key player in the reverse transcription and PIC maturation until the viral DNA integration into the host genome.


Assuntos
Capsídeo/metabolismo , Núcleo Celular/virologia , HIV-1/fisiologia , Transporte Ativo do Núcleo Celular , Capsídeo/química , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Núcleo Celular/metabolismo , HIV-1/química , HIV-1/metabolismo , Modelos Biológicos , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Transcrição Reversa , Integração Viral , Replicação Viral
6.
Viruses ; 13(1)2021 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-33477441

RESUMO

Integration of retroviral reverse transcripts into the chromosomes of the cells that they infect is required for efficient viral gene expression and the inheritance of viral genomes to daughter cells. Before integration can occur, retroviral reverse transcription complexes (RTCs) must access the nuclear environment where the chromosomes reside. Retroviral integration is non-random, with different types of virus-host interactions impacting where in the host chromatin integration takes place. Lentiviruses such as HIV efficiently infect interphase cells because their RTCs have evolved to usurp cellular nuclear import transport mechanisms, and research over the past decade has revealed specific interactions between the HIV capsid protein and nucleoporin (Nup) proteins such as Nup358 and Nup153. The interaction of HIV capsid with cleavage and polyadenylation specificity factor 6 (CPSF6), which is a component of the cellular cleavage and polyadenylation complex, helps to dictate nuclear import as well as post-nuclear RTC invasion. In the absence of the capsid-CPSF6 interaction, RTCs are precluded from reaching nuclear speckles and gene-rich regions of chromatin known as speckle-associated domains, and instead mis-target lamina-associated domains out at the nuclear periphery. Highlighting this area of research, small molecules that inhibit capsid-host interactions important for integration site targeting are highly potent antiviral compounds.


Assuntos
Proteínas do Capsídeo/metabolismo , Capsídeo/metabolismo , Infecções por HIV/virologia , HIV-1/fisiologia , Interações Hospedeiro-Patógeno , Integração Viral , Transporte Ativo do Núcleo Celular , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Cromatina , Infecções por HIV/genética , Infecções por HIV/metabolismo , Humanos , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Ligação Proteica , Relação Estrutura-Atividade
7.
Viruses ; 13(1)2021 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-33450892

RESUMO

The capsid structures of most Adeno-associated virus (AAV) serotypes, already assigned to an antigenic clade, have been previously determined. This study reports the remaining capsid structures of AAV7, AAV11, AAV12, and AAV13 determined by cryo-electron microscopy and three-dimensional image reconstruction to 2.96, 2.86, 2.54, and 2.76 Å resolution, respectively. These structures complete the structural atlas of the AAV serotype capsids. AAV7 represents the first clade D capsid structure; AAV11 and AAV12 are of a currently unassigned clade that would include AAV4; and AAV13 represents the first AAV2-AAV3 hybrid clade C capsid structure. These newly determined capsid structures all exhibit the AAV capsid features including 5-fold channels, 3-fold protrusions, 2-fold depressions, and a nucleotide binding pocket with an ordered nucleotide in genome-containing capsids. However, these structures have viral proteins that display clade-specific loop conformations. This structural characterization completes our three-dimensional library of the current AAV serotypes to provide an atlas of surface loop configurations compatible with capsid assembly and amenable for future vector engineering efforts. Derived vectors could improve gene delivery success with respect to specific tissue targeting, transduction efficiency, antigenicity or receptor retargeting.


Assuntos
Capsídeo/ultraestrutura , Dependovirus/classificação , Dependovirus/ultraestrutura , Modelos Moleculares , Vírion/ultraestrutura , Capsídeo/metabolismo , Proteínas do Capsídeo/química , Microscopia Crioeletrônica , Dependovirus/genética , Genoma Viral , Humanos , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Sorogrupo
8.
Viruses ; 13(1)2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33467558

RESUMO

Adenovirus (AdV) infection elicits a strong immune response with the production of neutralizing antibodies and opsonization by complement and coagulation factors. One anti-hexon neutralizing antibody, called 9C12, is known to activate the complement cascade, resulting in the deposition of complement component C4b on the capsid, and the neutralization of the virus. The mechanism of AdV neutralization by C4b is independent of downstream complement proteins and involves the blockage of the release of protein VI, which is required for viral escape from the endosome. To investigate the structural basis underlying how C4b blocks the uncoating of AdV, we built a model for the complex of human adenovirus type-5 (HAdV5) with 9C12, together with complement components C1 and C4b. This model positions C4b near the Arg-Gly-Asp (RGD) loops of the penton base. There are multiple amino acids in the RGD loop that might serve as covalent binding sites for the reactive thioester of C4b. Molecular dynamics simulations with a multimeric penton base and C4b indicated that stabilizing interactions may form between C4b and multiple RGD loops. We propose that C4b deposition on one RGD loop leads to the entanglement of C4b with additional RGD loops on the same penton base multimer and that this entanglement blocks AdV uncoating.


