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1.
Anal Bioanal Chem ; 412(7): 1563-1572, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31938845

RESUMO

Virus-like particles (VLPs) are widely used in medicine, but can be difficult to characterize and isolate from aggregates. In this research, primarily cyclical electrical field-flow fractionation (CyElFFF) coupled with multi-angle light scattering (MALS), and dynamic light scattering (DLS) detectors, was used for the first time to perform size and electrical characterization of three different types of Q beta bacteriophage virus-like particles (VLPs): a blank Q beta bacteriophage which is denoted as VLP and two conjugated ones with different peptides. The CyElFFF results were verified with transmission electron microscopy (TEM). Asymmetrical flow field-flow fractionation (AF4) coupled with MALS was also applied using conditions similar to those used in the CyElFFF experiments, and the results of the two techniques were compared to each other. Using these techniques, the size and electrophoretic characteristics of the fractionated VLPs in CyElFFF were obtained. The results indicate that CyElFFF can be used to obtain a clear distribution of electrophoretic mobilities for each type of VLP. Accordingly, CyElFFF was able to differentially retain and isolate VLPs with high surface electric charge/electrophoretic mobility from the ones with low electric charge/electrophoretic mobility. Regarding the size characterization, the size distribution of the eluted VLPs was obtained using both techniques. CyElFFF was able to identify subpopulations that did not appear in the AF4 results by generating a shoulder peak, whereas AF4 produced a single peak. Different size characteristics of the VLPs appearing in the shoulder peak and the main peak indicate that CyElFFF was able to isolate aggregated VLPs from the monomers partially. Graphical abstract.


Assuntos
Bacteriófagos/isolamento & purificação , Eletricidade , Fracionamento por Campo e Fluxo/métodos , Vírion/metabolismo , Sequência de Aminoácidos , Bacteriófagos/metabolismo , Eletroforese Capilar , Proteínas Virais/química
2.
Proc Natl Acad Sci U S A ; 117(2): 1181-1190, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31879355

RESUMO

Negative-stranded/ambisense RNA viruses (NSVs) include not only dangerous pathogens of medical importance but also serious plant pathogens of agronomic importance. Tomato spotted wilt virus (TSWV) is one of the most important plant NSVs, infecting more than 1,000 plant species, and poses major threats to global food security. The segmented negative-stranded/ambisense RNA genomes of TSWV, however, have been a major obstacle to molecular genetic manipulation. In this study, we report the complete recovery of infectious TSWV entirely from complementary DNA (cDNA) clones. First, a replication- and transcription-competent minigenome replication system was established based on 35S-driven constructs of the S(-)-genomic (g) or S(+)-antigenomic (ag) RNA template, flanked by the 5' hammerhead and 3' ribozyme sequence of hepatitis delta virus, a nucleocapsid (N) protein gene and codon-optimized viral RNA-dependent RNA polymerase (RdRp) gene. Next, a movement-competent minigenome replication system was developed based on M(-)-gRNA, which was able to complement cell-to-cell and systemic movement of reconstituted ribonucleoprotein complexes (RNPs) of S RNA replicon. Finally, infectious TSWV and derivatives carrying eGFP reporters were rescued in planta via simultaneous expression of full-length cDNA constructs coding for S(+)-agRNA, M(-)-gRNA, and L(+)-agRNA in which the glycoprotein gene sequence of M(-)-gRNA was optimized. Viral rescue occurred with the addition of various RNAi suppressors including P19, HcPro, and γb, but TSWV NSs interfered with the rescue of genomic RNA. This reverse genetics system for TSWV now allows detailed molecular genetic analysis of all aspects of viral infection cycle and pathogenicity.


Assuntos
DNA Complementar/genética , Tospovirus/genética , Tospovirus/fisiologia , Tospovirus/patogenicidade , RNA Polimerases Dirigidas por DNA/genética , Vírus Delta da Hepatite/genética , Proteínas do Nucleocapsídeo/genética , Doenças das Plantas/virologia , RNA Catalítico/genética , RNA Viral/genética , Replicon , Tabaco/virologia , Proteínas Virais/genética , Vírion/genética , Vírion/metabolismo , Replicação Viral
3.
Acta Virol ; 63(4): 450-458, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31802688

