RESUMEN
Previous results have shown that infection with the cytoplasmic-replicating parainfluenza virus 5 mutant P/V-CPI- sensitizes cells to DNA damaging agents, resulting in the enhanced killing of airway cancer cells. Here, we have tested the hypothesis that histone deacetylase (HDAC) inhibitors can also act with P/V-CPI- infection to enhance cancer cell killing. Using human small cell lung cancer and laryngeal cancer cell lines, 10 HDAC inhibitors were tested for their effect on viability of P/V-CPI- infected cells. HDAC inhibitors such as scriptaid enhanced caspase-3/7, -8 and -9 activity induced by P/V-CPI- and overall cell toxicity. Scriptaid-mediated enhanced killing was eliminated in lung cancer cells that were engineered to express a protein which sequesters double stranded RNA. Scriptaid also enhanced cancer cell killing by two other negative strand RNA viruses - the La Crosse virus and vesicular stomatitis virus. Scriptaid treatment enhanced the spread of the P/V-CPI- virus through a population of cancer cells, and suppressed interferon-beta induction through blocking phosphorylation and nuclear translocation of Interferon Regulatory Factor 3 (IRF-3). Taken together, these data support a role for combinations of a cytoplasmic-replicating RNA virus such as the P/V-CPI- mutant along with chemotherapeutic agents.
Asunto(s)
Inhibidores de Histona Desacetilasas/farmacología , Interferón beta/biosíntesis , Neoplasias/metabolismo , Virus Oncolíticos/fisiología , Virus de la Parainfluenza 5/fisiología , Caspasas/genética , Caspasas/metabolismo , Línea Celular Tumoral , Humanos , Hidroxilaminas/farmacología , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/metabolismo , Neoplasias/genética , Neoplasias/terapia , Neoplasias/virología , Virus Oncolíticos/genética , Virus de la Parainfluenza 5/genética , Quinolinas/farmacologíaRESUMEN
To define the links between paramyxovirus budding and cellular ESCRT machinery, we previously identified angiomotin-like 1 (AMOTL1) in a screen for host factors that bind to the matrix (M) protein of parainfluenza virus 5 (PIV5). This protein harbors three L/PPXY sequences, allowing it to interact with WW domain containing proteins including NEDD4 family members. We hypothesize that paramyxoviruses use AMOTL1 as a linker to indirectly recruit the same NEDD4 ubiquitin ligases for budding that other enveloped viruses recruit directly through their PPXY late domains. In support of this hypothesis, we found that AMOTL1 could link together M proteins and NEDD4 family proteins in three-way co-IP experiments. Both PIV5 and mumps virus M proteins could be linked to the NEDD4 family proteins NEDD4-1, NEDD4L, and NEDL1, provided that AMOTL1 was co-expressed as a bridging protein. AMOT and AMOTL2 could not substitute for AMOTL1, as they lacked the ability to bind with paramyxovirus M proteins. Attachment of a PPXY late domain sequence to PIV5 M protein obviated the need for AMOTL1 as a linker between M and NEDD4 proteins. Together, these results suggest a novel host factor recruitment strategy for paramyxoviruses to achieve particle release.
Asunto(s)
Proteínas de la Membrana/metabolismo , Ubiquitina-Proteína Ligasas Nedd4/metabolismo , Virus de la Parainfluenza 5/fisiología , Proteínas de la Matriz Viral/metabolismo , Liberación del Virus , Angiomotinas , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Células HEK293 , Interacciones Microbiota-Huesped , Humanos , Proteínas de la Membrana/genética , Ubiquitina-Proteína Ligasas Nedd4/genética , Unión Proteica , Infecciones por Rubulavirus , Ubiquitinación , Proteínas de la Matriz Viral/genéticaRESUMEN
UNLABELLED: The Paramyxoviridae comprise a large family of enveloped, negative-sense, single-stranded RNA viruses with significant economic and public health implications. For nearly all paramyxoviruses, infection is initiated by fusion of the viral and host cell plasma membranes in a pH-independent fashion. Fusion is orchestrated by the receptor binding protein hemagglutinin-neuraminidase (HN; also called H or G depending on the virus type) protein and a fusion (F) protein, the latter undergoing a major refolding process to merge the two membranes. Mechanistic details regarding the coupling of receptor binding to F activation are not fully understood. Here, we have identified the flexible loop region connecting the bulky enzymatically active head and the four-helix bundle stalk to be essential for fusion promotion. Proline substitution in this region of HN of parainfluenza virus 5 (PIV5) and Newcastle disease virus HN abolishes cell-cell fusion, whereas HN retains receptor binding and neuraminidase activity. By using reverse genetics, we engineered recombinant PIV5-EGFP viruses with mutations in the head-stalk linker region of HN. Mutations in this region abolished virus recovery and infectivity. In sum, our data suggest that the loop region acts as a "hinge" around which the bulky head of HN swings to-and-fro to facilitate timely HN-mediate F-triggering, a notion consistent with the stalk-mediated activation model of paramyxovirus fusion. IMPORTANCE: Paramyxovirus fusion with the host cell plasma membrane is essential for virus infection. Membrane fusion is orchestrated via interaction of the receptor binding protein (HN, H, or G) with the viral fusion glycoprotein (F). Two distinct models have been suggested to describe the mechanism of fusion: these include "the clamp" and the "provocateur" model of activation. By using biochemical and reverse genetics tools, we have obtained strong evidence in favor of the HN stalk-mediated activation of paramyxovirus fusion. Specifically, our data strongly support the notion that the short linker between the head and stalk plays a role in "conformational switching" of the head group to facilitate F-HN interaction and triggering.
