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1.
Virology ; 594: 110053, 2024 06.
Artigo em Inglês | MEDLINE | ID: mdl-38492518

RESUMO

Paramyxoviruses are reported to block apoptosis for their replication, but the mechanisms remain unclear. Furthermore, regulation of mitochondrial apoptosis by paramyxoviruses has been hardly reported. We investigated whether and how human parainfluenza virus type 2 (hPIV-2) counteracts apoptosis. Infection of recombinant hPIV-2 carrying mutated V protein showed higher caspase 3/7 activity and higher cytochrome c release from mitochondria than wild type hPIV-2 infection. This indicates that V protein controls mitochondrial apoptosis pathway. hPIV-2 V protein interacted with Bad, an apoptotic promoting protein, and this interaction inhibited the binding of Bad to Bcl-XL. V protein also bound to 14-3-3ε, which was essential for inhibition of 14-3-3ε cleavage. Our data collectively suggest that hPIV-2 V protein has two means of preventing mitochondrial apoptosis pathway: the inhibition of Bad-Bcl-XL interaction and the suppression of 14-3-3ε cleavage. This is the first report of the mechanisms behind how paramyxoviruses modulate mitochondrial apoptosis pathways.


Assuntos
Mitocôndrias , Vírus da Parainfluenza 2 Humana , Humanos , Vírus da Parainfluenza 2 Humana/metabolismo , Mitocôndrias/metabolismo , Apoptose , Proteínas de Transporte/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/metabolismo
2.
Microbiol Spectr ; 11(3): e0453722, 2023 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-37039701

RESUMO

To understand the molecular evolution of human parainfluenza virus type 2 (HPIV2), 21 Hemagglutinin-Neuraminidase (HN) gene sequences covering seven Chinese provinces in 2011 and 2017 to 2021 were combined with 90 published HN sequences worldwide for phylogenetic analysis. The result showed that global HPIV2 could be classified into two distinct clusters (I and II), five lineages (IA to IIE), and four sublineages (IB1 and 2, and IIE1 and 2). The minimum genetic distances between different clusters and lineages were 0.049 and 0.014, respectively. In the last decade, one lineage (IID) and three sublineages (IB1, IB2, and IIE1) have been cocirculating in China, with the sublineages IB2 and IIE1 dominating, while sublineages IB1 and IIE1 are dominant globally. In addition, the spread of HPIV2 had relative spatial clustering, and sublineage IB2 has only been detected in China thus far. The overall evolution rate of HPIV2 was relatively low, on the order of 10-4 substitutions/site/year, except for sublineage IB2 at 10-3 substitutions/site/year. Furthermore, human-animal transmission was observed, suggesting that the HPIV2 might have jumped out of animal reservoirs in approximately 1922, the predicted time of a common ancestor. The entire HN protein was under purifying/negative selection, and the specific amino acid changes and two novel N-glycosylation sites (N316 and N517) in sublineages IB1, IB2, and IIE1 were mostly located in the globular head region of the HN protein. In this study, preliminary evolutionary characteristics of HPIV2 based on the HN gene were obtained, increasing the recognition of the evolution and adaptation of HPIV2. IMPORTANCE The phylogenetic analysis showed that global HPIV2 could be classified into two distinct clusters (I and II) and five lineages (IA to IIE) with at least 0.049 and 0.014 genetic distances between clusters and lineages, respectively. Furthermore, lineages IB and IIE could be further divided into two sublineages (IB1-2 and IIE1-2). All China sequences belong to one lineage and three sublineages (IB1, IB2, IID, and IIE1), among which sublineages IB2 and IIE1 are predominant and cocirculating in China, while sublineages IB1 and IIE1 are dominant globally. The overall evolution rate of HPIV2 is on the order of 10-4 substitutions/site/year, with the highest rate of 2.18 × 10-3 for sublineage IB2. The entire HN protein is under purifying/negative selection, and the specific amino acid substitutions and two novel N-glycosylation sites (N316 and N517) in sublineages IB1, IB2, and IIE1 are mostly located in the globular head region of the HN protein.


Assuntos
Vírus da Parainfluenza 2 Humana , Neoplasias do Colo do Útero , Animais , Feminino , Humanos , Vírus da Parainfluenza 2 Humana/genética , Vírus da Parainfluenza 2 Humana/metabolismo , Filogenia , Neuraminidase , Hemaglutininas/metabolismo , Proteína HN/genética , Proteína HN/metabolismo , Proteínas Virais/genética , Evolução Molecular
3.
J Virol ; 95(6)2021 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-33408172