RESUMO

For successful infection, viruses must recognize their respective host cells. A common mechanism of host recognition by viruses is to utilize a portion of the host cell as a receptor. Bacteriophage Sf6, which infects Shigella flexneri, uses lipopolysaccharide as a primary receptor and then requires interaction with a secondary receptor, a role that can be fulfilled by either outer membrane proteins (Omp) A or C. Our previous work showed that specific residues in the loops of OmpA mediate Sf6 infection. To better understand Sf6 interactions with OmpA loop variants, we determined the kinetics of these interactions through the use of biolayer interferometry, an optical biosensing technique that yields data similar to surface plasmon resonance. Here, we successfully tethered whole Sf6 virions, determined the binding constant of Sf6 to OmpA to be 36 nM. Additionally, we showed that Sf6 bound to five variant OmpAs and the resulting kinetic parameters varied only slightly. Based on these data, we propose a model in which Sf6: Omp receptor recognition is not solely based on kinetics, but likely also on the ability of an Omp to induce a conformational change that results in productive infection. Keywords: Sf6; Shigella flexneri; OmpA; biolayer interferometry.


Assuntos
Proteínas da Membrana Bacteriana Externa , Bacteriófagos , Vírion , Proteínas da Membrana Bacteriana Externa/metabolismo , Bacteriófagos/metabolismo , Cinética , Ligação Proteica , Vírion/metabolismo
4.
PLoS Pathog ; 15(12): e1008209, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31790506

RESUMO

The processes of cell attachment and membrane fusion of Herpes Simplex Virus 1 involve many different envelope glycoproteins. Viral proteins gC and gD bind to cellular receptors. Upon binding, gD activates the gH/gL complex which in turn activates gB to trigger membrane fusion. Thus, these proteins must be located at the point of contact between cellular and viral envelopes to interact and allow fusion. Using super-resolution microscopy, we show that gB, gH/gL and most of gC are distributed evenly round purified virions. In contrast, gD localizes essentially as clusters which are distinct from gB and gH/gL. Upon cell binding, we observe that all glycoproteins, including gD, have a similar ring-like pattern, but the diameter of these rings was significantly smaller than those observed on cell-free viruses. We also observe that contrary to cell-free particles, gD mostly colocalizes with other glycoproteins on cell-bound particles. The differing patterns of localization of gD between cell-free and cell-bound viruses indicates that gD can be reorganized on the viral envelope following either a possible maturation of the viral particle or its adsorption to the cell. This redistribution of glycoproteins upon cell attachment could contribute to initiate the cascade of activations leading to membrane fusion.


Assuntos
Herpesvirus Humano 1/metabolismo , Proteínas do Envelope Viral/metabolismo , Vírion/metabolismo , Linhagem Celular , Glicoproteínas/metabolismo , Glicoproteínas/ultraestrutura , Herpesvirus Humano 1/ultraestrutura , Humanos , Microscopia/métodos , Proteínas do Envelope Viral/ultraestrutura , Vírion/ultraestrutura , Ligação Viral , Internalização do Vírus
5.
Int J Nanomedicine ; 14: 6601-6613, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31496701

RESUMO

Purpose: The primary goal of the present study was to explore and evaluate the highly conserved Neisserial surface protein A (NspA) molecule, fused with truncated HBV virus-like particles (VLPs), as a candidate vaccine against the virulent Neisseria meningitidis serogroup B (NMB). Methods: NspA was inserted into the major immunodominant region of the truncated hepatitis B virus core protein (HBc; amino acids 1-144). The chimeric protein, HBc-N144-NspA, was expressed from a prokaryotic vector and generated HBc-like particles, as determined by transmission electron microscopy. Further, the chimeric protein and control proteins were used to immunize mice and the resulting immune responses evaluated by flow cytometry, enzyme-linked immunosorbent assay, and analysis of serum bactericidal activity (SBA) titer. Results: Evaluation of the immunogenicity of the recombinant HBc-N144-NspA protein showed that it elicited the production of high levels of NspA-specific total IgG. The SBA titer of HBc-N144-NspA/F reached 1:16 2 weeks after the last immunization in BALB/c mice, when human serum complement was included in the vaccine. Immunization of HBc-N144-NspA, even without adjuvant, induced high levels of IL-4 and a high IgG1 to IgG2a ratio, confirming induction of an intense Th2 immune response. Levels of IL-17A increased rapidly in mice after the first immunization with HBc-N144-NspA, indicating the potential for this vaccine to induce a mucosal immune response. Meanwhile, the immunization of HBc-N144-NspA without adjuvant induced only mild inflammatory infiltration into the mouse muscle tissue. Conclusion: This study demonstrates that modification using HBc renders NspA a candidate vaccine, which can trigger protective immunity against NMB.