Asunto(s)
Proteína HN/metabolismo , Virus de la Enfermedad de Newcastle/fisiología , Virus de la Parainfluenza 5/fisiología , Acoplamiento Viral , Internalización del Virus , Animales , Línea Celular , Análisis Mutacional de ADN , Proteína HN/genética , Humanos , Mutagénesis Sitio-Dirigida , Virus de la Enfermedad de Newcastle/genética , Virus de la Parainfluenza 5/genéticaRESUMEN
Many enveloped RNA viruses recruit host cell proteins during assembly as a mechanism to limit antiviral effects of complement. Using viruses which incorporated CD46 alone, CD55 alone or both CD46 and CD55, we addressed the role of these two host cell regulators in limiting complement-mediated neutralization of Parainfluenza virus 5 (PIV5). PIV5 incorporated functional forms of both CD55 and CD46 into virions. PIV5 containing CD55 was highly resistant to complement-mediated neutralization, whereas CD46-containing PIV5 was as sensitive to neutralization as virus lacking both regulators. PIV5 infected cells had increased levels of cell surface CD55, which was further upregulated by exogenous treatment with tumor necrosis factor alpha. PIV5 derived from cells with higher CD55 levels was more resistant to complement-mediated neutralization in vitro than virus from control cells. We propose a role for virus induction of host cell complement inhibitors in defining virus growth and tissue tropism.
Asunto(s)
Antígenos CD55/genética , Proteínas del Sistema Complemento/inmunología , Regulación de la Expresión Génica , Virus de la Parainfluenza 5/fisiología , Infecciones por Rubulavirus/genética , Infecciones por Rubulavirus/virología , Replicación Viral , Animales , Antígenos CD55/metabolismo , Células CHO , Línea Celular , Activación de Complemento/inmunología , Cricetinae , Cricetulus , Humanos , Proteína Cofactora de Membrana/genética , Proteína Cofactora de Membrana/metabolismo , Pruebas de Neutralización , Infecciones por Rubulavirus/inmunologíaRESUMEN
UNLABELLED: Paramyxovirus particles are formed by a budding process coordinated by viral matrix (M) proteins. M proteins coalesce at sites underlying infected cell membranes and induce other viral components, including viral glycoproteins and viral ribonucleoprotein complexes (vRNPs), to assemble at these locations from which particles bud. M proteins interact with the nucleocapsid (NP or N) components of vRNPs, and these interactions enable production of infectious, genome-containing virions. For the paramyxoviruses parainfluenza virus 5 (PIV5) and mumps virus, M-NP interaction also contributes to efficient production of virus-like particles (VLPs) in transfected cells. A DLD sequence near the C-terminal end of PIV5 NP protein was previously found to be necessary for M-NP interaction and efficient VLP production. Here, we demonstrate that 15-residue-long, DLD-containing sequences derived from either the PIV5 or Nipah virus nucleocapsid protein C-terminal ends are sufficient to direct packaging of a foreign protein, Renilla luciferase, into budding VLPs. Mumps virus NP protein harbors DWD in place of the DLD sequence found in PIV5 NP protein, and consequently, PIV5 NP protein is incompatible with mumps virus M protein. A single amino acid change converting DLD to DWD within PIV5 NP protein induced compatibility between these proteins and allowed efficient production of mumps VLPs. Our data suggest a model in which paramyxoviruses share an overall common strategy for directing M-NP interactions but with important variations contained within DLD-like sequences that play key roles in defining M/NP protein compatibilities. IMPORTANCE: Paramyxoviruses are responsible for a wide range of diseases that affect both humans and animals. Paramyxovirus pathogens include measles virus, mumps virus, human respiratory syncytial virus, and the zoonotic paramyxoviruses Nipah virus and Hendra virus. Infectivity of paramyxovirus particles depends on matrix-nucleocapsid protein interactions which enable efficient packaging of encapsidated viral RNA genomes into budding virions. In this study, we have defined regions near the C-terminal ends of paramyxovirus nucleocapsid proteins that are important for matrix protein interaction and that are sufficient to direct a foreign protein into budding particles. These results advance our basic understanding of paramyxovirus genome packaging interactions and also have implications for the potential use of virus-like particles as protein delivery tools.
