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 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 Tilapia Lake Virus Does Not Hemagglutinate Avian and Piscine Erythrocytes and NH4Cl Does Not Inhibit Viral Replication In Vitro

Viruses ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 1152 ◽  
Author(s):  
Augustino Alfred Chengula ◽  
Stephen Mutoloki ◽  
Øystein Evensen ◽  
Hetron Mweemba Munang’andu
Keyword(s):  
Atlantic Salmon ◽  
Influenza A ◽  
Meleagris Gallopavo ◽  
Host Cells ◽  
Pcr Analysis ◽  
Infectious Salmon Anemia ◽  
Influenza C Virus ◽  
Infected Cells ◽  
Anemia Virus

Tilapia lake virus (TiLV) is a negative-sense single-stranded RNA (-ssRNA) icosahedral virus classified to be the only member in the family Amnoonviridae. Although TiLV segment-1 shares homology with the influenza C virus PB1 and has four conserved motifs similar to influenza A, B, and C polymerases, it is unknown whether there are other properties shared between TiLV and orthomyxovirus. In the present study, we wanted to determine whether TiLV agglutinated avian and piscine erythrocytes, and whether its replication was inhibited by lysosomotropic agents, such as ammonium chloride (NH4Cl), as seen for orthomyxoviruses. Our findings showed that influenza virus strain A/Puerto Rico/8 (PR8) was able to hemagglutinate turkey (Meleagris gallopavo), Atlantic salmon (Salmo salar L), and Nile tilapia (Oreochromis niloticus) red blood cells (RBCs), while infectious salmon anemia virus (ISAV) only agglutinated Atlantic salmon, but not turkey or tilapia, RBCs. In contrast to PR8 and ISAV, TiLV did not agglutinate turkey, Atlantic salmon, or tilapia RBCs. qRT-PCR analysis showed that 30 mM NH4Cl, a basic lysosomotropic agent, neither inhibited nor enhanced TiLV replication in E-11 cells. There was no difference in viral quantities in the infected cells with or without NH4Cl treatment during virus adsorption or at 1, 2, and 3 h post-infection. Given that hemagglutinin proteins that bind RBCs also serve as ligands that bind host cells during virus entry leading to endocytosis in orthomyxoviruses, the data presented here suggest that TiLV may use mechanisms that are different from orthomyxoviruses for entry and replication in host cells. Therefore, future studies should seek to elucidate the mechanisms used by TiLV for entry into host cells and to determine its mode of replication in infected cells.

scholarly journals Hsp70 Negatively Controls Rotavirus Protein Bioavailability in Caco-2 Cells Infected by the Rotavirus RF Strain

2006 ◽  
Vol 81 (3) ◽  
pp. 1297-1304 ◽  
Author(s):  
Alexis H. Broquet ◽  
Christelle Lenoir ◽  
Agnès Gardet ◽  
Catherine Sapin ◽  
Serge Chwetzoff ◽  
...  
Keyword(s):  
Viral Infection ◽  
Proteasome Inhibitors ◽  
Virus Production ◽  
Viral Proteins ◽  
Host Cells ◽  
Progeny Virus ◽  
Virus Protein ◽  
Interfering Rna ◽  
Hsp70 Induction

ABSTRACT Previous studies demonstrated that the induction of the heat shock protein Hsp70 in response to viral infection is highly specific and differs from one cell to another and for a given virus type. However, no clear consensus exists so far to explain the likely reasons for Hsp70 induction within host cells during viral infection. We show here that upon rotavirus infection of intestinal cells, Hsp70 is indeed rapidly, specifically, and transiently induced. Using small interfering RNA-Hsp70-transfected Caco-2 cells, we observed that Hsp70 silencing was associated with an increased virus protein level and enhanced progeny virus production. Upon Hsp70 silencing, we observed that the ubiquitination of the main rotavirus structural proteins was strongly reduced. In addition, the use of proteasome inhibitors in infected Caco-2 cells was shown to induce an accumulation of structural viral proteins. Together, these results are consistent with a role of Hsp70 in the control of the bioavailability of viral proteins within cells for virus morphogenesis.

