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1.
PLoS One ; 15(2): e0228572, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32045432

RESUMO

Human respiratory syncytial virus (RSV) and parainfluenza virus type 3 (HPIV3) are among the most common viral causes of childhood bronchiolitis and pneumonia worldwide, and lack effective antiviral drugs or vaccines. Recombinant (r) HPIV3 was modified to express the RSV fusion (F) glycoprotein, the major RSV neutralization and protective antigen, providing a live intranasal bivalent HPIV3/RSV vaccine candidate. This extends previous studies using a chimeric bovine-human PIV3 vector (rB/HPIV3). One advantage is that rHPIV3 expresses all of the HPIV3 antigens compared to only two for rB/HPIV3. In addition, the use of rHPIV3 as vector should avoid excessive attenuation following addition of the modified RSV F gene, which may occur with rB/HPIV3. To enhance its immunogenicity, RSV F was modified (i) to increase the stability of the prefusion (pre-F) conformation and (ii) by replacement of its transmembrane (TM) and cytoplasmic tail (CT) domains with those of HPIV3 F (H3TMCT) to increase incorporation in the vector virion. RSV F (+/- H3TMCT) was expressed from the first (F/preN) or the second (F/N-P) gene position of rHPIV3. The H3TMCT modification dramatically increased packaging of RSV F into the vector virion and, in hamsters, resulted in significant increases in the titer of high-quality serum RSV-neutralizing antibodies, in addition to the increase conferred by pre-F stabilization. Only F-H3TMCT/preN replication was significantly attenuated in the nasal turbinates by the RSV F insert. F-H3TMCT/preN, F/N-P, and F-H3TMCT/N-P provided complete protection against wt RSV challenge. F-H3TMCT/N-P exhibited the most stable and highest expression of RSV F, providing impetus for its further development.


Assuntos
Vacinas contra Parainfluenza/genética , Vírus da Parainfluenza 3 Humana/imunologia , Vacinas contra Vírus Sincicial Respiratório/genética , Proteínas Virais de Fusão/genética , Montagem de Vírus , Administração Intranasal , Animais , Chlorocebus aethiops , Cricetinae , Feminino , Humanos , Imunogenicidade da Vacina , Macaca mulatta , Mesocricetus , Vacinas contra Parainfluenza/administração & dosagem , Vacinas contra Parainfluenza/imunologia , Vírus da Parainfluenza 3 Humana/genética , Vírus da Parainfluenza 3 Humana/fisiologia , Estabilidade Proteica , Vacinas contra Vírus Sincicial Respiratório/administração & dosagem , Vacinas contra Vírus Sincicial Respiratório/imunologia , Células Vero , Proteínas Virais de Fusão/metabolismo
2.
mBio ; 10(6)2019 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-31822584

RESUMO

Human cytomegalovirus (HCMV) encodes an endoplasmic reticulum (ER)-resident glycoprotein, UL148, which activates the unfolded protein response (UPR) but is fully dispensable for viral replication in cultured cells. Hence, its previously ascribed roles in immune evasion and modulation of viral cell tropism are hypothesized to cause ER stress. Here, we show that UL148 is necessary and sufficient to drive the formation of prominent ER-derived structures that on average occupy 5% of the infected cell cytoplasm. The structures are sites where UL148 coalesces with cellular proteins involved in ER quality control, such as HRD1 and EDEM1. Electron microscopy revealed that cells infected with wild-type but not UL148-null HCMV show prominent accumulations of densely packed ruffled ER membranes which connect to distended cisternae of smooth and partially rough ER. During ectopic expression of UL148-green fluorescent protein (GFP) fusion protein, punctate signals traffic to accumulate at conspicuous structures. The structures exhibit poor recovery of fluorescence after photobleaching, which suggests that their contents are poorly mobile and do not efficiently exchange with the rest of the ER. Small-molecule blockade of the integrated stress response (ISR) prevents the formation of puncta, leading to a uniform reticular fluorescent signal. Accordingly, ISR inhibition during HCMV infection abolishes the coalescence of UL148 and HRD1 into discrete structures, which argues that UL148 requires the ISR to cause ER reorganization. Given that UL148 stabilizes immature forms of a receptor binding subunit for a viral envelope glycoprotein complex important for HCMV infectivity, our results imply that stress-dependent ER remodeling contributes to viral cell tropism.IMPORTANCE Perturbations to endoplasmic reticulum (ER) morphology occur during infection with various intracellular pathogens and in certain genetic disorders. We identify that a human cytomegalovirus (HCMV) gene product, UL148, profoundly reorganizes the ER during infection and is sufficient to do so when expressed on its own. Our results reveal that UL148-dependent reorganization of the ER is a prominent feature of HCMV-infected cells. Moreover, we find that this example of virally induced organelle remodeling requires the integrated stress response (ISR), a stress adaptation pathway that contributes to a number of disease states. Since ER reorganization accompanies roles of UL148 in modulation of HCMV cell tropism and in evasion of antiviral immune responses, our results may have implications for understanding the mechanisms involved. Furthermore, our findings provide a basis to utilize UL148 as a tool to investigate organelle responses to stress and to identify novel drugs targeting the ISR.