Asunto(s)
Secuencias de Aminoácidos , Virus de la Parotiditis/fisiología , Virus Nipah/fisiología , Proteínas de la Nucleocápside/metabolismo , Virus de la Parainfluenza 5/fisiología , Proteínas de la Matriz Viral/metabolismo , Ensamble de Virus , Línea Celular , Humanos , Luciferasas de Renilla/metabolismo , Virus de la Parotiditis/genética , Virus Nipah/genética , Proteínas de la Nucleocápside/química , Proteínas de la Nucleocápside/genética , Virus de la Parainfluenza 5/genética , Unión Proteica , Mapeo de Interacción de Proteínas , Proteínas de la Matriz Viral/química , Virosomas/metabolismo , Liberación del VirusRESUMEN
UNLABELLED: Paramyxoviruses include many important animal and human pathogens. The genome of parainfluenza virus 5 (PIV5), a prototypical paramyxovirus, encodes a V protein that inhibits viral RNA synthesis. In this work, the mechanism of inhibition was investigated. Using mutational analysis and a minigenome system, we identified regions in the N and C termini of the V protein that inhibit viral RNA synthesis: one at the very N terminus of V and the second at the C terminus of V. Furthermore, we determined that residues L16 and I17 are critical for the inhibitory function of the N-terminal region of the V protein. Both regions interact with the nucleocapsid protein (NP), an essential component of the viral RNA genome complex (RNP). Mutations at L16 and I17 abolished the interaction between NP and the N-terminal domain of V. This suggests that the interaction between NP and the N-terminal domain plays a critical role in V inhibition of viral RNA synthesis by the N-terminal domain. Both the N- and C-terminal regions inhibited viral RNA replication. The C terminus inhibited viral RNA transcription, while the N-terminal domain enhanced viral RNA transcription, suggesting that the two domains affect viral RNA through different mechanisms. Interestingly, V also inhibited the synthesis of the RNA of other paramyxoviruses, such as Nipah virus (NiV), human parainfluenza virus 3 (HPIV3), measles virus (MeV), mumps virus (MuV), and respiratory syncytial virus (RSV). This suggests that a common host factor may be involved in the replication of these paramyxoviruses. IMPORTANCE: We identified two regions of the V protein that interact with NP and determined that one of these regions enhances viral RNA transcription via its interaction with NP. Our data suggest that a common host factor may be involved in the regulation of paramyxovirus replication and could be a target for broad antiviral drug development. Understanding the regulation of paramyxovirus replication will enable the rational design of vaccines and potential antiviral drugs.
Asunto(s)
Virus de la Parainfluenza 5/genética , ARN Viral/biosíntesis , Proteínas Virales/metabolismo , Western Blotting , Cartilla de ADN/genética , Células HEK293 , Humanos , Inmunoprecipitación , Microscopía Confocal , Proteínas de la Nucleocápside/metabolismo , Virus de la Parainfluenza 5/fisiología , ARN Viral/antagonistas & inhibidores , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
UNLABELLED: In the extracellular environment, cell-free virions seek out naive host cells over long distances and between organisms. This is the primary mechanism of spread for most viruses. Here we provide evidence for an alternative pathway previously undescribed for orthomyxoviruses, whereby the spread of influenza A virus (IAV) infectious cores to neighboring cells can occur within intercellular connections. The formation of these connections requires actin dynamics and is enhanced by viral infection. Connected cells have contiguous membranes, and the core infectious viral machinery (RNP and polymerase) was present inside the intercellular connections. A live-cell movie of green fluorescent protein (GFP)-tagged NS1 of IAV shows viral protein moving from one cell to another through an intercellular connection. The movement of tagged protein was saltatory but overall traveled only in one direction. Infectious virus cores can move from one cell to another without budding and release of cell-free virions, as evidenced by the finding that whereas a neuraminidase inhibitor alone did not inhibit the development of IAV microplaques, the presence of a neuraminidase inhibitor together with drugs inhibiting actin dynamics or the microtubule stabilizer paclitaxel (originally named taxol) precluded microplaque formation. Similar results were also observed with parainfluenza virus 5 (PIV5), a paramyxovirus, when neutralizing antibody was used to block spread by cell-free virions. Intercellular spread of infectious core particles was unaffected or enhanced in the presence of nocodazole for IAV but inhibited for PIV5. The intercellular connections have a core of filamentous actin, which hints toward transport of virus particles through the use of a myosin motor. IMPORTANCE: Here we describe a new method by which influenza A virus (IAV) spreads from cell to cell: IAV uses intracellular connections. The formation of these connections requires actin dynamics and is enhanced by viral infection and the absence of microtubules. Connected cells appeared to have contiguous membranes, and the core infectious viral machinery (RNP and polymerase) was present inside the intercellular connections. Infectious virus cores can move from one cell to another without budding and release of cell-free virions. Similar results were also observed with parainfluenza virus 5 (PIV5).
