scholarly journals Trypsin increases pseudorabies virus production through activation of the ERK signalling pathway

2006 ◽  
Vol 87 (5) ◽  
pp. 1109-1112 ◽  
Author(s):  
Béatrice Riteau ◽  
Christiane de Vaureix ◽  
François Lefèvre
Keyword(s):  
Pseudorabies Virus ◽  
Virus Production ◽  
Signalling Pathway ◽  
Herpesvirus Infection ◽  
Extracellular Proteases ◽  
Extracellular Signal ◽  
Inflammatory Processes ◽  
Infected Cells

Extracellular proteases that are expressed in primary and secondary foci of viral infection are potentially important mediators of infectious inflammatory processes. For some viruses, such as influenza virus and rotaviruses, proteases such as trypsin enhance infectivity by a direct proteolytic effect on some virion proteins. By using an in vitro model of herpesvirus infection, the possibility that proteases modulate the viral cycle through signalling delivered to the infected cell was investigated. It is reported that exposure of pseudorabies virus-infected cells to trypsin increased virus production. Moreover, this treatment induced synergistic and sustained activation of the extracellular signal-regulated kinase (ERK) 1/2 signalling pathway, which appeared to be necessary for this increased viral production. These results suggest that herpesviruses could take advantage of the inflammatory context and particularly of the presence of proteases to increase their replication. Thus, these data point to a potentially important role of extracellular proteases in herpesvirus infection.

scholarly journals A CYTOCHEMICAL DESCRIPTION OF THE MULTIPLICATION OF MENGOVIRUS IN L-929 CELLS

1962 ◽  
Vol 12 (1) ◽  
pp. 1-15 ◽  
Author(s):  
Richard M. Franklin
Keyword(s):  
Phase Contrast ◽  
Basic Protein ◽  
Virus Production ◽  
Cytological Change ◽  
Virus Particles ◽  
Contrast Microscopy ◽  
Infected Cells ◽  
Cytological Changes ◽  
Perinuclear Area

A correlation of cytochemical changes with virus production has been studied in L cells infected with Mengovirus. After a latent period of about 2 hours, virus was produced rapidly, reaching maximum titers of up to 12,000 particles per cell in 6 to 8 hours. The earliest cytological change was in the nucleus and consisted of a slight condensation of chromatin. There is no evidence, however, for the multiplication of either the viral RNA or protein in the nucleus. RNA, of high molecular weight, accumulated in the perinuclear area of the cytoplasm and was later found in inclusions. The perinuclear RNA was digestible with RNase and may be located in or on ribosomes. The inclusion RNA was resistant to RNase but could be removed by pepsin or potassium permanganate; it is probably in completed virus particles. Viral antigen was first observed in a perinuclear location and later in the above-mentioned inclusions. Although the viral protein contains appreciable amounts of arginine and lysine, it is not a basic protein of the histone type. Phase-contrast microscopy of living cells clearly demonstrated the role of the inclusions in release of virus from infected cells. A comparison is made between these cytological changes in Mengo-infected cells and those which have been found by other workers in polio-infected cells. There are many very similar changes.

scholarly journals Functional analysis of the putative antiapoptotic genes, p49 and iap4, of Spodoptera litura nucleopolyhedrovirus with RNAi

2008 ◽  
Vol 89 (8) ◽  
pp. 1873-1880 ◽  
Author(s):  
Qian Yu ◽  
Tiehao Lin ◽  
Guozhong Feng ◽  
Kai Yang ◽  
Yi Pang
Keyword(s):  
Spodoptera Litura ◽  
Virus Production ◽  
Host Cells ◽  
Homology Search ◽  
Pcr Analysis ◽  
Viability Analysis ◽  
Infected Cells ◽  
Budded Virus ◽  
Almost All

A homology search of a public database revealed that Spodoptera litura nucleopolyhedrovirus (SpltNPV) possesses two putative, antiapoptotic genes, p49 and inhibitor of apoptosis 4 (iap4), but their function has not been investigated in its native host cells. In the present study, we used RNA interference (RNAi) to silence the expression of Splt-iap4 and Splt-p49, independently or together, to determine their roles during the SpltNPV life cycle. RT-PCR analysis and Western blot analysis showed the target gene expression had been knocked out in the SpltNPV-infected SpLi-221 cells after treatment with Splt-p49 or Splt-iap4 double-stranded RNA (dsRNA), respectively, confirming that the two genes were effectively silenced. In SpltNPV-infected cells treated with Splt-p49 dsRNA, apoptosis was observed beginning at 14 h, and almost all cells had undergone apoptosis by 48 h. In contrast, budded virus production and polyhedra formation progressed normally in infected cells treated with Splt-iap4 dsRNA. Cell viability analysis showed that Splt-IAP4 had no synergistic effect on the inhibition of apoptosis of SpLi-221 cells induced by SpltNPV infection. Interestingly, after Splt-iap4 dsRNA treatment, cells did not congregate like those infected with SpltNPV in the early infection phase, implying an unknown role of baculovirus iap4. Our results determine that Splt-p49 is necessary to prevent apoptosis; however, Splt-iap4 has no antiapoptotic function during SpltNPV infection.