Assuntos
Infecções por Citomegalovirus/metabolismo , Citomegalovirus/metabolismo , Retículo Endoplasmático/metabolismo , Glicoproteínas/metabolismo , Proteínas Virais de Fusão/metabolismo , Proteínas não Estruturais Virais/metabolismo , Linhagem Celular , Estresse do Retículo Endoplasmático/fisiologia , Humanos , Evasão da Resposta Imune/fisiologia , Proteínas de Membrana/metabolismo , Resposta a Proteínas não Dobradas/fisiologia , Proteínas do Envelope Viral/metabolismo , Tropismo Viral/fisiologia , Replicação Viral/fisiologia
3.
Proc Natl Acad Sci U S A ; 116(50): 25057-25067, 2019 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-31767754

RESUMO

Nipah virus (NiV) is a highly pathogenic paramyxovirus that causes frequent outbreaks of severe neurologic and respiratory disease in humans with high case fatality rates. The 2 glycoproteins displayed on the surface of the virus, NiV-G and NiV-F, mediate host-cell attachment and membrane fusion, respectively, and are targets of the host antibody response. Here, we provide a molecular basis for neutralization of NiV through antibody-mediated targeting of NiV-F. Structural characterization of a neutralizing antibody (nAb) in complex with trimeric prefusion NiV-F reveals an epitope at the membrane-distal domain III (DIII) of the molecule, a region that undergoes substantial refolding during host-cell entry. The epitope of this monoclonal antibody (mAb66) is primarily protein-specific and we observe that glycosylation at the periphery of the interface likely does not inhibit mAb66 binding to NiV-F. Further characterization reveals that a Hendra virus-F-specific nAb (mAb36) and many antibodies in an antihenipavirus-F polyclonal antibody mixture (pAb835) also target this region of the molecule. Integrated with previously reported paramyxovirus F-nAb structures, these data support a model whereby the membrane-distal region of the F protein is targeted by the antibody-mediated immune response across henipaviruses. Notably, our domain-specific sequence analysis reveals no evidence of selective pressure at this region of the molecule, suggestive that functional constraints prevent immune-driven sequence variation. Combined, our data reveal the membrane-distal region of NiV-F as a site of vulnerability on the NiV surface.


Assuntos
Anticorpos Neutralizantes , Vírus Hendra , Proteínas Virais de Fusão , Internalização do Vírus , Anticorpos Monoclonais , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/metabolismo , Linhagem Celular Tumoral , Glicosilação , Células HEK293 , Vírus Hendra/química , Vírus Hendra/imunologia , Vírus Hendra/metabolismo , Vírus Hendra/fisiologia , Humanos , Modelos Moleculares , Ligação Proteica , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/imunologia , Proteínas Virais de Fusão/metabolismo
4.
Nat Struct Mol Biol ; 26(10): 980-987, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31570878

RESUMO

Nipah virus (NiV) and Hendra virus (HeV) are zoonotic henipaviruses (HNVs) responsible for outbreaks of encephalitis and respiratory illness with fatality rates of 50-100%. No vaccines or licensed therapeutics currently exist to protect humans against NiV or HeV. HNVs enter host cells by fusing the viral and cellular membranes via the concerted action of the attachment (G) and fusion (F) glycoproteins, the main targets of the humoral immune response. Here, we describe the isolation and humanization of a potent monoclonal antibody cross-neutralizing NiV and HeV. Cryo-electron microscopy, triggering and fusion studies show the antibody binds to a prefusion-specific quaternary epitope, conserved in NiV F and HeV F glycoproteins, and prevents membrane fusion and viral entry. This work supports the importance of the HNV prefusion F conformation for eliciting a robust immune response and paves the way for using this antibody for prophylaxis and post-exposure therapy with NiV- and HeV-infected individuals.