Asunto(s)
Virus de la Influenza A/fisiología , Uniones Intercelulares/fisiología , Uniones Intercelulares/virología , Internalización del Virus , Actinas/metabolismo , Animales , Línea Celular , Humanos , Microscopía Fluorescente , Microscopía por Video , Virus de la Parainfluenza 5/fisiologíaRESUMEN
The African Green Monkey (AGM) model was used to analyze the role of complement in neutralization of parainfluenza virus. Parainfluenza virus 5 (PIV5) and human parainfluenza virus type 2 were effectively neutralized in vitro by naïve AGM sera, but neutralizing capacity was lost by heat-inactivation. The mechanism of neutralization involved formation of massive aggregates, with no evidence of virion lysis. Following inoculation of the respiratory tract with a PIV5 vector expressing HIV gp160, AGM produced high levels of serum and tracheal antibodies against gp120 and the viral F and HN proteins. However, in the absence of complement these anti-PIV5 antibodies had very poor neutralizing capacity. Virions showed extensive deposition of IgG and C1q with post- but not pre-immune sera. These results highlight the importance of complement in the initial antibody response to parainfluenza viruses, with implications for understanding infant immune responses and design of vaccine strategies for these pediatric pathogens.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Chlorocebus aethiops , Complemento C1q/inmunología , Virus de la Parainfluenza 2 Humana/inmunología , Virus de la Parainfluenza 5/inmunología , Infecciones por Paramyxoviridae/inmunología , Animales , Chlorocebus aethiops/inmunología , Chlorocebus aethiops/virología , Modelos Animales de Enfermedad , Humanos , Pruebas de Neutralización , Virus de la Parainfluenza 2 Humana/fisiología , Virus de la Parainfluenza 5/fisiología , Infecciones por Paramyxoviridae/virologíaRESUMEN
UNLABELLED: The strain diversity of a rubulavirus, parainfluenza virus 5 (PIV5), was investigated by comparing 11 newly determined and 6 previously published genome sequences. These sequences represent 15 PIV5 strains, of which 6 were isolated from humans, 1 was from monkeys, 2 were from pigs, and 6 were from dogs. Strain diversity is remarkably low, regardless of host, year of isolation, or geographical origin; a total of 7.8% of nucleotides are variable, and the average pairwise difference between strains is 2.1%. Variation is distributed unevenly across the PIV5 genome, but no convincing evidence of selection for antibody-mediated evasion in hemagglutinin-neuraminidase was found. The finding that some canine and porcine, but not primate, strains are mutated in the SH gene, and do not produce SH, raised the possibility that dogs (or pigs) may not be the natural host of PIV5. The genetic stability of PIV5 was also demonstrated during serial passage of one strain (W3) in Vero cells at a high multiplicity of infection, under conditions of competition with large proportions of defective interfering genomes. A similar observation was made for a strain W3 mutant (PIV5VΔC) lacking V gene function, in which the dominant changes were related to pseudoreversion in this gene. The mutations detected in PIV5VΔC during pseudoreversion, and also those characterizing the SH gene in canine and porcine strains, predominantly involved U-to-C transitions. This suggests an important role for biased hypermutation via an adenosine deaminase, RNA-specific (ADAR)-like activity. IMPORTANCE: Here we report the sequence variation of 16 different isolates of parainfluenza virus 5 (PIV5) that were isolated from a number of species, including humans, monkeys, dogs, and pigs, over 4 decades. Surprisingly, strain diversity was remarkably low, regardless of host, year of isolation, or geographical origin. Variation was distributed unevenly across the PIV5 genome, but no convincing evidence of immune or host selection was found. This overall genome stability of PIV5 was also observed when the virus was grown in the laboratory, and the genome stayed remarkably constant even during the selection of virus mutants. Some of the canine isolates had lost their ability to encode one of the viral proteins, termed SH, suggesting that although PIV5 commonly infects dogs, dogs may not be the natural host for PIV5.
Asunto(s)
Variación Genética , Inestabilidad Genómica , Secuenciación de Nucleótidos de Alto Rendimiento , Virus de la Parainfluenza 5/genética , Virus de la Parainfluenza 5/aislamiento & purificación , Infecciones por Rubulavirus/veterinaria , Infecciones por Rubulavirus/virología , Animales , Humanos , Datos de Secuencia Molecular , Virus de la Parainfluenza 5/fisiología , Pase Seriado , Cultivo de VirusRESUMEN
UNLABELLED: Paramyxoviruses are a large family of membrane-enveloped negative-stranded RNA viruses causing important diseases in humans and animals. Two viral integral membrane glycoproteins (fusion [F] and attachment [HN, H, or G]) mediate a concerted process of host receptor recognition, followed by the fusion of viral and cellular membranes, resulting in viral nucleocapsid entry into the cytoplasm. However, the sequence of events that closely links the timing of receptor recognition by HN, H, or G and the "triggering" interaction of the attachment protein with F is unclear. F activation results in F undergoing a series of irreversible conformational rearrangements to bring about membrane merger and virus entry. By extensive study of properties of multiple paramyxovirus HN proteins, we show that key features of F activation, including the F-activating regions of HN proteins, flexibility within this F-activating region, and changes in globular head-stalk interactions are highly conserved. These results, together with functionally active "headless" mumps and Newcastle disease virus HN proteins, provide insights into the F-triggering process. Based on these data and very recently published data for morbillivirus H and henipavirus G proteins, we extend our recently proposed "stalk exposure model" to other paramyxoviruses and propose an "induced fit" hypothesis for F-HN/H/G interactions as conserved core mechanisms of paramyxovirus-mediated membrane fusion. IMPORTANCE: Paramyxoviruses are a large family of membrane-enveloped negative-stranded RNA viruses causing important diseases in humans and animals. Two viral integral membrane glycoproteins (fusion [F] and attachment [HN, H, or G]) mediate a concerted process of host receptor recognition, followed by the fusion of viral and cellular membranes. We describe here the molecular mechanism by which HN activates the F protein such that virus-cell fusion is controlled and occurs at the right time and the right place. We extend our recently proposed "stalk exposure model" first proposed for parainfluenza virus 5 to other paramyxoviruses and propose an "induced fit" hypothesis for F-HN/H/G interactions as conserved core mechanisms of paramyxovirus-mediated membrane fusion.