Assuntos
Adenoviridae/imunologia , Complemento C4/química , Complemento C4/imunologia , Modelos Moleculares , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Sítios de Ligação , Capsídeo/química , Capsídeo/metabolismo , Capsídeo/ultraestrutura , Proteínas do Capsídeo/química , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/ultraestrutura , Humanos , Imunoglobulina G/química , Imunoglobulina G/imunologia , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade
9.
Viruses ; 13(1)2021 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-33435207

RESUMO

The structures of the four N-linked glycans from the prototype chlorovirus PBCV-1 major capsid protein do not resemble any other glycans in the three domains of life. All known chloroviruses and antigenic variants (or mutants) share a unique conserved central glycan core consisting of five sugars, except for antigenic mutant virus P1L6, which has four of the five sugars. A combination of genetic and structural analyses indicates that the protein coded by PBCV-1 gene a111/114r, conserved in all chloroviruses, is a glycosyltransferase with three putative domains of approximately 300 amino acids each. Here, in addition to in silico sequence analysis and protein modeling, we measured the hydrolytic activity of protein A111/114R. The results suggest that domain 1 is a galactosyltransferase, domain 2 is a xylosyltransferase and domain 3 is a fucosyltransferase. Thus, A111/114R is the protein likely responsible for the attachment of three of the five conserved residues of the core region of this complex glycan, and, if biochemically corroborated, it would be the second three-domain protein coded by PBCV-1 that is involved in glycan synthesis. Importantly, these findings provide additional support that the chloroviruses do not use the canonical host endoplasmic reticulum-Golgi glycosylation pathway to glycosylate their glycoproteins; instead, they perform glycosylation independent of cellular organelles using virus-encoded enzymes.


Assuntos
Glicosiltransferases/metabolismo , Phycodnaviridae/fisiologia , Polissacarídeos/biossíntese , Domínios Proteicos , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Glicosiltransferases/química , Ligação de Hidrogênio , Hidrólise , Modelos Moleculares , Conformação Proteica , Relação Estrutura-Atividade , Proteínas Virais/química
10.
Viruses ; 13(1)2021 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-33445703

RESUMO

Children in low-and middle-income countries, including Rwanda, experience a greater burden of rotavirus disease relative to developed countries. Evolutionary mechanisms leading to multiple reassortant rotavirus strains have been documented over time which influence the diversity and evolutionary dynamics of novel rotaviruses. Comprehensive rotavirus whole-genome analysis was conducted on 158 rotavirus group A (RVA) samples collected pre- and post-vaccine introduction in children less than five years in Rwanda. Of these RVA positive samples, five strains with the genotype constellations G4P[4]-I1-R2-C2-M2-A2-N2-T1-E1-H2 (n = 1), G9P[4]-I1-R2-C2-M2-A1-N1-T1-E1-H1 (n = 1), G12P[8]-I1-R2-C2-M1-A1-N2-T1-E2-H3 (n = 2) and G12P[8]-I1-R1-C1-M1-A2-N2-T2-E1-H1 (n = 1), with double and triple gene reassortant rotavirus strains were identified. Phylogenetic analysis revealed a close relationship between the Rwandan strains and cognate human RVA strains as well as the RotaTeq® vaccine strains in the VP1, VP2, NSP2, NSP4 and NSP5 gene segments. Pairwise analyses revealed multiple differences in amino acid residues of the VP7 and VP4 antigenic regions of the RotaTeq® vaccine strain and representative Rwandan study strains. Although the impact of such amino acid changes on the effectiveness of rotavirus vaccines has not been fully explored, this analysis underlines the potential of rotavirus whole-genome analysis by enhancing knowledge and understanding of intergenogroup reassortant strains circulating in Rwanda post vaccine introduction.