Assuntos
Proteínas da Membrana Bacteriana Externa/imunologia , Vírus da Hepatite B/metabolismo , Infecções Meningocócicas/imunologia , Infecções Meningocócicas/prevenção & controle , Neisseria meningitidis/patogenicidade , Sorogrupo , Vírion/metabolismo , Adjuvantes Imunológicos/farmacologia , Sequência de Aminoácidos , Animais , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/ultraestrutura , Citocinas/metabolismo , Escherichia coli/metabolismo , Feminino , Imunidade , Imunização , Inflamação/patologia , Ativação Linfocitária/imunologia , Infecções Meningocócicas/patologia , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/imunologia , Teste Bactericida do Soro , Baço/microbiologia , Linfócitos T/imunologia , Vacinação , Virulência
6.
DNA Cell Biol ; 38(11): 1170-1177, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31502877

RESUMO

Host response to viral infection is a highly regulated process involving engagement of various host factors, cytokines, chemokines, and stimulatory signals that pave the way for an antiviral immune response. The response is manifested in terms of viral sequestration, phagocytosis, and inhibition of genome replication, and, finally, if required, lymphocyte-mediated clearance of virally infected cells. During this process, cross-talk between viral and host factors can shape disease outcomes and immunopathology. Bone marrow stromal antigen 2 (BST-2), also know as tetherin, is induced by type I interferon produced in response to viral infections, as well as in certain cancers. BST-2 has been shown to be a host restriction factor of virus multiplication through its ability to physically tether budding virions and restrict viral spread. However, BST-2 has other roles in the host antiviral response. This review focuses on the diverse functions of BST-2 and its downstream signaling pathways in regulating host immune responses.


Assuntos
Antígenos CD/fisiologia , Imunomodulação/genética , Vírion/imunologia , Vírion/metabolismo , Imunidade Adaptativa/genética , Animais , Antígenos CD/genética , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/fisiologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunomodulação/imunologia , Viroses/genética , Viroses/imunologia
7.
Nucleic Acids Res ; 47(14): 7676-7689, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31424549

RESUMO

The potent antiretroviral protein APOBEC3G (A3G) specifically targets and deaminates deoxycytidine nucleotides, generating deoxyuridine, in single stranded DNA (ssDNA) intermediates produced during HIV replication. A non-catalytic domain in A3G binds strongly to RNA, an interaction crucial for recruitment of A3G to the virion; yet, A3G displays no deamination activity for cytidines in viral RNA. Here, we report NMR and molecular dynamics (MD) simulation analysis for interactions between A3Gctd and multiple substrate or non-substrate DNA and RNA, in combination with deamination assays. NMR ssDNA-binding experiments revealed that the interaction with residues in helix1 and loop1 (T201-L220) distinguishes the binding mode of substrate ssDNA from non-substrate. Using 2'-deoxy-2'-fluorine substituted cytidines, we show that a 2'-endo sugar conformation of the target deoxycytidine is favored for substrate binding and deamination. Trajectories of the MD simulation indicate that a ribose 2'-hydroxyl group destabilizes the π-π stacking of the target cytosine and H257, resulting in dislocation of the target cytosine base from the catalytic position. Interestingly, APOBEC3A, which can deaminate ribocytidines, retains the ribocytidine in the catalytic position throughout the MD simulation. Our results indicate that A3Gctd catalytic selectivity against RNA is dictated by both the sugar conformation and 2'-hydroxyl group.


Assuntos
Desaminase APOBEC-3G/metabolismo , DNA de Cadeia Simples/metabolismo , DNA/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Simulação de Dinâmica Molecular , RNA/metabolismo , Desaminase APOBEC-3G/química , Desaminase APOBEC-3G/genética , Biocatálise , Citidina/química , Citidina/metabolismo , DNA/química , DNA/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Desaminação , HIV-1/genética , HIV-1/metabolismo , Humanos , Ligação Proteica , RNA/química , RNA/genética , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo , Especificidade por Substrato , Vírion/genética , Vírion/metabolismo
8.
Tohoku J Exp Med ; 248(4): 285-296, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31447474