Assuntos
Anticorpos Neutralizantes/farmacologia , Antivirais/farmacologia , Vírus Hendra/efeitos dos fármacos , Infecções por Henipavirus/tratamento farmacológico , Vírus Nipah/efeitos dos fármacos , Proteínas Virais de Fusão/antagonistas & inibidores , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Células HEK293 , Vírus Hendra/metabolismo , Infecções por Henipavirus/metabolismo , Infecções por Henipavirus/virologia , Humanos , Modelos Moleculares , Vírus Nipah/metabolismo , Proteínas Virais de Fusão/metabolismo , Internalização do Vírus/efeitos dos fármacos
5.
Acta Virol ; 63(3): 253-260, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31507190

RESUMO

The human immunodeficiency virus (HIV) envelope, via a key extracellular amino acid sequence, may simulate the functionality of native undecapeptide substance P (SP) acting through the host's neurokinin 1 (SP preferring) receptor (NK-1R). Human monocytes and macrophages express both NK-1Rs and SP. In HIV/AIDS the NK-1R may function as a chemokine-like G-protein coupled co-receptor that: 1) fuses to the outer envelope of HIV; 2) enables intracellular entry of the envelope-capsid-NK-1R complex; 3) co-opts immune defence via its physiological interaction with the SP-like envelope; 4) may contribute to resistance of CD4/chemokine entry inhibitor type drugs; 5) relaxes the blood-brain barrier to support entry of the HIV into the central nervous system, and 6) mediates most of the common clinical sequelae of HIV/AIDS (encephalopathy and AIDS dementia complex). The data support the idea that NK-1R antagonists could be useful to treat HIV/AIDS. Keywords: human immunodeficiency virus; NK-1 receptor; NK-1 receptor antagonist; aprepitant; fusion protein; virus.


Assuntos
Infecções por HIV , Receptores da Neurocinina-1 , Substância P , Proteínas Virais de Fusão , Dipeptídeos/genética , Dipeptídeos/metabolismo , Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/tratamento farmacológico , Infecções por HIV/fisiopatologia , Humanos , Macrófagos/virologia , Monócitos/metabolismo , Monócitos/virologia , Antagonistas do Receptor de Neuroquinina-1/uso terapêutico , Receptores da Neurocinina-1/metabolismo , Substância P/metabolismo , Proteínas Virais de Fusão/metabolismo
6.
Virology ; 535: 102-110, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31299486

RESUMO

Severe fever with thrombocytopenia syndrome (SFTS) is an infectious disease with a high fatality rate, caused by SFTS virus (SFTSV). Because little is known about the nature of SFTSV, basic studies are required for the developments of vaccines and effective therapies. In the present study, we identified the amino acid residue important for membrane fusion induced by the SFTSV glycoprotein (GP). Syncytium formations were observed in cells expressing the GPs of SFTSV Japanese strain (YG-1 and SPL030). In contrast, no or only weak syncytium formations were induced in cells expressing GP of SFTSV Chinese strain (HB29). The replacement of arginine at amino acid residue 962 with serine in HB29 GP (R962S) induced membrane fusion, while the replacement of serine at residue 962 with arginine in YG1 GP (S962R) did not. These data indicate that serine at residue 962 in the SFTSV-GP is critical for inducing membrane fusion and viral infection.


Assuntos
Phlebovirus/fisiologia , Proteínas Virais de Fusão/metabolismo , Internalização do Vírus , Substituição de Aminoácidos , Fusão Celular , Células Gigantes/citologia , Células Gigantes/virologia , Mutagênese Sítio-Dirigida , Phlebovirus/genética , Proteínas Virais de Fusão/genética
7.
PLoS One ; 14(7): e0219312, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31276481

RESUMO

Ebolaviruses continue to inflict horrific disease and instill fear. The 2013-2016 outbreak in Western Africa caused unfathomable morbidity and mortality (over 11,000 deaths), and the second largest outbreak is on-going in the Democratic Republic of the Congo. The first stage of an Ebolavirus infection is entry, culminating in delivery of the viral genome into the cytoplasm to initiate replication. Among enveloped viruses, Ebolaviruses use a complex entry pathway: they bind to attachment factors on cell surfaces, are engulfed by macropinocytosis, and traffic through the endosomal system. En route, the receptor binding subunit of the glycoprotein (GP) is reduced from ~130 to ~19 kDa by cathepsins. This event allows cleaved GP (GPcl) to bind to Niemann-Pick C1 (NPC1), its endosomal receptor. The virus then fuses with a late endosomal membrane, but how this occurs remains a subject of debate. An early, but standing, observation is that entry of particles bearing GPcl is inhibited by agents that raise endosomal pH or inhibit cysteine proteases, suggesting the need for an additional factor(s). Yet, some have concluded that NPC1 is sufficient to trigger the fusion activity of GPcl. Here, we re-examined this question using sensitive cell-cell and pseudovirus-cell fusion assays. We did not observe detectable GPcl-mediated fusion with NPC1 or its GPcl binding domain at any pH tested, while robust fusion was consistently observed with GP from lymphocytic choriomeningitis virus at low pH. Addition of proposed fusion-enhancing factors-cations (Ca++ and K+), a reducing agent, the anionic lipid Bis(Monoacylglycero)Phosphate, and a mixture of cathepsins B and L-did not induce detectable fusion. Our findings are in line with the earlier proposal that an additional factor is required to trigger the full fusion activity of GPcl after binding to NPC1. We discuss caveats to our study and what the missing factor(s) might be.