RESUMO

Intracellular iron concentration is tightly controlled for cell viability. It is known to affect the growth of several viruses, but the molecular mechanisms are not well understood. We found that iron chelators inhibit growth of human parainfluenza virus type 2 (hPIV-2). Furthermore, infection with hPIV-2 alters ferritin localization from granules to a homogenous distribution within cytoplasm of iron-stimulated cells. The V protein of hPIV-2 interacts with ferritin heavy chain 1 (FTH1), a ferritin subunit. It also binds to nuclear receptor coactivator 4 (NCOA4), which mediates autophagic degradation of ferritin, so-called ferritinophagy. V protein consequently interferes with interaction between FTH1 and NCOA4. hPIV-2 growth is inhibited in FTH1 knockdown cell line where severe hPIV-2-induced apoptosis is shown. In contrast, NCOA4 knockdown results in the promotion of hPIV-2 growth and limited apoptosis. Our data collectively suggest that hPIV-2 V protein inhibits FTH1-NCOA4 interaction and subsequent ferritinophagy. This iron homeostasis modulation allows infected cells to avoid apoptotic cell death, resulting in effective growth of hPIV-2.IMPORTANCE hPIV-2 V protein interferes with interaction between FTH1 and NCOA4 and inhibits NCOA4-mediated ferritin degradation, leading to the inhibition of iron release to the cytoplasm. This iron homeostasis modulation allows infected cells to avoid apoptotic cell death, resulting in effective growth of hPIV-2.


Assuntos
Homeostase , Ferro/metabolismo , Vírus da Parainfluenza 2 Humana/fisiologia , Proteínas Estruturais Virais/metabolismo , Apoptose , Linhagem Celular , Ferritinas/genética , Ferritinas/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Coativadores de Receptor Nuclear/genética , Coativadores de Receptor Nuclear/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Vírus da Parainfluenza 2 Humana/crescimento & desenvolvimento , Vírus da Parainfluenza 2 Humana/metabolismo , Ligação Proteica
4.
Arch Virol ; 165(4): 799-807, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32100137

RESUMO

We previously found that infection with human parainfluenza virus type 2 (hPIV-2), a member of the genus Orthorubulavirus, family Paramyxoviridae, causes filamentous actin (F-actin) formation to promote viral growth. In the present study, we investigated whether similar regulation of F-actin formation is observed in infections with other rubulaviruses, such as parainfluenza virus type 5 (PIV-5) and simian virus 41 (SV41). Infection with these viruses caused F-actin formation and RhoA activation, which promoted viral growth. These results indicate that RhoA-induced F-actin formation is important for efficient growth of these rubulaviruses. Only SV41 and hPIV-2 V and P proteins bound to Graf1, while the V and P proteins of PIV-5, mumps virus, and hPIV-4 did not bind to Graf1. In contrast, the V proteins of these rubulaviruses bound to both inactive RhoA and profilin 2. These results suggest that there are common and unique mechanisms involved in regulation of F-actin formation by members of the genus Orthorubulavirus.


Assuntos
Actinas/metabolismo , Vírus da Parainfluenza 2 Humana/metabolismo , Vírus da Parainfluenza 5/metabolismo , Infecções por Rubulavirus/metabolismo , Rubulavirus/metabolismo , Actinas/química , Actinas/genética , Animais , Linhagem Celular , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Vírus da Parainfluenza 2 Humana/genética , Vírus da Parainfluenza 2 Humana/crescimento & desenvolvimento , Vírus da Parainfluenza 5/genética , Vírus da Parainfluenza 5/crescimento & desenvolvimento , Ligação Proteica , Rubulavirus/genética , Rubulavirus/crescimento & desenvolvimento , Infecções por Rubulavirus/genética , Infecções por Rubulavirus/virologia , Proteínas Virais/genética , Proteínas Virais/metabolismo , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo
5.
Virology ; 533: 108-114, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31150988

RESUMO

We previously reported that human parainfluenza virus type 2 (hPIV-2) promoted RhoA activation and subsequent filamentous actin (F-actin) formation. Actin-binding proteins, such as profilin and cofilin, are involved in the regulation of F-actin formation by RhoA signaling. In the present study, we identified profilin2 as a key molecule that is involved in hPIV-2-induced F-actin formation. Immunoprecipitation assays demonstrated that hPIV-2 V protein binds to profilin2 but not to profilin1. Mutation of Trp residues within C-terminal region of V protein abolished the binding capacity to profilin2. Depletion of profilin2 resulted in the inhibition of hPIV-2-induced F-actin formation and the suppression of hPIV-2 growth. Overexpression of wild type V but not Trp-mutated V protein reduced the quantity of actin co-immunoprecipitated with profilin2. Taken together, these results suggest that hPIV-2 V protein promotes F-actin formation by affecting actin-profilin2 interaction through its binding to profilin2.