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.

scholarly journals Role of Bcl-2 Family Members in Caspase-Independent Apoptosis during Chlamydia Infection

2002 ◽  
Vol 70 (1) ◽  
pp. 55-61 ◽  
Author(s):  
Jean-Luc Perfettini ◽  
John C. Reed ◽  
Nicole Israël ◽  
Jean-Claude Martinou ◽  
Alice Dautry-Varsat ◽  
...  
Keyword(s):  
Caspase 3 ◽  
Chlamydia Psittaci ◽  
Host Cells ◽  
Chlamydia Infection ◽  
Caspase Inhibitor ◽  
Obligate Intracellular Bacterium ◽  
Obligate Intracellular ◽  
Infected Cells ◽  
Induced Apoptosis

ABSTRACT Infection with an obligate intracellular bacterium, the Chlamydia trachomatis lymphogranuloma venereum (LGV/L2) strain or the guinea pig inclusion conjunctivitis serovar of Chlamydia psittaci, leads to apoptosis of host cells. The apoptosis is not affected by a broad-spectrum caspase inhibitor, and caspase-3 is not activated in infected cells, suggesting that apoptosis mediated by these two strains of Chlamydia is independent of known caspases. Overexpression of the proapoptotic Bcl-2 family member, Bax, was previously shown to induce caspase-independent apoptosis, and we find that Bax is activated and translocates from the cytosol to the mitochondria in C. psittaci-infected cells. C. psittaci-induced apoptosis is inhibited in host cells overexpressing Bax inhibitor-1 and is inhibited through overexpression of Bcl-2, which blocks both caspase-dependent and -independent apoptosis. As Bax and mitochondria are ideally located to sense stress-related metabolic changes emanating from the interior of an infected cell, it is likely that Bax-dependent apoptosis may also be observed in cells infected with other intracellular pathogens.

scholarly journals Trichoplusia ni Kinesin-1 Associates with Autographa californica Multiple Nucleopolyhedrovirus Nucleocapsid Proteins and Is Required for Production of Budded Virus

2016 ◽  
Vol 90 (7) ◽  
pp. 3480-3495 ◽  
Author(s):  
Siddhartha Biswas ◽  
Gary W. Blissard ◽  
David A. Theilmann
Keyword(s):  
Plasma Membrane ◽  
Trichoplusia Ni ◽  
Autographa Californica ◽  
Nucleocapsid Proteins ◽  
Content Type ◽  
Multiple Nucleopolyhedrovirus ◽  
Microtubule Transport ◽  
Infected Cells ◽  
Budded Virus

ABSTRACTThe mechanism by which nucleocapsids ofAutographa californicamultiple nucleopolyhedrovirus (AcMNPV) egress from the nucleus to the plasma membrane, leading to the formation of budded virus (BV), is not known. AC141 is a nucleocapsid-associated protein required for BV egress and has previously been shown to be associated with β-tubulin. In addition, AC141 and VP39 were previously shown by fluorescence resonance energy transfer by fluorescence lifetime imaging to interact directly with theDrosophila melanogasterkinesin-1 light chain (KLC) tetratricopeptide repeat (TPR) domain. These results suggested that microtubule transport systems may be involved in baculovirus nucleocapsid egress and BV formation. In this study, we investigated the role of lepidopteran microtubule transport using coimmunoprecipitation, colocalization, yeast two-hybrid, and small interfering RNA (siRNA) analyses. We show that nucleocapsid AC141 associates with the lepidopteranTrichoplusia niKLC and kinesin-1 heavy chain (KHC) by coimmunoprecipitation and colocalization. Kinesin-1, AC141, and microtubules colocalized predominantly at the plasma membrane. In addition, the nucleocapsid proteins VP39, FP25, and BV/ODV-C42 were also coimmunoprecipitated withT. niKLC. Direct analysis of the role ofT. nikinesin-1 by downregulation of KLC by siRNA resulted in a significant decrease in BV production. Nucleocapsids labeled with VP39 fused with three copies of the mCherry fluorescent protein also colocalized with microtubules. Yeast two-hybrid analysis showed no evidence of a direct interaction between kinesin-1 and AC141 or VP39, suggesting that either other nucleocapsid proteins or adaptor proteins may be required. These results further support the conclusion that microtubule transport is required for AcMNPV BV formation.IMPORTANCEIn two key processes of the replication cycle of the baculovirusAutographa californicamultiple nucleopolyhedrovirus (AcMNPV), nucleocapsids are transported through the cell. These include (i) entry of budded virus (BV) into the host cell and (ii) egress and budding of nucleocapsids newly produced from the plasma membrane. Prior studies have shown that the entry of nucleocapsids involves the polymerization of actin to propel nucleocapsids to nuclear pores and entry into the nucleus. For the spread of infection, progeny viruses must rapidly exit the infected cells, but the mechanism by which AcMNPV nucleocapsids traverse the cytoplasm is unknown. In this study, we examined whether nucleocapsids interact with lepidopteran kinesin-1 motor molecules and are potentially carried as cargo on microtubules to the plasma membrane in AcMNPV-infected cells. This study indicates that microtubule transport is utilized for the production of budded virus.