Assuntos
Genoma Viral , Genômica , Vírus Reordenados/genética , Infecções por Rotavirus/epidemiologia , Infecções por Rotavirus/virologia , Rotavirus/classificação , Rotavirus/genética , Sequência de Aminoácidos , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Bases de Dados de Ácidos Nucleicos , Genômica/métodos , Humanos , Modelos Moleculares , Filogenia , Conformação Proteica , Infecções por Rotavirus/prevenção & controle , Ruanda/epidemiologia , Análise de Sequência de DNA , Vacinação , Vacinas Virais/imunologia , Sequenciamento Completo do Genoma
11.
Methods Mol Biol ; 2183: 357-366, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32959253

RESUMO

Although adenovirus is a popular vector for delivering genes, there are several drawbacks that limit its effectiveness, including tropism and both the innate and adaptive immune responses. One approach that has been used to ameliorate these drawbacks is PEGylation of the virus with subsequent modification to add functional moieties for the purpose of cell targeting or enhancing infection. Here, we describe a general approach for PEGylating adenovirus and conjugating cell-penetrating peptides to the surface of the virus to impart the ability to transduce CAR-negative cells.


Assuntos
Adenoviridae/imunologia , Proteínas do Capsídeo/imunologia , Vetores Genéticos/efeitos adversos , Interações Hospedeiro-Patógeno/imunologia , Adenoviridae/genética , Sequência de Aminoácidos , Animais , Antígenos Virais/química , Antígenos Virais/imunologia , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Peptídeos Penetradores de Células/química , Técnicas de Transferência de Genes , Terapia Genética/efeitos adversos , Vetores Genéticos/genética , Camundongos , Estrutura Molecular , Células NIH 3T3 , Polietilenoglicóis/química , Transdução Genética
12.
Subcell Biochem ; 96: 451-470, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33252740

RESUMO

Non-enveloped Nackednaviridae and enveloped hepadnaviridae both have capsids that are formed by related small proteins which evolved more than 430 Mya. In Hepatitis B virus, which belongs to the enveloped hepadnaviridae, this small protein is termed Hepatitis B core protein (Hbc). Its function, as building block of a major human pathogen, triggered extensive research that elucidated the importance of almost every single amino acid for the structural integrity of the capsids and the orderly progression of the viral life cycle. In particular, encapsidation of the genome, envelopment of the capsid, uncoating of the genome and targeting of the different compartments during viral maturation have been a vivid focus of research. HBc has also been developed as a biotechnological tool for the design of nano-containers with tailored properties. These nano-containers can display foreign epitopes on their surfaces and induce a strong immune response, which is attractive for the development of vaccines against other pathogens. This chapter will discuss some of the unique properties of HBc and their significance for the formation of a functional macromolecular capsid.


Assuntos
Capsídeo/química , Vírus da Hepatite B/química , Proteínas do Core Viral/química , Proteínas do Capsídeo/química , Hepatite B/virologia , Humanos
13.
Subcell Biochem ; 96: 503-518, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33252742

RESUMO

Human adenoviruses (HAdVs) are large (150 MDa), complex, nonenveloped dsDNA viruses that cause self-limiting respiratory, ocular and enteric infections. They are significant health hazard in young, elderly and immuno-compromised populations. Moreover, various adenoviruses (AdVs) of mammalian origin are being used as vectors in gene, vaccine and cancer therapies. Multiple copies of at least 13 different proteins, all in all ~2800 protein molecules, come together to form an adenovirus virion packaging the ~36 Kbp geome. The details of structural organization of the adenovirus capsid and underlying network of protein-protein interactions provide clues into designing the modified and novel adenovirus vectors with desired functionalities and/or targeting specificities. The advancements in 3D structure determination by cryo-electron microscopy (cryo-EM) in the past decade have enabled unveiling of the complex organization of adenovirus architecture at near atomic resolution. Specifically, these studies revealed the structures and the network of interactions involving cement/minor proteins in stabilizing the AdV icosahedral architecture, which appear to be mostly conserved among human adenoviruses. In this chapter, we describe the current state of knowledge on the structure and organization of human adenoviruses.


Assuntos
Adenovírus Humanos/química , Proteínas do Capsídeo/química , Capsídeo/química , Adenovírus Humanos/ultraestrutura , Capsídeo/ultraestrutura , Proteínas do Capsídeo/ultraestrutura , Microscopia Crioeletrônica , Humanos , Modelos Moleculares , Ligação Proteica
14.
Methods Mol Biol ; 2208: 101-121, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32856258

RESUMO

Spherical viruses are unique nanocapsules formed by self-assembly of coat proteins (capsids). By mimicking natural spherical capsids, various artificial viral capsids are developed by using self-assembled proteins and peptides as building blocks. We developed an artificial viral capsid consisting of a ß-annulus peptide designed from natural viruses. The "ß-annulus capsid" can be functionalized by encapsulating guest molecules to the inside and decoration of exogenous molecules on the outside. Here, we describe the encapsulation and decoration on the ß-annulus capsids by connecting additional sequences to the ß-annulus peptide, conjugation with objective molecules, and subsequent self-assembly in aqueous solutions.