Assuntos
Ebolavirus/fisiologia , Doença pelo Vírus Ebola/virologia , Proteínas do Envelope Viral/metabolismo , Proteínas Virais de Fusão/metabolismo , Animais , Proteínas de Transporte/metabolismo , Fusão Celular , Linhagem Celular , Doença pelo Vírus Ebola/metabolismo , Humanos , Proteína C1 de Niemann-Pick , Ligação Proteica , Receptores Virais/metabolismo
8.
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
9.
J Gen Virol ; 100(7): 1112-1122, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31184573

RESUMO

Respiratory syncytial virus (RSV) remains a leading cause of infant mortality worldwide and exhaustive international efforts are underway to develop a vaccine. However, vaccine development has been hindered by a legacy of vaccine-enhanced disease, poor viral immunogenicity in infants, and genetic and physical instabilities. Natural infection with RSV does not prime for enhanced disease encouraging development of live-attenuated RSV vaccines for infants; however, physical instabilities of RSV may limit vaccine development. The role of RSV strain-specific differences on viral physical stability remains unclear. We have previously demonstrated that the RSV fusion (F) surface glycoprotein is responsible for mediating significant differences in thermostability between strains A2 and A2-line19F. In this study, we performed a more comprehensive analysis to characterize the replication and physical stability of recombinant RSV A and B strains that differed only in viral attachment (G) and/or F surface glycoprotein expression. We observed significant differences in thermal stability, syncytia size, pre-fusion F incorporation and viral growth kinetics in vitro, but limited variations to pH and freeze-thaw inactivation among several tested strains. Consistent with earlier studies, A2-line19F showed significantly enhanced thermal stability over A2, but also restricted growth kinetics in both HEp2 and Vero cells. As expected, no significant differences in susceptibility to UV inactivation were observed. These studies provide the first analysis of the physical stability of multiple strains of RSV, establish a key virus strain associated with enhanced thermal stability compared to conventional lab strain A2, and further support the pivotal role RSV F plays in virus stability.


Assuntos
Infecções por Vírus Respiratório Sincicial/virologia , Vacinas contra Vírus Sincicial Respiratório/química , Vírus Sincicial Respiratório Humano/fisiologia , Proteínas do Envelope Viral/química , Proteínas Virais de Fusão/química , Replicação Viral , Temperatura Alta , Humanos , Concentração de Íons de Hidrogênio , Estabilidade Proteica , Vacinas contra Vírus Sincicial Respiratório/genética , Vacinas contra Vírus Sincicial Respiratório/metabolismo , Vírus Sincicial Respiratório Humano/química , Vírus Sincicial Respiratório Humano/classificação , Vírus Sincicial Respiratório Humano/genética , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo
10.
Nat Commun ; 10(1): 2105, 2019 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-31068578

RESUMO

The respiratory syncytial virus (RSV) F glycoprotein is a class I fusion protein that mediates viral entry and is a major target of neutralizing antibodies. Structures of prefusion forms of RSV F, as well as other class I fusion proteins, have revealed compact trimeric arrangements, yet whether these trimeric forms can transiently open remains unknown. Here, we perform structural and biochemical studies on a recently isolated antibody, CR9501, and demonstrate that it enhances the opening of prefusion-stabilized RSV F trimers. The 3.3 Å crystal structure of monomeric RSV F bound to CR9501, combined with analysis of over 25 previously determined RSV F structures, reveals a breathing motion of the prefusion conformation. We also demonstrate that full-length RSV F trimers transiently open and dissociate on the cell surface. Collectively, these findings have implications for the function of class I fusion proteins, as well as antibody prophylaxis and vaccine development for RSV.


Assuntos
Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , Vírus Sincicial Respiratório Humano/fisiologia , Proteínas Virais de Fusão/metabolismo , Animais , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Linfócitos B/virologia , Chlorocebus aethiops , Simulação por Computador , Cristalografia por Raios X , Desenvolvimento de Medicamentos , Células HEK293 , Células HeLa , Humanos , Modelos Moleculares , Multimerização Proteica/fisiologia , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Infecções por Vírus Respiratório Sincicial/virologia , Vacinas contra Vírus Sincicial Respiratório/imunologia , Vírus Sincicial Respiratório Humano/isolamento & purificação , Células Vero , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/imunologia
11.
Biosci Trends ; 13(3): 225-233, 2019 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-31142702