RESUMO

In 1973, rotaviruses A (RVAs) were discovered as major causative agents of acute gastroenteritis in infants and young children worldwide. The infectious RV virion is an icosahedral particle composed of three concentric protein layers surrounding the 11 double-stranded (dsRNA) segments. An in vitro replication system for RVs in permanent cell lines was developed in 1982 and expanded to replication in intestinal organoids in 2015. However, the details of rotavirus (RV) entry into cells and particle maturation mechanisms at the molecular level remain incompletely understood. Slowing down human RVA replication in cell culture on ice allowed morphological visualization of virus particle entry and the assembly of triple-layered particles (virion). Although RVAs are non-enveloped viruses, after virus attachment to the cell membrane, the virus enters the cell by perforating the plasma membrane by a fusion mechanism involving VP5* of the cleaved VP4 protein, as the alternative virus entry route besides the receptor-mediated endocytosis which is generally accepted. After assembling double-layered particles (DLPs) in viroplasm or cytoplasm, they appear to be connected with the endoplasmic reticulum (ER) membrane and become coated with outer capsid proteins (VP4 and VP7) in a coating process. The perforation of the ER membrane is caused by an unknown mechanism following interaction between non-structural protein 4 (NSP4) and the inner capsid protein VP6 of the DLPs. The coating process is closely related to the formation of a hetero-oligomeric complex (NSP4, VP4 and VP7). These lines of evidence suggest the existence of novel mechanisms of RV morphogenesis.


Assuntos
Rotavirus/fisiologia , Internalização do Vírus , Replicação Viral/fisiologia , Endocitose , Rotavirus/ultraestrutura , Proteínas Virais/metabolismo , Vírion/metabolismo , Vírion/ultraestrutura
9.
Nat Microbiol ; 4(10): 1636-1644, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31285583

RESUMO

To achieve efficient binding and subsequent fusion, most enveloped viruses encode between one and five proteins1. For many viruses, the clustering of fusion proteins-and their distribution on virus particles-is crucial for fusion activity2,3. Poxviruses, the most complex mammalian viruses, dedicate 15 proteins to binding and membrane fusion4. However, the spatial organization of these proteins and how this influences fusion activity is unknown. Here, we show that the membrane of vaccinia virus is organized into distinct functional domains that are critical for the efficiency of membrane fusion. Using super-resolution microscopy and single-particle analysis, we found that the fusion machinery of vaccinia virus resides exclusively in clusters at virion tips. Repression of individual components of the fusion complex disrupts fusion-machinery polarization, consistent with the reported loss of fusion activity5. Furthermore, we show that displacement of functional fusion complexes from virion tips disrupts the formation of fusion pores and infection kinetics. Our results demonstrate how the protein architecture of poxviruses directly contributes to the efficiency of membrane fusion, and suggest that nanoscale organization may be an intrinsic property of these viruses to assure successful infection.


Assuntos
Fusão de Membrana/fisiologia , Vírus Vaccinia/fisiologia , Vírion/metabolismo , Animais , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Células Cultivadas , Células HeLa , Humanos , Modelos Moleculares , Vaccinia/virologia , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo , Vírion/química , Vírion/genética , Vírion/ultraestrutura , Internalização do Vírus
10.
Virology ; 534: 45-53, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31176063

RESUMO

Tailed dsDNA bacteriophages and herpesviruses form capsids using coat proteins that have the HK97 fold. In these viruses, the coat proteins first assemble into procapsids, which subsequently mature during DNA packaging. Generally interactions between the coat protein E-loop of one subunit and the P-domain of an adjacent subunit help stabilize both capsomers and capsids. Based on a recent 3.3 Šcryo-EM structure of the bacteriophage P22 virion, E-loop amino acids E52, E59 and E72 were suggested to stabilize the capsid through intra-capsomer salt bridges with the P-domain residues R102, R109 and K118. The glutamic acid residues were each mutated to alanine to test this hypothesis. The substitutions resulted in a WT phenotype and did not destabilize capsids; rather, the alanine substituted coat proteins increased the stability of procapsids and virions. These results indicate that different types of interactions must be used between the E-loop and P-domain to stabilize phage P22 procapsids and virions.