Assuntos
Nanocápsulas/química , Peptídeos/química , Sequência de Aminoácidos , Capsídeo/química , Proteínas do Capsídeo/química
15.
Viruses ; 12(12)2020 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-33352888

RESUMO

The viral protein 1 unique region (VP1u) of human parvovirus B19 (B19V) is a multifunctional capsid protein with essential roles in virus tropism, uptake, and subcellular trafficking. These functions reside on hidden protein domains, which become accessible upon interaction with cell membrane receptors. A receptor-binding domain (RBD) in VP1u is responsible for the specific targeting and uptake of the virus exclusively into cells of the erythroid lineage in the bone marrow. A phospholipase A2 domain promotes the endosomal escape of the incoming virus. The VP1u is also the immunodominant region of the capsid as it is the target of neutralizing antibodies. For all these reasons, the VP1u has raised great interest in antiviral research and vaccinology. Besides the essential functions in B19V infection, the remarkable erythroid specificity of the VP1u makes it a unique erythroid cell surface biomarker. Moreover, the demonstrated capacity of the VP1u to deliver diverse cargo specifically to cells around the proerythroblast differentiation stage, including erythroleukemic cells, offers novel therapeutic opportunities for erythroid-specific drug delivery. In this review, we focus on the multifunctional role of the VP1u in B19V infection and explore its potential in diagnostics and erythroid-specific therapeutics.


Assuntos
Biotecnologia , Proteínas do Capsídeo/fisiologia , Sítios de Ligação , Proteínas do Capsídeo/química , Proteínas do Capsídeo/imunologia , Epitopos Imunodominantes , Sinais de Localização Nuclear , Parvovirus B19 Humano/fisiologia , Fosfolipases A2/química , Receptores Virais , Tropismo Viral , Vírion/fisiologia
16.
Int J Mol Sci ; 21(24)2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-33333826

RESUMO

The epidermal growth factor receptor (EGFR) plays a central role in the progression of many solid tumors. We used this validated target to analyze the de novo design of EGFR-binding peptides and their application for the delivery of complex payloads via rational design of a viral vector. Peptides were computationally designed to interact with the EGFR dimerization interface. Two new peptides and a reference (EDA peptide) were chemically synthesized, and their binding ability characterized. Presentation of these peptides in each of the 60 capsid proteins of recombinant adeno-associated viruses (rAAV) via a genetic based loop insertion enabled targeting of EGFR overexpressing tumor cell lines. Furthermore, tissue distribution and tumor xenograft specificity were analyzed with systemic injection in chicken egg chorioallantoic membrane (CAM) assays. Complex correlations between the targeting of the synthetic peptides and the viral vectors to cells and in ovo were observed. Overall, these data demonstrate the potential of computational design in combination with rational capsid modification for viral vector targeting opening new avenues for viral vector delivery and specifically suicide gene therapy.


Assuntos
Dependovirus/metabolismo , Vírus Oncolíticos/química , Peptídeos/química , Engenharia de Proteínas/métodos , Animais , Capsídeo/química , Capsídeo/metabolismo , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Linhagem Celular Tumoral , Embrião de Galinha , Membrana Corioalantoide/metabolismo , Dicroísmo Circular , Biologia Computacional , Dependovirus/química , Dimerização , Receptores ErbB/química , Receptores ErbB/genética , Receptores ErbB/metabolismo , Terapia Genética , Vetores Genéticos , Humanos , Microscopia de Fluorescência , Vírus Oncolíticos/genética , Vírus Oncolíticos/metabolismo , Peptídeos/síntese química , Ligação Proteica , Transplante Heterólogo , Regulação para Cima , Cicatrização/efeitos dos fármacos
17.
Nat Commun ; 11(1): 5253, 2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-33067459