Assuntos
Actinas/metabolismo , Vírus da Parainfluenza 2 Humana/metabolismo , Profilinas/metabolismo , Infecções por Rubulavirus/metabolismo , Infecções por Rubulavirus/virologia , Actinas/genética , Interações Hospedeiro-Patógeno , Humanos , Vírus da Parainfluenza 2 Humana/genética , Profilinas/genética , Ligação Proteica , Infecções por Rubulavirus/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo
6.
Virology ; 528: 54-63, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30576860

RESUMO

Human parainfluenza virus type 2 phosphoprotein (P) is an essential component of viral polymerase. The P gene encodes both P and accessory V proteins by a specific gene editing mechanism. Therefore, the N-terminal 164 amino acids of P protein are common to V protein. Interestingly, while P protein is located in the cytoplasm, V protein is found mainly in the nucleus. Using deletion mutants, we show the presence of a nuclear localization signal (NLS) in the P/V common domain, and a nuclear export signal (NES) in the C-terminal P specific region. The NLS region makes a complex with importin α5 or 7. In the presence of leptomycin B, P protein is retained in the nucleus, indicating that it contains a CRM1-dependent NES. We identified the NLS (65PVKPRRKK72) and the NES (225IIELLKGLDL234) using ß-galactosidase fusion proteins. Moreover, nucleocytoplasmic shuttling of P protein appears to be important for efficient viral polymerase activity.


Assuntos
Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Vírus da Parainfluenza 2 Humana/metabolismo , Fosfoproteínas/metabolismo , Proteínas Virais/metabolismo , Animais , Linhagem Celular , Chlorocebus aethiops , Ácidos Graxos Insaturados/farmacologia , Células HeLa , Humanos , Carioferinas/metabolismo , Sinais de Exportação Nuclear , Sinais de Localização Nuclear/metabolismo , Fosfoproteínas/genética , Transporte Proteico , Células Vero , Proteínas Virais/genética
7.
J Gen Virol ; 99(4): 501-511, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29485395

RESUMO

The multifunctional V protein of human parainfluenza virus type 2 (hPIV2) plays important roles in controlling viral genome replication, inhibiting the host interferon response and promoting virus growth. We screened a yeast two-hybrid library using V protein as bait to identify host factors that are important for other functions of V. One of several positive clones isolated from HeLa cell-derived cDNA library encodes caspase-1. We found that the C-terminal region of V interacts with the C-terminal region of caspase-1 in mammalian cells. Moreover, the V protein repressed caspase-1 activity and the formation of interleukin-1ß (IL-1ß) in a dose-dependent manner. IL-1ß secretion induced by wild-type hPIV2 infection in human monocytic THP-1 cells was significantly lower than that induced by recombinant hPIV2 lacking V protein or having a mutant V. These data suggest that hPIV2 V protein inhibits caspase-1-mediated maturation of IL-1ß via its interaction with caspase-1.


Assuntos
Caspase 1/metabolismo , Vírus da Parainfluenza 2 Humana/metabolismo , Infecções por Rubulavirus/enzimologia , Proteínas Virais/metabolismo , Motivos de Aminoácidos , Caspase 1/química , Caspase 1/genética , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Vírus da Parainfluenza 2 Humana/química , Vírus da Parainfluenza 2 Humana/genética , Ligação Proteica , Infecções por Rubulavirus/genética , Infecções por Rubulavirus/virologia , Proteínas Virais/química , Proteínas Virais/genética , Replicação Viral
8.
J Virol ; 92(7)2018 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-29343567

RESUMO

A parainfluenza virus 5 (PIV5) with mutations in the P/V gene (P/V-CPI-) is restricted for spread in normal cells but not in cancer cells in vitro and is effective at reducing tumor burdens in mouse model systems. Here we show that P/V-CPI- infection of HEp-2 human laryngeal cancer cells results in the majority of the cells dying, but unexpectedly, over time, there is an emergence of a population of cells that survive as P/V-CPI- persistently infected (PI) cells. P/V-CPI- PI cells had elevated levels of basal caspase activation, and viability was highly dependent on the activity of cellular inhibitor-of-apoptosis proteins (IAPs) such as Survivin and XIAP. In challenge experiments with external inducers of apoptosis, PI cells were more sensitive to cisplatin-induced DNA damage and cell death. This increased cisplatin sensitivity correlated with defects in DNA damage signaling pathways such as phosphorylation of Chk1 and translocation of damage-specific DNA binding protein 1 (DDB1) to the nucleus. Cisplatin-induced killing of PI cells was sensitive to the inhibition of wild-type (WT) p53-inducible protein 1 (WIP1), a phosphatase which acts to terminate DNA damage signaling pathways. A similar sensitivity to cisplatin was seen with cells during acute infection with P/V-CPI- as well as during acute infections with WT PIV5 and the related virus human parainfluenza virus type 2 (hPIV2). Our results have general implications for the design of safer paramyxovirus-based vectors that cannot establish PI as well as the potential for combining chemotherapy with oncolytic RNA virus vectors.IMPORTANCE There is intense interest in developing oncolytic viral vectors with increased potency against cancer cells, particularly those cancer cells that have gained resistance to chemotherapies. We have found that infection with cytoplasmically replicating parainfluenza virus can result in increases in the killing of cancer cells by agents that induce DNA damage, and this is linked to alterations to DNA damage signaling pathways that balance cell survival versus death. Our results have general implications for the design of safer paramyxovirus-based vectors that cannot establish persistent infection, the repurposing of drugs that target cellular IAPs as antivirals, and the combined use of DNA-damaging chemotherapy agents in conjunction with oncolytic RNA virus vectors.