RESUMO

Newcastle disease virus (NDV), an avian paramyxovirus, causes Newcastle disease (ND) which is a highly contagious and fatal viral disease affecting poultry and most species of birds. The fusion (F) protein of NDV mediates membrane fusion, which is essential to the processes of viral entry, replication, and dissemination. Although several domains of NDV F are known to have important effects on regulating the membrane fusion activity, the role of the region around domain III (DIII) and domain I (DI) still remains ill-defined. Site-directed mutagenesis was utilized to change the conserved amino acids at 269, 274, 277, 286, 287, 290, 295, and 297 to alanine in order to investigate the effects of these conserved amino acids around the DIII and DI linker region of the NDV F protein on fusion activity. It was found that five of these substitutions almost abolished fusion activity except for mutants I269A, Q286A, and N297A, which showed 57.1%, 161.1%, and 97.7% of the wt F level, respectively. Four (I274A, D277A, V287A, and P290A) of these five mutants likely result in interfering with folding or transporting of the molecule since these proteins were minimally expressed at the cell surface, formed aggregates, or not proteolytically cleaved. However, mutant L295A almost abolished fusion activity even with a similar level of cell surface expression. These data indicated that conserved amino acids around the DIII-DI linker region are critical for the folding of the F protein and have an important influence on fusion activity.


Assuntos
Vírus da Doença de Newcastle/metabolismo , Proteínas Virais de Fusão/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Animais , Western Blotting , Cricetinae , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Dobramento de Proteína
12.
Virology ; 531: 248-254, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30946995

RESUMO

The human metapneumovirus (HMPV) fusion protein (F) mediates fusion of the viral envelope and cellular membranes to establish infection. HMPV F from some, but not all, viral strains promotes fusion only after exposure to low pH. Previous studies have identified several key residues involved in low pH triggering, including H435 and a proposed requirement for glycine at position 294. We analyzed the different levels of fusion activity, protein expression and cleavage of three HMPV F proteins not previously examined. Interestingly, low pH-triggered fusion in the absence of G294 was identified in one F protein, while a novel histidine residue (H434) was identified that enhanced low pH promoted fusion in another. The third F protein failed to promote cell-to-cell fusion, suggesting other requirements for F protein triggering. Our results demonstrate HMPV F triggering is more complex than previously described and suggest a more intricate mechanism for fusion protein function and activation.


Assuntos
Metapneumovirus/metabolismo , Infecções por Paramyxoviridae/virologia , Proteínas Virais de Fusão/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Chlorocebus aethiops , Humanos , Metapneumovirus/química , Metapneumovirus/genética , Estabilidade Proteica , Alinhamento de Sequência , Células Vero , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/genética
13.
Virus Res ; 266: 58-68, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31004621

RESUMO

Respiratory syncytial virus (RSV) is a leading cause of infant hospitalization worldwide each year and there is presently no licensed vaccine to prevent severe RSV infections. Two major RSV glycoproteins, attachment (G) and fusion (F) protein, regulate viral replication and both proteins contain potential glycosylation sites which are highly variable for the G protein and conserved for the F protein among virus isolates. The RSV F sequence possesses five N-glycosylation sites located in the F2 subunit (N27 and N70), the p27 peptide (N116 and N126) and the F1 subunit (N500). The importance of RSV F N-glycosylation in virus replication and immunogenicity is not yet fully understood, and a better understanding may provide new insights for vaccine development. By using a BAC-based reverse genetics system, recombinant viruses expressing F proteins with loss of N-glycosylation sites were made. Mutant viruses with single N-glycosylation sites removed could be recovered, while this was not possible with the mutant with all N-glycosylation sites removed. Although the individual RSV F N-glycosylation sites were shown not to be essential for viral replication, they do contribute to the efficiency of in vitro and in vivo viral infection. To evaluate the role of N-glycosylation sites on RSV F antigenicity, serum antibody titers were determined after infection of BALB/c mice with RSV expressing the glycomutant F proteins. Infection with recombinant virus lacking the N-glycosylation site at position N116 (RSV F N116Q) resulted in significant higher neutralizing antibody titers compared to RSV F WT infection, which is surprising since this N-glycan is present in the p27 peptide which is assumed to be absent from the mature F protein in virions. Thus, single or combined RSV F glycomutations which affect virus replication and fusogenicity, and which may induce enhanced antibody responses upon immunization could have the potential to improve the efficacy of RSV LAV approaches.


Assuntos
Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/fisiologia , Vírus Sincicial Respiratório Humano/patogenicidade , Proteínas Virais de Fusão/metabolismo , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Linhagem Celular Tumoral , Chlorocebus aethiops , Feminino , Células Gigantes/virologia , Glicosilação , Humanos , Imunização , Imunogenicidade da Vacina , Camundongos Endogâmicos BALB C , Mutação , Infecções por Vírus Respiratório Sincicial/metabolismo , Infecções por Vírus Respiratório Sincicial/patologia , Vírus Sincicial Respiratório Humano/crescimento & desenvolvimento , Vírus Sincicial Respiratório Humano/imunologia , Células Vero , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/imunologia , Replicação Viral
14.
PLoS Pathog ; 15(4): e1007675, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-31022290