Assuntos
Bacteriófago P22/metabolismo , Proteínas do Capsídeo/química , Capsídeo/química , Bacteriófago P22/química , Bacteriófago P22/genética , Bacteriófago P22/ultraestrutura , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Modelos Moleculares , Domínios Proteicos , Estabilidade Proteica , Vírion/química , Vírion/genética , Vírion/metabolismo
11.
Artigo em Inglês | MEDLINE | ID: mdl-31192164

RESUMO

Previous studies have shown that the endoplasmic reticulum (ER)-anchored protein VAP is strictly required by human papillomavirus type 16 (HPV-16) for successful infectious entry. Entry appeared to be mediated in part through the induction of endosomal tubulation and subsequent transport of the virion to the trans-Golgi network (TGN). In this study, we were interested in investigating whether this mechanism of infectious entry is conserved across multiple Papillomavirus types. To do this, we analyzed the role of VAP and endosomal tubulation following infection with Pseudovirions (PsVs) derived from the alpha, beta, delta, kappa, and pi papillomavirus genera, reflecting viruses that are important human and animal pathogens. We demonstrate that VAP is essential for infection with all PV types analyzed. Furthermore, we find that VAP and EGFR-dependent endosomal tubulation is also induced by all these different Papillomaviruses. These results indicate an evolutionarily conserved requirement for VAP-induced endocytic tubulation during Papillomavirus infectious entry.


Assuntos
Endossomos/metabolismo , Endossomos/virologia , Infecções por Papillomavirus/virologia , Internalização do Vírus , Alphapapillomavirus/patogenicidade , Animais , Transporte Biológico Ativo , Proteínas do Capsídeo/metabolismo , Endocitose , Retículo Endoplasmático/virologia , Endossomos/genética , Técnicas de Inativação de Genes , Células HEK293 , Células HeLa , Humanos , Proteínas dos Microfilamentos/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Vírion/metabolismo , Rede trans-Golgi/genética , Rede trans-Golgi/metabolismo , Rede trans-Golgi/virologia
12.
PLoS Biol ; 17(6): e3000316, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31199794

RESUMO

Infections with human herpesviruses are ubiquitous and a public health concern worldwide. Current treatments reduce the severity of some symptoms associated to herpetic infections but neither remove the viral reservoir from the infected host nor protect from the recurrent symptom outbreaks that characterise herpetic infections. The difficulty in therapeutically tackling these viral systems stems in part from their remarkably large proteomes and the complex networks of physical and functional associations that they tailor. This study presents our efforts to unravel the complexity of the interactome of herpes simplex virus type 1 (HSV1), the prototypical herpesvirus species. Inspired by our previous work, we present an improved and more integrative computational pipeline for the protein-protein interaction (PPI) network reconstruction in HSV1, together with a newly developed consensus clustering framework, which allowed us to extend the analysis beyond binary physical interactions and revealed a system-level layout of higher-order functional associations in the virion proteome. Additionally, the analysis provided new functional annotation for the currently undercharacterised protein pUS10. In-depth bioinformatics sequence analysis unravelled structural features in pUS10 reminiscent of those observed in some capsid-associated proteins in tailed bacteriophages, with which herpesviruses are believed to share a common ancestry. Using immunoaffinity purification (IP)-mass spectrometry (MS), we obtained additional support for our bioinformatically predicted interaction between pUS10 and the inner tegument protein pUL37, which binds cytosolic capsids, contributing to initial tegumentation and eventually virion maturation. In summary, this study unveils new, to our knowledge, insights at both the system and molecular levels that can help us better understand the complexity behind herpesvirus infections.


Assuntos
Biologia Computacional/métodos , Herpesvirus Humano 1/metabolismo , Herpesvirus Humano 1/ultraestrutura , Animais , Capsídeo/química , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Bases de Dados Factuais , Herpes Simples/metabolismo , Humanos , Hidroliases/metabolismo , Ligação Proteica , Mapas de Interação de Proteínas , Relação Estrutura-Atividade , Proteínas Virais/metabolismo , Proteínas Estruturais Virais/metabolismo , Vírion/metabolismo , Montagem de Vírus
13.
Nat Microbiol ; 4(9): 1558-1570, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31160823

RESUMO

Several Ebola viruses cause outbreaks of lethal haemorrhagic fever in humans, but developing therapies tackle only Zaire Ebola virus. Dendritic cells (DCs) are targets of this infection in vivo. Here, we found that Ebola virus entry into activated DCs requires the sialic acid-binding Ig-like lectin 1 (Siglec-1/CD169), which recognizes sialylated gangliosides anchored to viral membranes. Blockage of the Siglec-1 receptor by anti-Siglec-1 monoclonal antibodies halted Ebola viral uptake and cytoplasmic entry, offering cross-protection against other ganglioside-containing viruses such as human immunodeficiency virus type 1.