Assuntos
Dano ao DNA , Neoplasias/terapia , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/metabolismo , Vírus da Parainfluenza 2 Humana/metabolismo , Transdução de Sinais , Animais , Chlorocebus aethiops , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Camundongos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Vírus Oncolíticos/genética , Vírus da Parainfluenza 2 Humana/genética , Survivina , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Células Vero , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo
9.
Drug Discov Ther ; 11(5): 246-252, 2017 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-29070744

RESUMO

The effect of glycyrrhizin on the replication of human parainfluenza virus type 2 (hPIV-2) was examined. Cell fusion induced by hPIV-2 was inhibited by glycyrrhizin, and glycyrrhizin reduced the number of viruses released from the cells. Glycyrrhizin did not change cell morphology at 1 day of culture, but caused some damage at 4 days, as determined by the effect on actin microfilaments. However, it affected the cell viability at 1 day: about 20% of the cells were not alive by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at 1 day of culture. Real-time polymerase chain reaction (PCR) and PCR showed that virus genome synthesis was largely inhibited. mRNA synthesis was also inhibited by glycyrrhizin. Viral protein synthesis was largely inhibited as observed by an indirect immunofluorescence study. Multinucleated giant cell formation was studied using a recombinant green fluorescence protein (GFP)-expressing hPIV-2 without matrix protein (rhPIV-2ΔMGFP). A few single cells with fluorescence were observed, but the formation of giant cells was completely blocked. Taken together, it was shown that viral genome, mRNA and protein syntheses, including F and HN proteins, were inhibited by glycyrrhizin, and consequently multinucleated giant cell formation was not observed and the infectious virus was not detected in the culture medium.


Assuntos
Ácido Glicirrízico/farmacologia , Vírus da Parainfluenza 2 Humana/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , RNA Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Citoesqueleto de Actina/efeitos dos fármacos , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Genoma Viral/efeitos dos fármacos , Células Gigantes/efeitos dos fármacos , Proteína HN/biossíntese , Proteína HN/efeitos dos fármacos , Macaca mulatta , Vírus da Parainfluenza 2 Humana/genética , Vírus da Parainfluenza 2 Humana/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Virais de Fusão/biossíntese , Proteínas Virais de Fusão/efeitos dos fármacos , Proteínas Virais/biossíntese , Proteínas Virais/efeitos dos fármacos , Replicação Viral/genética
10.
J Virol ; 90(20): 9394-405, 2016 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-27512058

RESUMO

UNLABELLED: Rho GTPases are involved in a variety of cellular activities and are regulated by guanine nucleotide exchange factors and GTPase-activating proteins (GAPs). We found that the activation of Rho GTPases by lysophosphatidic acid promotes the growth of human parainfluenza virus type 2 (hPIV-2). Furthermore, hPIV-2 infection causes activation of RhoA, a Rho GTPase. We hypothesized that Graf1 (also known as ARHGAP26), a GAP, regulates hPIV-2 growth by controlling RhoA signaling. Immunofluorescence analysis showed that hPIV-2 infection altered Graf1 localization from a homogenous distribution within the cytoplasm to granules. Graf1 colocalized with hPIV-2 P, NP, and L proteins. Graf1 interacts with P and V proteins via their N-terminal common region, and the C-terminal Src homology 3 domain-containing region of Graf1 is important for these interactions. In HEK293 cells constitutively expressing Graf1, hPIV-2 growth was inhibited, and RhoA activation was not observed during hPIV-2 infection. In contrast, Graf1 knockdown restored hPIV-2 growth and RhoA activation. Overexpression of hPIV-2 P and V proteins enhanced hPIV-2-induced RhoA activation. These results collectively suggested that hPIV-2 P and V proteins enhanced hPIV-2 growth by binding to Graf1 and that Graf1 inhibits hPIV-2 growth through RhoA inactivation. IMPORTANCE: Robust growth of hPIV-2 requires Rho activation. hPIV-2 infection causes RhoA activation, which is suppressed by Graf1. Graf1 colocalizes with viral RNP (vRNP) in hPIV-2-infected cells. We found that Graf1 interacts with hPIV-2 P and V proteins. We also identified regions in these proteins which are important for this interaction. hPIV-2 P and V proteins enhanced the hPIV-2 growth via binding to Graf1, while Graf1 inhibited hPIV-2 growth through RhoA inactivation.