RESUMO

Fusogenic reoviruses encode fusion-associated small transmembrane (FAST) protein, which induces cell-cell fusion. FAST protein is the only known fusogenic protein in non-enveloped viruses, and its role in virus replication is not yet known. We generated replication-competent, FAST protein-deficient pteropine orthoreovirus and demonstrated that FAST protein was not essential for viral replication, but enhanced viral replication in the early phase of infection. Addition of recombinant FAST protein enhanced replication of FAST-deficient virus and other non-fusogenic viruses in a fusion-dependent and FAST-species-independent manner. In a mouse model, replication and pathogenicity of FAST-deficient virus were severely impaired relative to wild-type virus, indicating that FAST protein is a major determinant of the high pathogenicity of fusogenic reovirus. FAST-deficient virus also conferred effective protection against challenge with lethal homologous virus strains in mice. Our results demonstrate a novel role of a viral fusogenic protein and the existence of a cell-cell fusion-dependent replication system in non-enveloped viruses.


Assuntos
Fusão Celular , Infecções por Reoviridae/virologia , Reoviridae/genética , Reoviridae/patogenicidade , Proteínas Virais de Fusão/metabolismo , Virulência , Replicação Viral , Animais , Masculino , Camundongos , Camundongos Endogâmicos C3H , Mutação , Infecções por Reoviridae/genética , Infecções por Reoviridae/metabolismo , Proteínas Virais de Fusão/genética
15.
Cell ; 176(6): 1420-1431.e17, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30849373

RESUMO

Respiratory syncytial virus (RSV) is a worldwide public health concern for which no vaccine is available. Elucidation of the prefusion structure of the RSV F glycoprotein and its identification as the main target of neutralizing antibodies have provided new opportunities for development of an effective vaccine. Here, we describe the structure-based design of a self-assembling protein nanoparticle presenting a prefusion-stabilized variant of the F glycoprotein trimer (DS-Cav1) in a repetitive array on the nanoparticle exterior. The two-component nature of the nanoparticle scaffold enabled the production of highly ordered, monodisperse immunogens that display DS-Cav1 at controllable density. In mice and nonhuman primates, the full-valency nanoparticle immunogen displaying 20 DS-Cav1 trimers induced neutralizing antibody responses ∼10-fold higher than trimeric DS-Cav1. These results motivate continued development of this promising nanoparticle RSV vaccine candidate and establish computationally designed two-component nanoparticles as a robust and customizable platform for structure-based vaccine design.


Assuntos
Anticorpos Neutralizantes/imunologia , Vírus Sinciciais Respiratórios/imunologia , Vacinação/métodos , Animais , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/imunologia , Caveolina 1 , Linhagem Celular , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/uso terapêutico , Cultura Primária de Células , Vírus Sinciciais Respiratórios/patogenicidade , Vacinas/imunologia , Proteínas Virais de Fusão/imunologia , Proteínas Virais de Fusão/metabolismo , Proteínas Virais de Fusão/fisiologia
16.
mBio ; 10(1)2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30782664

RESUMO

Paramyxoviruses, specifically, the childhood pathogen human parainfluenza virus type 3, are internalized into host cells following fusion between the viral and target cell membranes. The receptor binding protein, hemagglutinin (HA)-neuraminidase (HN), and the fusion protein (F) facilitate viral fusion and entry into the cell through a coordinated process involving HN activation by receptor binding, which triggers conformational changes in the F protein to activate it to reach its fusion-competent state. Interfering with this process through premature activation of the F protein has been shown to be an effective antiviral strategy in vitro. Conformational changes in the F protein leading to adoption of the postfusion form of the protein-prior to receptor engagement of HN at the host cell membrane-render the virus noninfectious. We previously identified a small compound (CSC11) that implements this antiviral strategy through an interaction with HN, causing HN to activate F in an untimely process. To assess the functionality of such compounds, it is necessary to verify that the postfusion state of F has been achieved. As demonstrated by Melero and colleagues, soluble forms of the recombinant postfusion pneumovirus F proteins and of their six helix bundle (6HB) motifs can be used to generate postfusion-specific antibodies. We produced novel anti-HPIV3 F conformation-specific antibodies that can be used to assess the functionality of compounds designed to induce F activation. In this study, using systematic chemical modifications of CSC11, we synthesized a more potent derivative of this compound, CM9. Much like CSC11, CM9 causes premature triggering of the F protein through an interaction with HN prior to receptor engagement, thereby preventing fusion and subsequent infection. In addition to validating the potency of CM9 using plaque reduction, fusion inhibition, and binding avidity assays, we confirmed the transition to a postfusion conformation of F in the presence of CM9 using our novel anti-HPIV3 conformation-specific antibodies. We present both CM9 and these newly characterized postfusion antibodies as novel tools to explore and develop antiviral approaches. In turn, these advances in both our molecular toolset and our understanding of HN-F interaction will support development of more-effective antivirals. Combining the findings described here with our recently described physiologically relevant ex vivo system, we have the potential to inform the development of therapeutics to block viral infection.IMPORTANCE Paramyxoviruses, including human parainfluenza virus type 3, are internalized into host cells by fusion between viral and target cell membranes. The receptor binding protein, hemagglutinin-neuraminidase (HN), and the fusion protein (F) facilitate viral fusion and entry into cells through a process involving HN activation by receptor binding, which triggers conformational changes in F to activate it to reach its fusion-competent state. Interfering with this process through premature activation of the F protein may be an effective antiviral strategy in vitro We identified and optimized small compounds that implement this antiviral strategy through an interaction with HN, causing HN to activate F in an untimely fashion. To address that mechanism, we produced novel anti-HPIV3 F conformation-specific antibodies that can be used to assess the functionality of compounds designed to induce F activation. Both the novel antiviral compounds that we present and these newly characterized postfusion antibodies are novel tools for the exploration and development of antiviral approaches.