Assuntos
Anticorpos Monoclonais/farmacologia , Citoplasma/virologia , Ebolavirus/fisiologia , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/antagonistas & inibidores , Ligação Viral/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Células Dendríticas/virologia , Gangliosídeos/metabolismo , HIV-1/fisiologia , Doença pelo Vírus Ebola/virologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Interferon-alfa/farmacologia , Lipopolissacarídeos/farmacologia , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Vírion/metabolismo
14.
PLoS Pathog ; 15(6): e1007860, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31181126

RESUMO

Influenza A virus (IAV) neuraminidase (NA) receptor-destroying activity and hemagglutinin (HA) receptor-binding affinity need to be balanced with the host receptor repertoire for optimal viral fitness. NAs of avian, but not human viruses, contain a functional 2nd sialic acid (SIA)-binding site (2SBS) adjacent to the catalytic site, which contributes to sialidase activity against multivalent substrates. The receptor-binding specificity and potentially crucial contribution of the 2SBS to the HA-NA balance of virus particles is, however, poorly characterized. Here, we elucidated the receptor-binding specificity of the 2SBS of N2 NA and established an important role for this site in the virion HA-NA-receptor balance. NAs of H2N2/1957 pandemic virus with or without a functional 2SBS and viruses containing this NA were analysed. Avian-like N2, with a restored 2SBS due to an amino acid substitution at position 367, was more active than human N2 on multivalent substrates containing α2,3-linked SIAs, corresponding with the pronounced binding-specificity of avian-like N2 for these receptors. When introduced into human viruses, avian-like N2 gave rise to altered plaque morphology and decreased replication compared to human N2. An opposite replication phenotype was observed when N2 was combined with avian-like HA. Specific bio-layer interferometry assays revealed a clear effect of the 2SBS on the dynamic interaction of virus particles with receptors. The absence or presence of a functional 2SBS affected virion-receptor binding and receptor cleavage required for particle movement on a receptor-coated surface and subsequent NA-dependent self-elution. The contribution of the 2SBS to virus-receptor interactions depended on the receptor-binding properties of HA and the identity of the receptors used. We conclude that the 2SBS is an important and underappreciated determinant of the HA-NA-receptor balance. The rapid loss of a functional 2SBS in pandemic viruses may have served to balance the novel host receptor-repertoire and altered receptor-binding properties of the corresponding HA protein.


Assuntos
Vírus da Influenza A Subtipo H2N2 , Vírus da Influenza A Subtipo H3N2 , Neuraminidase , Receptores Virais , Proteínas Virais , Vírion , Animais , Sítios de Ligação , Cães , Humanos , Vírus da Influenza A Subtipo H2N2/química , Vírus da Influenza A Subtipo H2N2/genética , Vírus da Influenza A Subtipo H2N2/metabolismo , Vírus da Influenza A Subtipo H3N2/química , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/metabolismo , Células Madin Darby de Rim Canino , Ácido N-Acetilneuramínico/genética , Ácido N-Acetilneuramínico/metabolismo , Neuraminidase/química , Neuraminidase/genética , Neuraminidase/metabolismo , Receptores Virais/química , Receptores Virais/genética , Receptores Virais/metabolismo , Células Vero , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo , Vírion/química , Vírion/genética , Vírion/metabolismo
15.
Cells ; 8(6)2019 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-31212706

RESUMO

The translation of selenoprotein mRNAs involves a non-canonical ribosomal event in which an in-frame UGA is recoded as a selenocysteine (Sec) codon instead of being read as a stop codon. The recoding machinery is centered around two dedicated RNA components: The selenocysteine insertion sequence (SECIS) located in the 3' UTR of the mRNA and the selenocysteine-tRNA (Sec-tRNA[Ser]Sec). This translational UGA-selenocysteine recoding event by the ribosome is a limiting stage of selenoprotein expression. Its efficiency is controlled by the SECIS, the Sec-tRNA[Ser]Sec and their interacting protein partners. In the present work, we used a recently developed CRISPR strategy based on murine leukemia virus-like particles (VLPs) loaded with Cas9-sgRNA ribonucleoproteins to inactivate the Sec-tRNA[Ser]Sec gene in human cell lines. We showed that these CRISPR-Cas9-VLPs were able to induce efficient genome-editing in Hek293, HepG2, HaCaT, HAP1, HeLa, and LNCaP cell lines and this caused a robust reduction of selenoprotein expression. The alteration of selenoprotein expression was the direct consequence of lower levels of Sec-tRNA[Ser]Sec and thus a decrease in translational recoding efficiency of the ribosome. This novel strategy opens many possibilities to study the impact of selenoprotein deficiency in hard-to-transfect cells, since these CRISPR-Cas9-VLPs have a wide tropism.