Asunto(s)
Proteína HN/química , Proteína HN/metabolismo , Enfermedad de Newcastle/virología , Virus de la Enfermedad de Newcastle/fisiología , Virus de la Parainfluenza 5/fisiología , Infecciones por Rubulavirus/virología , Proteínas Virales de Fusión/metabolismo , Internalización del Virus , Secuencia de Aminoácidos , Animales , Pollos , Secuencia Conservada , Proteína HN/genética , Humanos , Datos de Secuencia Molecular , Virus de la Enfermedad de Newcastle/química , Virus de la Enfermedad de Newcastle/genética , Virus de la Parainfluenza 5/química , Virus de la Parainfluenza 5/genética , Estructura Terciaria de Proteína , Proteínas Virales de Fusión/genética , Acoplamiento ViralRESUMEN
Paramyxovirus membrane glycoproteins F (fusion protein) and HN, H, or G (attachment protein) are critical for virus entry, which occurs through fusion of viral and cellular envelopes. The F protein folds into a homotrimeric, metastable prefusion form that can be triggered by the attachment protein to undergo a series of structural rearrangements, ultimately folding into a stable postfusion form. In paramyxovirus-infected cells, the F protein is activated in the Golgi apparatus by cleavage adjacent to a hydrophobic fusion peptide that inserts into the target membrane, eventually bringing the membranes together by F refolding. However, it is not clear how the attachment protein, known as HN in parainfluenza virus 5 (PIV5), interacts with F and triggers F to initiate fusion. To understand the roles of various F protein domains in fusion triggering and metastability, single point mutations were introduced into the PIV5 F protein. By extensive study of F protein cleavage activation, surface expression, and energetics of fusion triggering, we found a role for an immunoglobulin-like (Ig-like) domain, where multiple hydrophobic residues on the PIV5 F protein may mediate F-HN interactions. Additionally, destabilizing mutations of PIV5 F that resulted in HN trigger-independent mutant F proteins were identified in a region along the border of F trimer subunits. The positions of the potential HN-interacting region and the region important for F stability in the lower part of the PIV5 F prefusion structure provide clues to the receptor-binding initiated, HN-mediated F trigger.
Asunto(s)
Virus de la Parainfluenza 5/fisiología , Infecciones por Rubulavirus/virología , Proteínas Virales de Fusión/química , Proteínas Virales de Fusión/metabolismo , Internalización del Virus , Animales , Línea Celular , Proteína HN/química , Proteína HN/genética , Proteína HN/metabolismo , Humanos , Mutación , Virus de la Parainfluenza 5/química , Virus de la Parainfluenza 5/genética , Unión Proteica , Estabilidad Proteica , Estructura Terciaria de Proteína , Proteínas Virales de Fusión/genéticaRESUMEN
A novel porcine parainfluenza 5 (pPIV5), KNU-11, in the genus Rubulavirus of the subfamily Paramyxovirinae, was isolated from the lung of a piglet in Korea in 2011. To understand the importance of this virus as an infectious agent, in vitro and in vivo characteristics of KNU-11 virus was investigated. KNU-11 was remarkably cytopathogenic, showing distinct cell rounding and clumping evident in porcine alveolar macrophage (PAM), porcine kidney (PK-15), and swine testicle (ST) cells within 12h postinfection and capable of hemagglutinating guinea pig red blood cells. Interestingly, this cytopathology was found to be absent in cell lines from other mammalian species. To evaluate the in vitro immunity of the pPIV5 isolate, we sought to explore alteration of inflammatory cytokine and chemokine expression in PAM cells infected with KNU-11 by using quantitative real-time RT-PCR. Most cytokine and chemokine genes including type 1 interferons (IFN-α/ß) and IFN-related antiviral genes were found to be significantly elevated in KNU-11 virus-infected PAM cells. A serum neutralization test-based serosurvey demonstrated that neutralizing antibodies against KNU-11 are readily detected in domestic swine populations, suggesting high prevalence of pPIV5 in Korean pig farms. Animal studies showed that KNU-11 fails to establish an acute respiratory illness, indicating that pPIV5 is non- or very mildly pathogenic to pigs.