RESUMO

Enterovirus 71 (EV71)-neutralizing antibodies correlate with protection and have potential as therapeutic agents. We isolate and characterize a panel of plasmablast-derived monoclonal antibodies from an infected child whose antibody response focuses on the plateau epitope near the icosahedral 3-fold axes. Eight of a total of 19 antibodies target this epitope and three of these potently neutralize the virus. Representative neutralizing antibodies 38-1-10A and 38-3-11A both confer effective protection against lethal EV71 challenge in hSCARB2-transgenic mice. The cryo-electron microscopy structures of the EV71 virion in complex with Fab fragments of these potent and protective antibodies reveal the details of a conserved epitope formed by residues in the BC and HI loops of VP2 and the BC and HI loops of VP3 spanning the region around the 3-fold axis. Remarkably, the two antibodies interact with the epitope in quite distinct ways. These plateau-binding antibodies provide templates for promising candidate therapeutics.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Enterovirus Humano A/imunologia , Infecções por Enterovirus/imunologia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Enterovirus Humano A/química , Enterovirus Humano A/genética , Infecções por Enterovirus/virologia , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Testes de Neutralização
18.
Nat Commun ; 11(1): 4693, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32943634

RESUMO

The alphavirus capsid protein (Cp) selectively packages genomic RNA (gRNA) into the viral nucleocapsid to produce infectious virus. Using photoactivatable ribonucleoside crosslinking and an innovative biotinylated Cp retrieval method, here we comprehensively define binding sites for Semliki Forest virus (SFV) Cp on the gRNA. While data in infected cells demonstrate Cp binding to the proposed genome packaging signal (PS), mutagenesis experiments show that PS is not required for production of infectious SFV or Chikungunya virus. Instead, we identify multiple Cp binding sites that are enriched on gRNA-specific regions and promote infectious SFV production and gRNA packaging. Comparisons of binding sites in cytoplasmic vs. viral nucleocapsids demonstrate that budding causes discrete changes in Cp-gRNA interactions. Notably, Cp's top binding site is maintained throughout virus assembly, and specifically binds and assembles with Cp into core-like particles in vitro. Together our data suggest a model for selective alphavirus genome recognition and assembly.


Assuntos
Alphavirus/metabolismo , Proteínas do Capsídeo/metabolismo , Capsídeo/metabolismo , Genômica , RNA Viral/genética , Alphavirus/genética , Alphavirus/ultraestrutura , Animais , Sítios de Ligação , Capsídeo/química , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Vírus Chikungunya/genética , Chlorocebus aethiops , Modelos Moleculares , Nucleocapsídeo/metabolismo , Ligação Proteica , RNA Viral/química , Vírus da Floresta de Semliki/metabolismo , Células Vero , Montagem de Vírus , Replicação Viral
19.
Nat Commun ; 11(1): 4795, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32963252

RESUMO

Varicella-zoster virus (VZV), a member of the Alphaherpesvirinae subfamily, causes severe diseases in humans of all ages. The viral capsids play critical roles in herpesvirus infection, making them potential antiviral targets. Here, we present the 3.7-Å-resolution structure of the VZV A-capsid and define the molecular determinants underpinning the assembly of this complicated viral machinery. Overall, the VZV capsid has a similar architecture to that of other known herpesviruses. The major capsid protein (MCP) assembles into pentons and hexons, forming extensive intra- and inter-capsomer interaction networks that are further secured by the small capsid protein (SCP) and the heterotriplex. The structure reveals a pocket beneath the floor of MCP that could potentially be targeted by antiviral inhibitors. In addition, we identified two alphaherpesvirus-specific structural features in SCP and Tri1 proteins. These observations highlight the divergence of different herpesviruses and provide an important basis for developing antiviral drugs.


Assuntos
Proteínas do Capsídeo/química , Capsídeo/química , Microscopia Crioeletrônica/métodos , Herpesvirus Humano 3/metabolismo , Linhagem Celular , Humanos , Modelos Moleculares , Conformação Proteica , Domínios Proteicos
20.
Nat Struct Mol Biol ; 27(9): 863-869, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32901160

RESUMO

HIV-1 capsid plays multiple key roles in viral replication, and inhibition of capsid assembly is an attractive target for therapeutic intervention. Here, we report the atomic-resolution structure of capsid protein (CA) tubes, determined by magic-angle spinning NMR and data-guided molecular dynamics simulations. Functionally important regions, including the NTD ß-hairpin, the cyclophilin A-binding loop, residues in the hexamer central pore, and the NTD-CTD linker region, are well defined. The structure of individual CA chains, their arrangement in the pseudo-hexameric units of the tube and the inter-hexamer interfaces are consistent with those in intact capsids and substantially different from the organization in crystal structures, which feature flat hexamers. The inherent curvature in the CA tubes is controlled by conformational variability of residues in the linker region and of dimer and trimer interfaces. The present structure reveals atomic-level detail in capsid architecture and provides important guidance for the design of novel capsid inhibitors.


Assuntos
Proteínas do Capsídeo/química , Capsídeo/química , Infecções por HIV/virologia , HIV-1/química , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Multimerização Proteica
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