scholarly journals Transcriptional role of p53 in interferon-mediated antiviral immunity

2008 ◽  
Vol 205 (8) ◽  
pp. 1929-1938 ◽  
Author(s):  
César Muñoz-Fontela ◽  
Salvador Macip ◽  
Luis Martínez-Sobrido ◽  
Lauren Brown ◽  
Joseph Ashour ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Viral Replication ◽  
Viral Infections ◽  
Suppressor Gene ◽  
Central Component ◽  
Type I ◽  
Infected Cells

Tumor suppressor p53 is activated by several stimuli, including DNA damage and oncogenic stress. Previous studies (Takaoka, A., S. Hayakawa, H. Yanai, D. Stoiber, H. Negishi, H. Kikuchi, S. Sasaki, K. Imai, T. Shibue, K. Honda, and T. Taniguchi. 2003. Nature. 424:516–523) have shown that p53 is also induced in response to viral infections as a downstream transcriptional target of type I interferon (IFN) signaling. Moreover, many viruses, including SV40, human papillomavirus, Kaposi's sarcoma herpesvirus, adenoviruses, and even RNA viruses such as polioviruses, have evolved mechanisms designated to abrogate p53 responses. We describe a novel p53 function in the activation of the IFN pathway. We observed that infected mouse and human cells with functional p53 exhibited markedly decreased viral replication early after infection. This early inhibition of viral replication was mediated both in vitro and in vivo by a p53-dependent enhancement of IFN signaling, specifically the induction of genes containing IFN-stimulated response elements. Of note, p53 also contributed to an increase in IFN release from infected cells. We established that this p53-dependent enhancement of IFN signaling is dependent to a great extent on the ability of p53 to activate the transcription of IFN regulatory factor 9, a central component of the IFN-stimulated gene factor 3 complex. Our results demonstrate that p53 contributes to innate immunity by enhancing IFN-dependent antiviral activity independent of its functions as a proapoptotic and tumor suppressor gene.

scholarly journals Functional Role of N-Linked Glycosylation in Pseudorabies Virus Glycoprotein gH

2018 ◽  
Vol 92 (9) ◽  
pp. e00084-18 ◽  
Author(s):  
Melina Vallbracht ◽  
Sascha Rehwaldt ◽  
Barbara G. Klupp ◽  
Thomas C. Mettenleiter ◽  
Walter Fuchs
Keyword(s):  
Pseudorabies Virus ◽  
Viral Envelope ◽  
Fusion Activity ◽  
Virus Particles ◽  
Glycosylation Sites ◽  
Functional Relevance ◽  
And Function ◽  
Cell Spread

ABSTRACTMany viral envelope proteins are modified by asparagine (N)-linked glycosylation, which can influence their structure, physicochemical properties, intracellular transport, and function. Here, we systematically analyzed the functional relevance of N-linked glycans in the alphaherpesvirus pseudorabies virus (PrV) glycoprotein H (gH), which is an essential component of the conserved core herpesvirus fusion machinery. Upon gD-mediated receptor binding, the heterodimeric complex of gH and gL activates gB to mediate fusion of the viral envelope with the host cell membrane for viral entry. gH contains five potential N-linked glycosylation sites at positions 77, 162, 542, 604, and 627, which were inactivated by conservative mutations (asparagine to glutamine) singly or in combination. The mutated proteins were tested for correct expression and fusion activity. Additionally, the mutated gH genes were inserted into the PrV genome for analysis of function during virus infection. Our results demonstrate that all five sites are glycosylated. Inactivation of the PrV-specific N77 or the conserved N627 resulted in significantly reducedin vitrofusion activity, delayed penetration kinetics, and smaller virus plaques. Moreover, substitution of N627 greatly affected transport of gH in transfected cells, resulting in endoplasmic reticulum (ER) retention and reduced surface expression. In contrast, mutation of N604, which is conserved in theVaricellovirusgenus, resulted in enhancedin vitrofusion activity and viral cell-to-cell spread. These results demonstrate a role of the N-glycans in proper localization and function of PrV gH. However, even simultaneous inactivation of all five N-glycosylation sites of gH did not severely inhibit formation of infectious virus particles.IMPORTANCEHerpesvirus infection requires fusion of the viral envelope with cellular membranes, which involves the conserved fusion machinery consisting of gB and the heterodimeric gH/gL complex. The bona fide fusion protein gB depends on the presence of the gH/gL complex for activation. Viral envelope glycoproteins, such as gH, usually contain N-glycans, which can have a strong impact on their folding, transport, and functions. Here, we systematically analyzed the functional relevance of all five predicted N-linked glycosylation sites in the alphaherpesvirus pseudorabies virus (PrV) gH. Despite the fact that mutation of specific sites affected gH transport,in vitrofusion activity, and cell-to-cell spread and resulted in delayed penetration kinetics, even simultaneous inactivation of all five N-glycosylation sites of gH did not severely inhibit formation of infectious virus particles. Thus, our results demonstrate a modulatory but nonessential role of N-glycans for gH function.