Asunto(s)
Virus de la Parainfluenza 5/aislamiento & purificación , Virus de la Parainfluenza 5/fisiología , Infecciones por Rubulavirus/veterinaria , Enfermedades de los Porcinos/virología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Línea Celular , Citocinas/biosíntesis , Citocinas/genética , Efecto Citopatogénico Viral , Perfilación de la Expresión Génica , Cobayas , Hemaglutinación , Pulmón/virología , Pruebas de Neutralización , Virus de la Parainfluenza 5/genética , Virus de la Parainfluenza 5/patogenicidad , Reacción en Cadena en Tiempo Real de la Polimerasa , República de Corea/epidemiología , Infecciones por Rubulavirus/epidemiología , Infecciones por Rubulavirus/virología , Estudios Seroepidemiológicos , Porcinos , Enfermedades de los Porcinos/epidemiologíaRESUMEN
Ubiquitin is important for the budding of many retroviruses and other enveloped viruses, but the precise role of ubiquitin in virus budding remains unclear. Here, we characterized the ubiquitination of the matrix (M) protein of a paramyxovirus, parainfluenza virus 5 (PIV5). The PIV5 M protein (but not the PIV5 nucleocapsid protein) was found to be targeted for monoubiquitination in transfected mammalian cells. Major sites of ubiquitin attachment identified by mass spectrometry analysis were lysine residues at amino acid positions 79/80, 130, and 247. The cumulative mutation of lysine residues 79, 80, and 130 to arginines led to an altered pattern of M protein ubiquitination and impaired viruslike particle (VLP) production. However, the cumulative mutation of lysine residues 79, 80, 130, and 247 to arginines restored M protein ubiquitination and VLP production, suggesting that ubiquitin is attached to alternative sites on the M protein when the primary ones have been removed. Additional lysine residues were targeted for mutagenesis based on the UbiPred algorithm. An M protein with seven lysine residues changed to arginines exhibited altered ubiquitination and poor VLP production. A recombinant virus encoding an M protein with seven lysines mutated was generated, and this virus exhibited a 6-fold-reduced maximum titer, with the defect being attributed mainly to the budding of noninfectious particles. The recombinant virus was assembly deficient, as judged by the redistribution of viral M and hemagglutinin-neuraminidase proteins in infected cells. Similar assembly defects were observed for the wild-type (wt) virus after treatment with a proteasome inhibitor. Collectively, these findings suggest that the monoubiquitination of the PIV5 M protein is important for proper virus assembly and for the budding of infectious particles.
Asunto(s)
Virus de la Parainfluenza 5/fisiología , Infecciones por Rubulavirus/virología , Ubiquitina/metabolismo , Proteínas de la Matriz Viral/metabolismo , Ensamble de Virus , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Línea Celular , Humanos , Datos de Secuencia Molecular , Virus de la Parainfluenza 5/química , Virus de la Parainfluenza 5/genética , Ubiquitina/genética , Ubiquitinación , Proteínas de la Matriz Viral/química , Proteínas de la Matriz Viral/genética , Liberación del VirusRESUMEN
Paramyxovirus matrix (M) proteins organize virus assembly, linking viral glycoproteins and viral ribonucleoproteins together at virus assembly sites on cellular membranes. Using a yeast two-hybrid screening approach, we identified 14-3-3 as a binding partner for the M protein of parainfluenza virus 5 (PIV5). Binding in both transfected and PIV5-infected cells was confirmed by coimmunoprecipitation and was mapped to a C-terminal region within the M protein, namely, 366-KTKSLP-371. This sequence resembles known 14-3-3 binding sites, in which the key residue for binding is a phosphorylated serine residue. Mutation of S369 within the PIV5 M protein disrupted 14-3-3 binding and improved the budding of both virus-like particles (VLPs) and recombinant viruses, suggesting that 14-3-3 binding impairs virus budding. 14-3-3 protein overexpression reduced the budding of VLPs. Using (33)P labeling, phosphorylated M protein was detected in PIV5-infected cells, and this phosphorylation was nearly absent in cells infected with a recombinant virus harboring an S369A mutation within the M protein. Assembly of the M protein into clusters and filaments at infected cell surfaces was enhanced in cells infected with a recombinant virus defective in 14-3-3 binding. These findings support a model in which a portion of M protein within PIV5-infected cells is phosphorylated at residue S369, binds the 14-3-3 protein, and is held away from sites of virus budding.
Asunto(s)
Proteínas 14-3-3/metabolismo , Regulación hacia Abajo , Virus de la Parainfluenza 5/fisiología , Infecciones por Rubulavirus/metabolismo , Proteínas de la Matriz Viral/metabolismo , Virión/fisiología , Ensamble de Virus , Proteínas 14-3-3/genética , Secuencia de Aminoácidos , Línea Celular , Humanos , Datos de Secuencia Molecular , Virus de la Parainfluenza 5/química , Virus de la Parainfluenza 5/genética , Fosforilación , Unión Proteica , Infecciones por Rubulavirus/genética , Infecciones por Rubulavirus/virología , Alineación de Secuencia , Proteínas de la Matriz Viral/química , Proteínas de la Matriz Viral/genética , Virión/química , Virión/genéticaRESUMEN
Enveloped virus particles are formed by budding from infected-cell membranes. For paramyxoviruses, viral matrix (M) proteins are key drivers of virus assembly and budding. However, other paramyxovirus proteins, including glycoproteins, nucleocapsid (NP or N) proteins, and C proteins, are also important for particle formation in some cases. To investigate the role of NP protein in parainfluenza virus 5 (PIV5) particle formation, NP protein truncation and substitution mutants were analyzed. Alterations near the C-terminal end of NP protein completely disrupted its virus-like particle (VLP) production function and significantly impaired M-NP protein interaction. Recombinant viruses with altered NP proteins were generated, and these viruses acquired second-site mutations. Recombinant viruses propagated in Vero cells acquired mutations that mainly affected components of the viral polymerase, while recombinant viruses propagated in MDBK cells acquired mutations that mainly affected the viral M protein. Two of the Vero-propagated viruses acquired the same mutation, V/P(S157F), found previously to be responsible for elevated viral gene expression induced by a well-characterized variant of PIV5, P/V-CPI(-). Vero-propagated viruses caused elevated viral protein synthesis and spread rapidly through infected monolayers by direct cell-cell fusion, bypassing the need to bud infectious virions. Both Vero- and MDBK-propagated viruses exhibited infectivity defects and altered polypeptide composition, consistent with poor incorporation of viral ribonucleoprotein complexes (RNPs) into budding virions. Second-site mutations affecting M protein restored interaction with altered NP proteins in some cases and improved VLP production. These results suggest that multiple avenues are available to paramyxoviruses for overcoming defects in M-NP protein interaction.