Assuntos
Proteínas Ativadoras de GTPase/metabolismo , Vírus da Parainfluenza 2 Humana/metabolismo , Transdução de Sinais/fisiologia , Proteínas rho de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Células COS , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Citoplasma/metabolismo , Citoplasma/virologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HEK293 , Células HeLa , Humanos , Células Vero , Domínios de Homologia de src/fisiologia
11.
J Gen Virol ; 97(3): 561-570, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26675672

RESUMO

Tetherin (BST-2/CD317/HM1.24) is an antiviral membrane protein that prevents the release of enveloped viruses from the cell surface. We found that the growth of human parainfluenza virus type 2 (hPIV-2), but not that of V protein-deficient recombinant hPIV-2, was inhibited by tetherin. V protein immunoprecipitates with tetherin, and this interaction requires its C-terminal Trp residues. The glycosyl phosphatidylinositol attachment signal of tetherin, but not its cytoplasmic tail, was necessary for its binding with V. The distribution of the V protein clearly changed when co-expressed with tetherin in plasmid-transfected cells. hPIV-2 infection of HeLa cells reduced cell surface tetherin without affecting total cellular tetherin. This reduction also occurred in HeLa cells constitutively expressing V, whereas mutated V protein did not affect the cell surface tetherin. Our results suggest that hPIV-2 V protein antagonizes tetherin by binding it and reducing its presence at the cell surface.


Assuntos
Antígenos CD/metabolismo , Crupe/metabolismo , Vírus da Parainfluenza 2 Humana/metabolismo , Proteínas Virais/metabolismo , Motivos de Aminoácidos , Antígenos CD/química , Antígenos CD/genética , Crupe/genética , Crupe/virologia , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Vírus da Parainfluenza 2 Humana/química , Vírus da Parainfluenza 2 Humana/genética , Ligação Proteica , Proteínas Virais/química , Proteínas Virais/genética
12.
J Virol ; 89(24): 12374-87, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26423949

RESUMO

UNLABELLED: Virus-specific interaction between the attachment protein (HN) and the fusion protein (F) is prerequisite for the induction of membrane fusion by parainfluenza viruses. This HN-F interaction presumably is mediated by particular amino acids in the HN stalk domain and those in the F head domain. We found in the present study, however, that a simian virus 41 (SV41) F-specific chimeric HPIV2 HN protein, SCA, whose cytoplasmic, transmembrane, and stalk domains were derived from the SV41 HN protein, could not induce cell-cell fusion of BHK-21 cells when coexpressed with an SV41 HN-specific chimeric PIV5 F protein, no. 36. Similarly, a headless form of the SV41 HN protein failed to induce fusion with chimera no. 36, whereas it was able to induce fusion with the SV41 F protein. Interestingly, replacement of 13 amino acids of the SCA head domain, which are located at or around the dimer interface of the head domain, with SV41 HN counterparts resulted in a chimeric HN protein, SCA-RII, which induced fusion with chimera no. 36 but not with the SV41 F protein. More interestingly, retroreplacement of 11 out of the 13 amino acids of SCA-RII with the SCA counterparts resulted in another chimeric HN protein, IM18, which induced fusion either with chimera no. 36 or with the SV41 F protein, similar to the SV41 HN protein. Thus, we conclude that the F protein specificity of the HN protein that is observed in the fusion event is not solely defined by the primary structure of the HN stalk domain. IMPORTANCE: It is appreciated that the HN head domain initially conceals the HN stalk domain but exposes it after the head domain has bound to the receptors, which allows particular amino acids in the stalk domain to interact with the F protein and trigger it to induce fusion. However, other regulatory roles of the HN head domain in the fusion event have been ill defined. We have shown in the current study that removal of the head domain or amino acid substitutions in a particular region of the head domain drastically change the F protein specificity of the HN protein, suggesting that the ability of a given HN protein to interact with an F protein is defined not only by the primary structure of the HN stalk domain but also by its conformation. This notion seems to account for the unidirectional substitutability among rubulavirus HN proteins in triggering noncognate F proteins.


Assuntos
Proteína HN/metabolismo , Vírus da Parainfluenza 2 Humana/metabolismo , Proteínas Virais de Fusão/metabolismo , Proteínas Virais/metabolismo , Animais , Linhagem Celular , Cricetinae , Proteína HN/química , Proteína HN/genética , Vírus da Parainfluenza 2 Humana/química , Vírus da Parainfluenza 2 Humana/genética , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/genética , Proteínas Virais/química , Proteínas Virais/genética
13.
Microbiol Immunol ; 59(11): 676-83, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26446904

RESUMO

Gene expression of nonsegmented negative-strand RNA viruses (nsNSVs) such as parainfluenza viruses requires the RNA synthesis activity of their polymerase L protein; however, the detailed mechanism of this process is poorly understood. In this study, a parainfluenza minireplicon assay expressing secretory Gaussia luciferase (Gluc) was established to analyze large protein (L) activity. Measurement of Gluc expression in the culture medium of cells transfected with the minigenome and viral polymerase components enabled quick and concise calculation of L activity. By comparing the amino acid sequences in conserved region III (CRIII), a putative polymerase-active domain of the L protein, two strictly conserved aspartates were identified in all families of nsNSV. A series of L mutants from human parainfluenza virus type 2 and parainfluenza virus type 5 showed that these aspartates are necessary for reporter gene expression. It was also confirmed that these aspartates are important for the production of viral mRNA and antigenome cRNA, but not for a polymerase-complex formation. These findings suggest that these two aspartates are key players in the nucleotidyl transfer reaction using two metal ions.