Plasminogen: an enigmatic zymogen

Blood ◽  
2021 ◽  
Author(s):  
Charithani B Keragala ◽  
Robert L Medcalf
Keyword(s):  
Animal Studies ◽  
Wound Repair ◽  
Active Form ◽  
Cell Behaviour ◽  
Surface Receptors ◽  
Enzymatic Cascades ◽  
Inflammatory Processes ◽  
Over 40 Years

Plasminogen is an abundant plasma protein that exists in various zymogenic forms. Plasmin, the proteolytically active form of plasminogen, is known for its essential role in fibrinolysis. The therapeutic targeting of the fibrinolytic system to date has been for two purposes: to promote plasmin generation for thromboembolic conditions, or to stop plasmin to reduce bleeding. However, both plasmin and plasminogen serve other important functions, some of which are unrelated to fibrin removal. Indeed, for over 40 years, the anti-fibrinolytic agent, tranexamic acid, has been administered for its serendipitously discovered skin whitening properties. Plasmin also plays an important role in the removal of misfolded/aggregated proteins and can trigger other enzymatic cascades including complement. In addition, plasminogen, via binding to one of its dozen cell-surface receptors, can modulate cell behaviour and further influence immune and inflammatory processes. Plasminogen administration itself has been reported to improve thrombolysis and to accelerate wound repair. While many of these more recent findings have been derived from in vitro or animal studies, the use of anti-fibrinolytics to reduce bleeding in humans has revealed additional clinically relevant consequences, particularly in relation to reducing infection risk that is independent of its haemostatic effects. The finding that many viruses harness the host plasminogen to aid infectivity has suggested that anti-fibrinolytic agents may have anti-viral benefits. Here we review the broadening role of the plasminogen activating system in physiology and pathophysiology and how manipulation of this system may be harnessed for benefits unrelated to its conventional application in thrombosis and haemostasis.

Advances in microglia cellular models: focus on extracellular vesicle production

2021 ◽  
Author(s):  
Lorenzo Ceccarelli ◽  
Laura Marchetti ◽  
Chiara Giacomelli ◽  
Claudia Martini
Keyword(s):  
Cell Communication ◽  
Extracellular Vesicle ◽  
Cellular Models ◽  
Advantages And Disadvantages ◽  
Inflammatory Processes ◽  
The Central Nervous System ◽  
Harmful Effects ◽  
Brain Homeostasis

Microglia are the major component of the innate immune system in the central nervous system. They promote the maintenance of brain homeostasis as well as support inflammatory processes that are often related to pathological conditions such as neurodegenerative diseases. Depending on the stimulus received, microglia cells dynamically change their phenotype releasing specific soluble factors and largely modify the cargo of their secreted extracellular vesicles (EVs). Despite the mechanisms at the basis of microglia actions have not been completely clarified, the recognized functions exerted by their EVs in patho-physiological conditions represent the proof of the crucial role of these organelles in tuning cell-to-cell communication, promoting either protective or harmful effects. Consistently, in vitro cell models to better elucidate microglia EV production and mechanisms of their release have been increased in the last years. In this review, the main microglial cellular models that have been developed and validated will be described and discussed, with particular focus on those used to produce and derive EVs. The advantages and disadvantages of their use will be evidenced too. Finally, given the wide interest in applying EVs in diagnosis and therapy too, the heterogeneity of available models for producing microglia EVs is here underlined, to prompt a cross-check or comparison among them.

scholarly journals In Vitro Action of Meconium on Bronochomotor Tonus of Newborns with Meconium Aspiration Syndrome

2018 ◽  
Vol 6 (6) ◽  
pp. 992-996 ◽  
Author(s):  
Arsim Haliti ◽  
Lirim Mustafa ◽  
Sadi Bexheti ◽  
Drita Islami ◽  
Adnan Bozalija ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Respiratory System ◽  
Meconium Aspiration Syndrome ◽  
Control Group ◽  
Meconium Aspiration ◽  
Smooth Musculature ◽  
Inflammatory Processes ◽  
Tracheobronchial System