Asunto(s)
Proteínas de la Cápside/metabolismo , Virus de la Parainfluenza 5/fisiología , Infecciones por Rubulavirus/metabolismo , Proteínas de la Matriz Viral/metabolismo , Virión/fisiología , Ensamble de Virus , Secuencia de Aminoácidos , Animales , Proteínas de la Cápside/genética , Bovinos , Chlorocebus aethiops , Genoma Viral , Células Gigantes/fisiología , Humanos , Riñón/citología , Riñón/metabolismo , Riñón/virología , Datos de Secuencia Molecular , Mutación/genética , Infecciones por Rubulavirus/genética , Infecciones por Rubulavirus/virología , Células Vero , Proteínas de la Matriz Viral/genéticaRESUMEN
The PIV-5 hemagglutinin-neuraminidase (HN) protein is a multifunctional protein with sialic acid binding, neuraminidase and fusion promotion activity. HN is internalized by clathrin-mediated endocytosis and degraded. HN lacks internalization signals in its cytoplasmic tail but a single glutamic acid present at residue 37 at the putative transmembrane/ectodomain boundary is critical. We rescued rPIV-5 with mutations E37D or E37K, which have been shown to impair or abolish HN internalization, respectively. These viruses exhibited growth properties similar to wild-type (wt) virus but are impaired for fitness in tissue culture. Biochemical analysis of HN activities showed differences between HN E37D and HN E37K in fusion promotion and incorporation of HN and F into virions. Furthermore, oligomeric analyses indicate that HN E37 mutants perturb the tetrameric organization of HN, probably by destabilizing the dimer-of-dimers interface.
Asunto(s)
Endocitosis , Proteína HN/genética , Proteína HN/metabolismo , Mutación Missense , Virus de la Parainfluenza 5/fisiología , Internalización del Virus , Animales , Línea Celular , Chlorocebus aethiops , Dimerización , Perros , Proteína HN/química , Virus de la Parainfluenza 5/química , Virus de la Parainfluenza 5/genética , Células VeroRESUMEN
Caveolin 1 (Cav-1) is an integral membrane protein that forms the coat structure of plasma membrane caveolae and regulates caveola-dependent functions. Caveolae are enriched in cholesterol and sphingolipids and are related to lipid rafts. Many studies implicate rafts as sites of assembly and budding of enveloped virus. We show that Cav-1 colocalizes with the paramyxovirus parainfluenza virus 5 (PIV-5) nucleocapsid (NP), matrix (M), and hemagglutinin-neuraminidase (HN) proteins. Moreover, electron microscopy shows that Cav-1 is clustered at sites of viral budding. HN, M, and F(1)/F(2) are associated with detergent-resistant membranes, and these proteins float on sucrose gradients with Cav-1-rich fractions. A complex containing Cav-1 with M, NP, and HN from virus-infected cells and a complex containing Cav-1 and M from M-transfected cells were found on coimmunoprecipitation. A role of Cav-1 in the PIV-5 life cycle was investigated by utilizing MCF-7 human breast cancer cells that stably express Cav-1 (MCF-7/Cav-1). PIV-5 entry into MCF-7 and MCF-7/Cav-1 was found to be Cav-1 independent. However, the interaction between HN and M proteins was dramatically reduced in the Cav-1 null MCF-7 cells, and PIV-5 grown in MCF-7 cells had a reduced infectivity. Similarly, when PIV-5 was grown in MDCK cells that stably expressed dominant negative Cav-1 (MDCK/P132LCav-1), the virus showed a reduced infectivity. Virions lacking Cav-1 were defective and contained high levels of host cellular proteins and reduced levels of HN and M. These data suggest that Cav-1 affects assembly and/or budding, and this is supported by the finding that Cav-1 is incorporated into mature viral particles.
Asunto(s)
Caveolina 1/fisiología , Virus de la Parainfluenza 5/fisiología , Secuencia de Aminoácidos , Animales , Caveolina 1/genética , Línea Celular , Perros , Proteína HN/fisiología , Células HeLa , Interacciones Huésped-Patógeno/fisiología , Humanos , Microdominios de Membrana/virología , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Virus de la Parainfluenza 5/genética , Dominios y Motivos de Interacción de Proteínas , Homología de Secuencia de Aminoácido , Proteínas Virales de Fusión/fisiología , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/fisiología , Ensamble de Virus/fisiología , Liberación del Virus/fisiologíaRESUMEN
Although the replication cycle of parainfluenza virus type 5 (PIV5) is initially severely impaired in cells in an interferon (IFN)-induced antiviral state, the virus still targets STAT1 for degradation. As a consequence, the cells can no longer respond to IFN and after 24-48 h, they go out of the antiviral state and normal virus replication is established. Following infection of cells in an IFN-induced antiviral state, viral nucleocapsid proteins are initially localized within small cytoplasmic bodies, and appearance of these cytoplasmic bodies correlates with the loss of STAT1 from infected cells. In situ hybridization, using probes specific for the NP and L genes, demonstrated the presence of virus genomes within these cytoplasmic bodies. These viral cytoplasmic bodies do not co-localize with cellular markers for stress granules, cytoplasmic P-bodies or autophagosomes. Furthermore, they are not large insoluble aggregates of viral proteins and/or nucleocapsids, as they can simply and easily be dispersed by 'cold-shocking' live cells, a process that disrupts the cytoskeleton. Given that during in vivo infections, PIV5 will inevitably infect cells in an IFN-induced antiviral state, we suggest that these cytoplasmic bodies are areas in which PIV5 genomes reside whilst the virus dismantles the antiviral state of the cells. Consequently, viral cytoplasmic bodies may play an important part in the strategy that PIV5 uses to circumvent the IFN system.