Assuntos
Sistemas CRISPR-Cas/genética , Códon de Terminação/genética , RNA de Transferência Aminoácido-Específico/genética , Ribossomos/metabolismo , Selenocisteína/metabolismo , Vírion/metabolismo , Sequência de Bases , Edição de Genes , Células HEK293 , Células HeLa , Humanos , Mutação INDEL/genética , Conformação de Ácido Nucleico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência Aminoácido-Específico/química , Selênio/metabolismo , Selenoproteínas/genética , Selenoproteínas/metabolismo
16.
Nat Commun ; 10(1): 1997, 2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-31040288

RESUMO

Human G protein-coupled receptors (GPCRs) respond to various ligands and stimuli. However, GPCRs rely on membrane for proper folding, making their biochemical properties difficult to study. By displaying GPCRs in viral envelopes, we fabricated a Virion Display (VirD) array containing 315 non-olfactory human GPCRs for functional characterization. Using this array, we found that 10 of 20 anti-GPCR mAbs were ultra-specific. We further demonstrated that those failed in the mAb assays could recognize their canonical ligands, suggesting proper folding. Next, using two peptide ligands on the VirD-GPCR array, we identified expected interactions and novel interactions. Finally, we screened the array with group B Streptococcus, a major cause of neonatal meningitis, and demonstrated that inhibition of a newly identified target, CysLTR1, reduced bacterial penetration both in vitro and in vivo. We believe that the VirD-GPCR array holds great potential for high-throughput screening for small molecule drugs, affinity reagents, and ligand deorphanization.


Assuntos
Receptores Acoplados a Proteínas-G/metabolismo , Vírion/metabolismo , Animais , Western Blotting , Imunofluorescência , Células HEK293 , Células HeLa , Humanos , Proteômica/métodos , Streptococcus/metabolismo , Células Vero , Virologia/métodos
17.
Nat Commun ; 10(1): 2098, 2019 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-31068585

RESUMO

Hepatitis D virus (HDV) doesn't encode envelope proteins for packaging of its ribonucleoprotein (RNP) and typically relies on the surface glycoproteins (GPs) from hepatitis B virus (HBV) for virion assembly, envelopment and cellular transmission. HDV RNA genome can efficiently replicate in different tissues and species, raising the possibility that it evolved, and/or is still able to transmit, independently of HBV. Here we show that alternative, HBV-unrelated viruses can act as helper viruses for HDV. In vitro, envelope GPs from several virus genera, including vesiculovirus, flavivirus and hepacivirus, can package HDV RNPs, allowing efficient egress of HDV particles in the extracellular milieu of co-infected cells and subsequent entry into cells expressing the relevant receptors. Furthermore, HCV can propagate HDV infection in the liver of co-infected humanized mice for several months. Further work is necessary to evaluate whether HDV is currently transmitted by HBV-unrelated viruses in humans.


Assuntos
Coinfecção/transmissão , Hepatite D/transmissão , Vírus Delta da Hepatite/fisiologia , Montagem de Vírus , Animais , Linhagem Celular Tumoral , Coinfecção/virologia , Flavivirus/metabolismo , Hepacivirus/metabolismo , Hepacivirus/patogenicidade , Hepatite D/virologia , Vírus Delta da Hepatite/isolamento & purificação , Vírus Delta da Hepatite/patogenicidade , Hepatócitos/transplante , Hepatócitos/virologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Cultura Primária de Células , RNA Viral/isolamento & purificação , Ribonucleoproteínas/metabolismo , Vesiculovirus/metabolismo , Proteínas do Envelope Viral/metabolismo , Vírion/metabolismo
18.
Virology ; 531: 269-279, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30974383

RESUMO

The study evaluated the effects of nucleoprotein viral and the infectious virus in SHK-1 cells. The results show a strong respiratory burst activation and the induction of p47phox, SOD, GLURED, and apoptotic genes. Additionally, the cells alter the profile of SUMOylated proteins by the effect of transfection and infection experiments. In silico analyses show a set of structural motifs in NP susceptible of post-translational modification by the SUMO protein. Interestingly, the inhibition of the NADPH oxidase complex blocked the production of reactive oxygen species and the high level of cellular ROS due to the nucleoprotein and the ISAv. At the same time, the blocking of the p38MAPK signaling pathway and the use of Aristotelia chilensis, decreased viral progeny production. These results suggest that the NP triggers a strong production of ROS and modifying the post-translational profile mediated by SUMO-2/3, a phenomenon that favors the production of new virions.