scholarly journals Identification of a Role for Nucleolin in Rabies Virus Infection

2014 ◽  
Vol 89 (3) ◽  
pp. 1939-1943 ◽  
Author(s):  
S. Oksayan ◽  
J. Nikolic ◽  
C. T. David ◽  
D. Blondel ◽  
D. A. Jans ◽  
...  
Keyword(s):  
Rabies Virus ◽  
Infectious Virus ◽  
Virus Production ◽  
Host Cells ◽  
Nucleocytoplasmic Trafficking ◽  
P Protein ◽  
Rabies Virus Infection ◽  
Infected Cells ◽  
Protein Isoform ◽  
Nucleolin Expression

Rabies virus replicates in the cytoplasm of host cells, but rabies virus phosphoprotein (P-protein) undergoes active nucleocytoplasmic trafficking. Here we show that the largely nuclear P-protein isoform P3 can localize to nucleoli and forms specific interactions with nucleolin. Importantly, depletion of nucleolin expression inhibits viral protein expression and infectious virus production by infected cells. This provides the first evidence that lyssaviruses interact with nucleolin and that nucleolin is important to lyssavirus infection.

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 Characterization of AcMNPV with a deletion of ac69 gene

2011 ◽  
Vol 2 (1) ◽  
pp. 4
Author(s):  
Jianhao Ke ◽  
Jinwen Wang ◽  
Riqiang Deng ◽  
Lin Lin ◽  
Bei Jinlong ◽  
...  
Keyword(s):  
Spodoptera Frugiperda ◽  
Trichoplusia Ni ◽  
Virus Production ◽  
Viral Infectivity ◽  
Methyltransferase Activity ◽  
Budded Virus

<p>ORF69 (Ac69) of <em>Autographa californica</em> multiple nucleopolyhedrovirus (Ac<em>M</em>NPV) is conserved in some baculovirus genomes. Although it has been shown that Ac69 has cap 0-dependent methyltransferase activity and is not required for budded virus production in <em>Spodoptera frugiperda</em> Sf-9 cells, its role in occlusion-derived virus synthesis and virus oral infectivity is not known. This paper describes generation of an <em>ac69</em> knockout Ac<em>M</em>NPV bacmid mutant and analyses of the influence of <em>ac69</em> deletion on the viral infectivity in Sf-9 cells and <em>Trichoplusia ni</em> larvae so as to investigate the role of <em>ac69 in the viral life cycle. Results indicated that ac69</em> deletion has little effect on the production rates and morphogenesis of budded virus and occlusion-derived virus in Sf-9 cells. In addition, animal experiment revealed that the deletion mutant did not affect Ac<em>M</em>NPV infectivity for <em>Trichoplusia ni</em> larvae in LD<sub>50</sub> and LT<sub>50</sub> bioassay when administered orally. These results suggest that <em>ac69</em> may be dispensable for viral infectivity both in vitro and in vivo.</p>

scholarly journals Bombyx mori nucleopolyhedrovirus BmP95 plays an essential role in budded virus production and nucleocapsid assembly

2013 ◽  
Vol 94 (7) ◽  
pp. 1669-1679 ◽  
Author(s):  
Xingwei Xiang ◽  
Yunwang Shen ◽  
Rui Yang ◽  
Lin Chen ◽  
Xiaolong Hu ◽  
...  
Keyword(s):  
Bombyx Mori ◽  
Virus Production ◽  
Full Length ◽  
Tubular Structures ◽  
Bombyx Mori Nucleopolyhedrovirus ◽  
Budded Virus ◽  
Conserved Gene ◽  
Fluorescence And Light Microscopy

Bombyx mori nucleopolyhedrovirus (BmNPV) BmP95 is a highly conserved gene that is found in all of the baculovirus genomes sequenced to date and is also found in nudiviruses. To investigate the role of BmP95 in virus infection in vitro, a BmP95 deletion virus (vBmP95-De) was generated by homologous recombination in Escherichia coli. Fluorescence and light microscopy and titration analysis indicated that the BmP95 deletion bacmid led to a defect in production of infectious budded virus (BV). However, deletion of BmP95 did not affect viral DNA replication. Electron microscopy showed that masses of aberrant tubular structures were present in cells transfected with the BmP95 deletion bacmid, indicating that deletion of BmP95 affected assembly of the nucleocapsid. This defect could be rescued by insertion of full-length BmP95 into the polyhedrin locus of the BmP95-knockout bacmid but not the N-terminal domain of BmP95. Together, these results showed that full-length BmP95 is essential for BV production and is required for nucleocapsid assembly.

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