Assuntos
Antivirais/farmacologia , Proteína HN/metabolismo , Vírus da Parainfluenza 3 Humana/efeitos dos fármacos , Vírus da Parainfluenza 3 Humana/fisiologia , Proteínas Virais de Fusão/metabolismo , Internalização do Vírus/efeitos dos fármacos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/isolamento & purificação , Antivirais/síntese química , Linhagem Celular , Chlorocebus aethiops , Humanos , Ligação Proteica , Conformação Proteica , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/imunologia , Ensaio de Placa Viral
17.
J Virol ; 93(8)2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30728259

RESUMO

A clinical isolate of measles virus (MeV) bearing a single amino acid alteration in the viral fusion protein (F; L454W) was previously identified in two patients with lethal sequelae of MeV central nervous system (CNS) infection. The mutation dysregulated the viral fusion machinery so that the mutated F protein mediated cell fusion in the absence of known MeV cellular receptors. While this virus could feasibly have arisen via intrahost evolution of the wild-type (wt) virus, it was recently shown that the same mutation emerged under the selective pressure of small-molecule antiviral treatment. Under these conditions, a potentially neuropathogenic variant emerged outside the CNS. While CNS adaptation of MeV was thought to generate viruses that are less fit for interhost spread, we show that two animal models can be readily infected with CNS-adapted MeV via the respiratory route. Despite bearing a fusion protein that is less stable at 37°C than the wt MeV F, this virus infects and replicates in cotton rat lung tissue more efficiently than the wt virus and is lethal in a suckling mouse model of MeV encephalitis even with a lower inoculum. Thus, either during lethal MeV CNS infection or during antiviral treatment in vitro, neuropathogenic MeV can emerge, can infect new hosts via the respiratory route, and is more pathogenic (at least in these animal models) than wt MeV.IMPORTANCE Measles virus (MeV) infection can be severe in immunocompromised individuals and lead to complications, including measles inclusion body encephalitis (MIBE). In some cases, MeV persistence and subacute sclerosing panencephalitis (SSPE) occur even in the face of an intact immune response. While they are relatively rare complications of MeV infection, MIBE and SSPE are lethal. This work addresses the hypothesis that despite a dysregulated viral fusion complex, central nervous system (CNS)-adapted measles virus can spread outside the CNS within an infected host.


Assuntos
Sistema Nervoso Central/virologia , Encefalite Viral , Corpos de Inclusão Viral , Pulmão/virologia , Vírus do Sarampo/fisiologia , Sarampo , Mutação de Sentido Incorreto , Proteínas Virais de Fusão , Replicação Viral , Substituição de Aminoácidos , Animais , Sistema Nervoso Central/metabolismo , Chlorocebus aethiops , Modelos Animais de Doenças , Encefalite Viral/genética , Encefalite Viral/metabolismo , Encefalite Viral/transmissão , Humanos , Corpos de Inclusão Viral/genética , Corpos de Inclusão Viral/metabolismo , Pulmão/metabolismo , Sarampo/metabolismo , Sarampo/transmissão , Camundongos , Camundongos Transgênicos , Sigmodontinae , Células Vero , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo
18.
PLoS Pathog ; 15(1): e1007508, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30615658

RESUMO

We have investigated the molecular-level structure of the Vaccinia virion in situ by protein-protein chemical crosslinking, identifying 4609 unique-mass crosslink ions at an effective FDR of 0.33%, covering 2534 unique pairs of crosslinked protein positions, 625 of which were inter-protein. The data were statistically non-random and rational in the context of known structures, and showed biological rationality. Crosslink density strongly tracked the individual proteolytic maturation products of p4a and p4b, the two major virion structural proteins, and supported the prediction of transmembrane domains within membrane proteins. A clear sub-network of four virion structural proteins provided structural insights into the virion core wall, and proteins VP8 and A12 formed a strongly-detected crosslinked pair with an apparent structural role. A strongly-detected sub-network of membrane proteins A17, H3, A27 and A26 represented an apparent interface of the early-forming virion envelope with structures added later during virion morphogenesis. Protein H3 seemed to be the central hub not only for this sub-network but also for an 'attachment protein' sub-network comprising membrane proteins H3, ATI, CAHH(D8), A26, A27 and G9. Crosslinking data lent support to a number of known interactions and interactions within known complexes. Evidence is provided for the membrane targeting of genome telomeres. In covering several orders of magnitude in protein abundance, this study may have come close to the bottom of the protein-protein crosslinkome of an intact organism, namely a complex animal virus.