Asunto(s)
Citoplasma/inmunología , Genoma Viral , Cuerpos de Inclusión Viral/inmunología , Interferones/inmunología , Virus de la Parainfluenza 5/genética , Infecciones por Rubulavirus/inmunología , Animales , Línea Celular , Chlorocebus aethiops , Citoplasma/genética , Citoplasma/virología , Humanos , Cuerpos de Inclusión Viral/genética , Interferones/genética , Virus de la Parainfluenza 5/inmunología , Virus de la Parainfluenza 5/fisiología , Infecciones por Rubulavirus/genética , Infecciones por Rubulavirus/virología , Células Vero , Replicación ViralRESUMEN
P/V gene substitutions convert the non-cytopathic paramyxovirus Simian Virus 5 (SV5), which is a poor inducer of host cell responses in human tissue culture cells, into a mutant (P/V-CPI-) that induces high levels of apoptosis, interferon (IFN)-beta, and proinflammatory cytokines. However, the effect of SV5-P/V gene mutations on virus growth and adaptive immune responses in animals has not been determined. Here, we used two distinct animal model systems to test the hypothesis that SV5-P/V mutants which are more potent activators of innate responses in tissue culture will also elicit higher antiviral antibody responses. In mouse cells, in vitro studies identified a panel of SV5-P/V mutants that ranged in their ability to limit IFN responses. Intranasal infection of mice with these WT and P/V mutant viruses elicited equivalent anti-SV5 IgG responses at all doses tested, and viral titers recovered from the respiratory tract were indistinguishable. In primary cultures of ferret lung fibroblasts, WT rSV5 and P/V-CPI- viruses had phenotypes similar to those established in human cell lines, including differential induction of IFN secretion, IFN signaling and apoptosis. Intranasal infection of ferrets with a low dose of WT rSV5 elicited approximately 500 fold higher anti-SV5 serum IgG responses compared to the P/V-CPI- mutant, and this correlated with overall higher viral titers for the WT virus in tracheal tissues. There was a dose-dependent increase in antibody response to infection of ferrets with P/V-CPI-, but not with WT rSV5. Together our data indicate that WT rSV5 and P/V mutants can elicit distinct innate and adaptive immunity phenotypes in the ferret animal model system, but not in the mouse system. We present a model for the effect of P/V gene substitutions on SV5 growth and immune responses in vivo.
Asunto(s)
Anticuerpos Antivirales/sangre , Virus de la Parainfluenza 5/fisiología , Fosfoproteínas/genética , Infecciones por Rubulavirus/sangre , Infecciones por Rubulavirus/virología , Proteínas Virales/genética , Proteínas Estructurales Virales/genética , Animales , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Hurones , Fibroblastos/inmunología , Fibroblastos/metabolismo , Fibroblastos/virología , Inmunoglobulina G/sangre , Interferones/biosíntesis , Pulmón/virología , Ratones , Ratones Endogámicos BALB C , Pruebas de Neutralización , Nariz/virología , Mutación Puntual , Proteínas de Unión al ARN , Infecciones por Rubulavirus/metabolismoRESUMEN
The order Mononegavirales (comprised of nonsegmented negative-stranded RNA viruses or NNSVs) contains many important pathogens. Parainfluenza virus 5 (PIV5), formerly known as simian virus 5, is a prototypical paramyxovirus and encodes a V protein, which has a cysteine-rich C terminus that is conserved among all paramyxoviruses. The V protein of PIV5, like that of many other paramyxoviruses, plays an important role in regulating viral RNA synthesis. In this work, we show that V interacts with Akt, a serine/threonine kinase, also known as protein kinase B. Both pharmacological inhibitors and small interfering RNA against Akt1 reduced PIV5 replication, indicating that Akt plays a critical role in PIV5 replication. Furthermore, treatment with Akt inhibitors also reduced the replication of several other paramyxoviruses, as well as vesicular stomatitis virus, the prototypical rhabdovirus, indicating that Akt may play a more universal role in NNSV replication. The phosphoproteins (P proteins) of NNSVs are essential cofactors for the viral RNA polymerase complex and require heavy phosphorylation for their activity. Inhibition of Akt activity reduced the level of P phosphorylation, suggesting that Akt is involved in regulating viral RNA synthesis. In addition, Akt1 phosphorylated a recombinant P protein of PIV5 purified from bacteria. The finding that Akt plays a critical role in replication of NNSV will lead to a better understanding of how these viruses replicate, as well as novel strategies to treat infectious diseases caused by NNSVs.