Assuntos
Ácido Aspártico/genética , Copépodes/enzimologia , Luciferases/metabolismo , Vírus da Parainfluenza 2 Humana/enzimologia , Vírus da Parainfluenza 2 Humana/metabolismo , Transfecção/métodos , Proteínas Virais/genética , Proteínas Virais/fisiologia , Replicação Viral/fisiologia , Animais , Células Cultivadas , Sequência Conservada , Humanos
14.
J Virol ; 87(14): 7966-76, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23678181

RESUMO

Paramyxovirus V proteins block Toll-like receptor 7 (TLR7)- and TLR9-dependent signaling leading to alpha interferon production. Our recent study has provided evidence that interaction of the V proteins with IRF7 is important for the blockade. However, the detailed mechanisms still remain unclear. Here we reexamined the interaction of the human parainfluenza virus type 2 (HPIV2) V protein with signaling molecules involved in TLR7/9-dependent signaling. Immunoprecipitation experiments in HEK293T cells transfected with V protein and one of the signaling molecules revealed that the V protein interacted with not only IRF7 but also TRAF6, IKKα, and MyD88. Whereas overexpression of TRAF6 markedly enhanced the level of V protein associating with IRF7, IKKα, and MyD88 in HEK293T cells, the level of V protein associating with TRAF6 was little affected by overexpression of IRF7, IKKα, and MyD88. Moreover, knockdown or knockout of endogenous TRAF6 in HEK293T or mouse embryonic fibroblast cells resulted in dissociation of the V protein from IRF7, IKKα, and MyD88. These results demonstrate that binding of the V protein to IRF7, IKKα, and MyD88 is largely indirect and mediated by endogenous TRAF6. It was found that the V protein inhibited TRAF6-mediated lysine 63 (K63)-linked polyubiquitination of IRF7, which is prerequisite for IRF7 activation. Disruption of the tryptophan-rich motif of the V protein significantly affected its TRAF6-binding efficiency, which correlated well with the magnitude of inhibition of K63-linked polyubiquitination and the resultant activation of IRF7. Taken together, these results suggest that the HPIV2 V protein prevents TLR7/9-dependent interferon induction by inhibiting TRAF6-mediated K63-linked polyubiquitination of IRF7.


Assuntos
Fator Regulador 7 de Interferon/metabolismo , Interferon-alfa/metabolismo , Vírus da Parainfluenza 2 Humana/metabolismo , Transdução de Sinais/fisiologia , Fator 6 Associado a Receptor de TNF/metabolismo , Proteínas Virais/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Quinase I-kappa B/metabolismo , Imunoprecipitação , Fator 88 de Diferenciação Mieloide/metabolismo , Fator 6 Associado a Receptor de TNF/genética , Receptor 7 Toll-Like/metabolismo , Receptor Toll-Like 9/metabolismo , Ubiquitinação/efeitos dos fármacos , Proteínas Virais/farmacologia
15.
J Virol ; 85(23): 12146-59, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21917945

RESUMO

The hemagglutinin-neuraminidase (HN) protein of human parainfluenza viruses (hPIVs) both binds (H) and cleaves (N) oligosaccharides that contain N-acetylneuraminic acid (Neu5Ac). H is thought to correspond to receptor binding and N to receptor-destroying activity. At present, N's role in infection remains unclear: does it destroy only receptors, or are there other targets? We previously demonstrated that hPIV1 and 3 HNs bind to oligosaccharides containing the motif Neu5Acα2-3Galß1-4GlcNAc (M. Amonsen, D. F. Smith, R. D. Cummings, and G. M. Air, J. Virol. 81:8341-8345, 2007). In the present study, we tested the binding specificity of hPIV2 on the Consortium for Functional Glycomics' glycan array and found that hPIV2 binds to oligosaccharides containing the same motif. We determined the specificities of N on red blood cells, soluble small-molecule and glycoprotein substrates, and the glycan array and compared them to the specificities of H. hPIV2 and -3, but not hPIV1, cleaved their ligands on red blood cells. hPIV1, -2, and -3 cleaved their NeuAcα2-3 ligands on the glycan array; hPIV2 and -3 also cleaved NeuAcα2-6 ligands bound by influenza A virus. While all three HNs exhibited similar affinities for all cleavable soluble substrates, their activities were 5- to 10-fold higher on small molecules than on glycoproteins. In addition, some soluble glycoproteins were not cleaved, despite containing oligosaccharides that were cleaved on the glycan array. We conclude that the susceptibility of an oligosaccharide substrate to N increases when the substrate is fixed to a surface. These findings suggest that HN may undergo a conformational change that activates N upon receptor binding at a cell surface.