Assuntos
Vírus Vaccinia/ultraestrutura , Vírion/metabolismo , Biologia Computacional/métodos , Proteínas de Membrana/metabolismo , Espectrometria de Massas em Tandem/métodos , Vírus Vaccinia/genética , Vírus Vaccinia/metabolismo , Proteínas Virais de Fusão/metabolismo , Vírion/ultraestrutura
19.
Microb Pathog ; 128: 414-422, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30597256

RESUMO

Exosomes are micro messengers encapsulating RNA, DNA, and proteins for intercellular communication associated with various physiological and pathological reactions. Several viral infection processes have been reported to pertain to exosomal pathways. However, because of the difficulty in obtaining avian-sourced exosomes, avian virus-related exosomes are scarcely investigated. In this study, we developed a protein A/G-correlated method and successfully obtained the Newcastle disease virus-related exosome (NDV Ex). These exosomes promoted NDV propagation, proven by both GW4869-mediated deprivation and exosomal supplementation. Viral structural proteins NP and F were detected in the NDV Ex and further investigation indicated that the NP protein can be transferred to DF-1 cells through exosomes. The intracellular NP protein exhibited viral replication-promoting and cytokine-suppressing abilities. Therefore, NDV infection produces exosomes, which transfer viral NP protein and promote NDV infection, emphasizing the importance of exosomes in an NDV infection.


Assuntos
Exossomos/metabolismo , Vírus da Doença de Newcastle/fisiologia , Vírus da Doença de Newcastle/patogenicidade , Estruturas Virais/isolamento & purificação , Estruturas Virais/metabolismo , Replicação Viral , Animais , Linhagem Celular , Galinhas , Citocinas/metabolismo , Humanos , Vírus da Doença de Newcastle/crescimento & desenvolvimento , Nucleoproteínas/isolamento & purificação , Nucleoproteínas/metabolismo , Proteínas Recombinantes , Tetraspanina 28/genética , Tetraspanina 28/metabolismo , Tetraspanina 30/genética , Tetraspanina 30/metabolismo , Proteínas Virais de Fusão/isolamento & purificação , Proteínas Virais de Fusão/metabolismo , Proteínas Virais/genética , Proteínas Virais/isolamento & purificação , Proteínas Virais/metabolismo
20.
Viruses ; 11(1)2019 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-30621148

RESUMO

Recently, we found that the cytidine deaminase APOBEC3G (A3G) inhibits measles (MV) replication. Using a microarray, we identified differential regulation of several host genes upon ectopic expression of A3G. One of the up-regulated genes, the endoplasmic reticulum (ER) protein retention receptor KDELR2, reduced MV replication ~5 fold when it was over-expressed individually in Vero and CEM-SS T cells. Silencing of KDELR2 in A3G-expressing Vero cells abrogated the antiviral activity induced by A3G, confirming its role as an A3G-regulated antiviral host factor. Recognition of the KDEL (Lys-Asp-Glu-Leu) motif by KDEL receptors initiates the retrograde transport of soluble proteins that have escaped the ER and play an important role in ER quality control. Although KDELR2 over-expression reduced MV titers in cell cultures, we observed no interaction between KDELR2 and the MV hemagglutinin (H) protein. Instead, KDELR2 retained chaperones in the ER, which are required for the correct folding and transport of the MV envelope glycoproteins H and fusion protein (F) to the cell surface. Our data indicate that KDELR2 competes with MV envelope proteins for binding to calnexin and GRP78/Bip, and that this interaction limits the availability of the chaperones for MV proteins, causing the reduction of virus spread and titers.


Assuntos
Hemaglutininas Virais/metabolismo , Interações entre Hospedeiro e Microrganismos , Vírus do Sarampo/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas Virais de Fusão/metabolismo , Animais , Calnexina/metabolismo , Chlorocebus aethiops , Regulação da Expressão Gênica , Inativação Gênica , Células HEK293 , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Hemaglutininas Virais/genética , Humanos , Vírus do Sarampo/fisiologia , Células Vero , Proteínas de Transporte Vesicular/genética , Proteínas Virais de Fusão/genética , Carga Viral
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