AIM: Here we studied the role of meconium in the respiratory system on live and exited newborns (weight 250-3000 g). Throughout this study is followed the response of tracheal rings in acetylcholine and histamine in different molar concentrations (10-1, 10-2, 10-3, 10-4 mol/dm3).METHODS: To study the smooth tracheal musculature we used 23 tracheal preparations obtained from the newborns exited from meconium aspiration.RESULTS: Based on the functional analysis of the tracheal specimen we have concluded that the meconium aspiration did not change the smooth musculature response on acetylcholine and histamine when compared to control group, exited from lung inflammatory processes (e.g., pneumonia, bronchopneumonia, atelectasis, cerebral hemorrhage), where tracheal smooth musculature response is significant (P for other causes is not significant (P > 0.01).CONCLUSION: The conclusions suggest that meconium did not potentiate the constrictor action of acetylcholine and histamine in the tracheobronchial system and did not cause modulation of bronchomotor tonus in case of his aspiration. Meconium causes mild relaxation of smooth tracheal musculature with a mechanism which is not mediated by cyclooxygenase products, from tracheal epithelium or proteins. Also, direct activity in the smooth musculature of several tested acids seems to have no significant impact in increasing the tonus of respiratory airway of smooth tracheal musculature.

scholarly journals An Unbiased Approach to Mapping the Signaling Network of the Pseudorabies Virus US3 Protein

Pathogens ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 916
Author(s):  
Robert J. J. Jansens ◽  
Sandra Marmiroli ◽  
Herman W. Favoreel
Keyword(s):  
Gene Expression ◽  
Protein Kinase ◽  
Pseudorabies Virus ◽  
Signaling Network ◽  
Cellular Proteins ◽  
Future Research ◽  
Nuclear Egress ◽  
Infected Cells ◽  
Important Virulence Factor

The US3 serine/threonine protein kinase is conserved among the alphaherpesvirus family and represents an important virulence factor. US3 plays a role in viral nuclear egress, induces dramatic alterations of the cytoskeleton, represses apoptosis, enhances gene expression and modulates the immune response. Although several substrates of US3 have been identified, an unbiased screen to identify US3 phosphorylation targets has not yet been described. Here, we perform a shotgun and phosphoproteomics analysis of cells expressing the US3 protein of pseudorabies virus (PRV) to identify US3 phosphorylation targets in an unbiased way. We identified several cellular proteins that are differentially phosphorylated upon US3 expression and validated the phosphorylation of lamin A/C at serine 404, both in US3-transfected and PRV-infected cells. These results provide new insights into the signaling network of the US3 protein kinase and may serve as a basis for future research into the role of the US3 protein in the viral replication cycle.

scholarly journals Bombyx mori nucleopolyhedrovirus ORF56 encodes an occlusion-derived virus protein and is not essential for budded virus production

2008 ◽  
Vol 89 (5) ◽  
pp. 1212-1219 ◽  
Author(s):  
Hai-Jun Xu ◽  
Zhang-Nv Yang ◽  
Jin-Fang Zhao ◽  
Cai-Hong Tian ◽  
Jun-Qing Ge ◽  
...  
Keyword(s):  
Bombyx Mori ◽  
Cultured Cells ◽  
Virus Production ◽  
Core Gene ◽  
Virus Protein ◽  
Infected Cells ◽  
Budded Virus ◽  
Larval Bioassay

Bombyx mori nucleopolyhedrovirus ORF56 (Bm56) is a baculovirus core gene that is highly conserved in all baculoviruses that have had their genomes sequenced to date. Its transcripts in BmNPV-infected cells could be detected from 12 h post-infection (p.i.) and the encoded protein could be detected at 16 h p.i. by using a polyclonal antibody against glutathione S-transferase–Bm56 fusion protein. Western blot analysis showed that Bm56 is a structural component of the occlusion-derived virus nucleocapsid. Subsequent confocal microscopy revealed that Bm56 was distributed in the outer nuclear membrane and the intranuclear region of infected cells. To investigate the role of Bm56 in virus replication, a Bm56-knockout bacmid of BmNPV was constructed via homologous recombination in Escherichia coli. The Bm56 deletion had no effect on budded virus (BV) production in cultured cells; however, the deletion affected occlusion-body morphogenesis. A larval bioassay demonstrated that the Bm56 deletion did not reduce infectivity, whereas it resulted in a 50 % lethal time that was 16–18 h longer than that of the wild-type bacmid at every dose used in this study. These results indicate that Bm56 facilitates efficient virus production in vivo; however, it is not essential for BV production in vitro.

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