Assuntos
Hemaglutininas/metabolismo , Neuraminidase/metabolismo , Oligossacarídeos/metabolismo , Vírus da Parainfluenza 1 Humana/metabolismo , Vírus da Parainfluenza 2 Humana/metabolismo , Vírus da Parainfluenza 3 Humana/metabolismo , Receptores de Superfície Celular/metabolismo , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Galinhas , Eritrócitos/metabolismo , Glicosilação , Testes de Hemaglutinação , Humanos , Análise em Microsséries , Dados de Sequência Molecular , Vírus da Parainfluenza 1 Humana/isolamento & purificação , Vírus da Parainfluenza 2 Humana/isolamento & purificação , Vírus da Parainfluenza 3 Humana/isolamento & purificação , Filogenia , Polissacarídeos/metabolismo , Receptores Virais/metabolismo , Infecções por Respirovirus/genética , Infecções por Respirovirus/metabolismo , Infecções por Respirovirus/virologia , Homologia de Sequência de Aminoácidos , Turquia
16.
Biomed Res ; 29(6): 331-4, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19129677

RESUMO

The effects of fucoidan and L-fucose, a fundamental major component of fucoidan, on the growth of human parainfluenza virus type 2 (hPIV-2) in LLCMK(2) cells were investigated. Fucoidan inhibited cell fusion and hemadsorption, but L-fucose only partly inhibited both. Virus RNA was not detected in the hPIV-2 infected cells cultured with fucoidan. However, L-fucose did not inhibit virus RNA synthesis. Indirect immunofluorescence study showed that virus protein synthesis was inhibited by fucoidan, but not by L-fucose. Furthermore, using a recombinant, green fluorescence protein-expressing hPIV-2, it was found that virus entry was inhibited by fucoidan, but not by L-fucose. These results suggested that fucoidan inhibited virus adsorption to the surface of the cells by binding to the cell surface and prevented infection, indicating that the sulfated polysaccharide form was important for the inhibition by fucoidan.


Assuntos
Vírus da Parainfluenza 2 Humana/efeitos dos fármacos , Polissacarídeos/farmacologia , Infecções por Rubulavirus/metabolismo , Linhagem Celular , Criança , Fucose/metabolismo , Humanos , Vírus da Parainfluenza 2 Humana/genética , Vírus da Parainfluenza 2 Humana/metabolismo , Proteínas Virais/metabolismo
17.
Microbiol Immunol ; 51(6): 601-8, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17579271

RESUMO

When K562 cells were infected with Newcastle disease virus (NDV) or human parainfluenza type 2 virus (hPIV-2), polykaryocyte formation could not be detected. Failure of multinucleated giant cell formation in K562 cells infected with either NDV or hPIV-2 is due to disturbance of the viral envelope-cell fusion step or to defect in the cell-cell fusion step, respectively. Especially, NDV completely replicated in K562 cells, and the hemagglutinin-neuraminidase and fusion proteins expressed on the cell surface of NDV-infected K562 cell were fully functional for fusion inducing activity. Therefore, the cell membranes of K562 cells are considered to be resistant to virus-induced cell fusion. Membrane fusion is regulated by many host factors including membrane fluidity, cytoskeletal systems, and fusion regulatory proteins system. An unknown regulatory mechanism of virus-induced cell fusion may function on the cell surface of K562 cells.


Assuntos
Células Gigantes/virologia , Vírus da Doença de Newcastle/fisiologia , Vírus da Parainfluenza 2 Humana/fisiologia , Infecções por Paramyxoviridae/patologia , Eletroforese em Gel de Poliacrilamida , Citometria de Fluxo , Células Gigantes/patologia , Células HeLa , Humanos , Células K562 , Vírus da Doença de Newcastle/metabolismo , Vírus da Parainfluenza 2 Humana/genética , Vírus da Parainfluenza 2 Humana/metabolismo , Infecções por Paramyxoviridae/metabolismo , Infecções por Paramyxoviridae/virologia , RNA Viral/química , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células U937 , Proteínas Virais/metabolismo , Replicação Viral
18.
Virology ; 362(1): 85-98, 2007 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-17250865

RESUMO

Our previous results have shown that some residues of V protein-specific domain in human parainfluenza virus type 2 (hPIV2) are essential not only for STAT protein degradation but also for promoting virus growth. Here, we demonstrated that the virus growth of these recombinant hPIV2s (rPIV2) expressing mutated V proteins were improved in HeLa cell transiently expressing the wild-type V protein, but not in the cells constitutively expressing it. Consequently, we identified the region of the V protein that is essential for its oligomerization and for complex formation with NP protein. We also identified a host protein, AlP1/Alix, involved in apoptosis and efficient budding of several enveloped viruses as an interacting partner of the V and NP proteins. Depletion of AIP1/Alix by small interfering RNA suppressed virus growth. These data suggest that the conserved carboxyl terminus of the V protein plays an important role in virus growth.