Assuntos
Doenças dos Peixes/metabolismo , Proteínas de Peixes/metabolismo , Isavirus/metabolismo , NADPH Oxidases/metabolismo , Nucleoproteínas/metabolismo , Infecções por Orthomyxoviridae/veterinária , Estresse Oxidativo , Proteínas Virais/metabolismo , Animais , Doenças dos Peixes/genética , Doenças dos Peixes/virologia , Proteínas de Peixes/genética , Interações Hospedeiro-Patógeno , Isavirus/genética , NADPH Oxidases/genética , Nucleoproteínas/genética , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/virologia , Espécies Reativas de Oxigênio/metabolismo , Explosão Respiratória , Salmão , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação , Proteínas Virais/genética , Vírion/genética , Vírion/metabolismo
19.
Biochim Biophys Acta Biomembr ; 1861(6): 1204-1212, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30951702

RESUMO

There is emerging evidence of the utility of virus-like particles (VLPs) as a novel model for the study of receptor-ligand interactions in a native plasma membrane environment. VLPs consist of a viral core protein encapsulated by portions of the cell membrane with membrane proteins and receptors expressed in their native conformation. VLPs can be generated in mammalian cells by transfection with the retroviral core protein (gag). In this study, we used Chinese hamster ovary (CHO T10) cells stably overexpressing the insulin receptor (IR) to generate IR bearing VLPs. The diameter and size uniformity of VLPs were estimated by dynamic light scattering and morphological features examined by scanning electron microscopy. The presence of high affinity IR on VLPs was demonstrated by competitive binding assays (KD: 2.3 ±â€¯0.4 nM, n = 3), which was similar to that on the parental CHO T10 cells (KD: 2.1 ±â€¯0.4 nM, n = 3). We also report that increases or decreases in membrane cholesterol content by treatment with methyl-ß-cyclodextrin (MBCD) or cholesterol pre-loaded methyl-ß-cyclodextrin (cMBCD), respectively, substantially decreased insulin binding (> 30%) to both VLPs and cells, and we speculate this is due to a change in receptor disposition. We suggest that this novel finding of decreases in insulin binding in response to changes in membrane cholesterol content may largely account for the unexplained decreases in insulin signalling events previously reported elsewhere. Finally, we propose VLPs as a viable membrane model for the study of insulin-IR interactions in a native membrane environment.


Assuntos
Insulina/metabolismo , Receptor de Insulina/metabolismo , Vírion/metabolismo , Animais , Ligação Competitiva , Células CHO , Colesterol/metabolismo , Cricetulus , Ligação Proteica
20.
Nat Cell Biol ; 21(4): 452-461, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30936472

RESUMO

Particles that bud off from the cell surface, including viruses and microvesicles, typically have a unique membrane protein composition distinct from that of the originating plasma membrane. This selective protein composition enables viruses to evade the immune response and infect other cells. But how membrane proteins sort into budding viruses such as human immunodeficiency virus (HIV) remains unclear. Proteins could passively distribute into HIV-assembly-site membranes producing compositions resembling pre-existing plasma-membrane domains. Here, we demonstrate that proteins instead sort actively into HIV-assembly-site membranes, generating compositions enriched in cholesterol and sphingolipids that undergo continuous remodelling. Proteins are recruited into and removed from the HIV assembly site through lipid-based partitioning, initiated by oligomerization of the HIV structural protein Gag. Changes in membrane curvature at the assembly site further amplify this sorting process. Thus, a lipid-based sorting mechanism, aided by increasing membrane curvature, generates the unique membrane composition of the HIV surface.


Assuntos
HIV/metabolismo , Proteínas do Vírus da Imunodeficiência Humana/metabolismo , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Vírion/metabolismo , Animais , Antígeno 2 do Estroma da Médula Óssea/metabolismo , Células COS , Membrana Celular/ultraestrutura , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Células HeLa , Humanos , Vírion/química
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