Assuntos
Vírus da Parainfluenza 2 Humana/crescimento & desenvolvimento , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Chlorocebus aethiops , Complexos Endossomais de Distribuição Requeridos para Transporte , Teste de Complementação Genética , Células HeLa , Humanos , Dados de Sequência Molecular , Mutação , Nucleoproteínas/metabolismo , Vírus da Parainfluenza 2 Humana/metabolismo , Ligação Proteica , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Interferência de RNA , Células Vero , Proteínas Virais/genética
19.
Virology ; 317(2): 208-19, 2003 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-14698661

RESUMO

The V protein of SV41 targets STAT1, while a specific loss of STAT2 is induced by the hPIV2 V protein. We established HeLa cells constitutively expressing various chimeric proteins between the hPIV2 and SV41 V proteins, and which STAT (STAT1 or 2) was expressed in these cells was analyzed. Both the P-V common domain and the V specific domain of hPIV2 V protein are necessary for STAT2 lowering. The internal domain (aa145-173) containing a large number of nonidentical amino acids between hPIV2 and SV41 does not direct STAT tropism, and the regions necessary for STAT2 lowering are discontinuous. The N-terminal domain (aa1-104) and the internal domain (aa126-196) of the hPIV2 V protein do not determine STAT tropism. HeLa cells expressing A105E or H108P show distinct expression of STAT2, but do show low expression or a loss of STAT1, indicating that the amino acid residues 105 and 108 of the hPIV2 V protein are essential for STAT2 lowering. Interestingly, there is an important amino acid(s) in the region (aa121-125) for STAT2 lowering, and the presence of either amino acid residue 123 or 125 of the hPIV2 V protein is necessary for lowering of STAT2. In addition, HeLa cells expressing S216D or 1217R expressed STAT2, but no STAT1, indicating that the amino acid residues 216 and 217 of the hPIV2 V protein are indispensable for STAT2 lowering. HeLa/hPIV2V cells and HeLa/S104/P are resistant to IFN-beta, while they are sensitive to IFN-gamma. On the other hand, HeLa/SV41V, HeLa/S216D, and HeLa1217R cells are resistant to both IFNs. Intriguingly, HeLa/A105E and HeLa/H108P cells were found to be sensitive to IFN-gamma.


Assuntos
Sequência de Aminoácidos , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Vírus da Parainfluenza 2 Humana/patogenicidade , Transativadores/metabolismo , Proteínas Virais/química , Células HeLa , Humanos , Mutagênese Sítio-Dirigida , Mutação , Vírus da Parainfluenza 2 Humana/genética , Vírus da Parainfluenza 2 Humana/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Rubulavirus/genética , Rubulavirus/metabolismo , Fator de Transcrição STAT2 , Relação Estrutura-Atividade , Proteínas Virais/genética , Proteínas Virais/metabolismo
20.
Virology ; 283(2): 230-9, 2001 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-11336548

RESUMO

Type I interferon (IFN) induces antiviral responses through the activation of the ISGF3 transcription factor complex that contains the subunit proteins STAT1, STAT2, and p48/ISGF3 gamma/IRF9. The ability of some human paramyxoviruses to overcome IFN actions by specific proteolysis of STAT proteins has been examined. Infection of cells with type 2, but not type 1 or type 3 human parainfluenza virus (HPIV) leads to a loss of cellular STAT2 protein. Expression of a single HPIV2 protein derived from the V open reading frame blocks IFN-dependent transcriptional responses in the absence of other viral proteins. The loss of IFN response is due to V-protein-induced proteolytic degradation of STAT2. Expression of HPIV2 V causes the normally stable STAT2 protein to be rapidly degraded, and this proteolytic activity can be partially alleviated by proteasome inhibition. No V-protein-specific effects on STAT2 mRNA levels were observed. The results indicate that the V protein of HPIV2 is sufficient to recognize and target a specific cellular transcription factor for destruction by cellular machinery.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Interferon Tipo I/imunologia , Vírus da Parainfluenza 2 Humana/patogenicidade , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Virais , Proteínas Estruturais Virais/metabolismo , Linhagem Celular , Cisteína Endopeptidases/metabolismo , DNA Complementar , Humanos , Fator Gênico 3 Estimulado por Interferon , Fator Gênico 3 Estimulado por Interferon, Subunidade gama , Complexos Multienzimáticos/antagonistas & inibidores , Complexos Multienzimáticos/metabolismo , Vírus da Parainfluenza 2 Humana/genética , Vírus da Parainfluenza 2 Humana/metabolismo , Complexo de Endopeptidases do Proteassoma , Processamento de Proteína Pós-Traducional , RNA Mensageiro/metabolismo , Infecções por Rubulavirus/fisiopatologia , Infecções por Rubulavirus/virologia , Fator de Transcrição STAT1 , Fator de Transcrição STAT2 , Transfecção , Proteínas Estruturais Virais/genética
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