scholarly journals Generation and analysis of recombinant Bunyamwera orthobunyaviruses expressing V5 epitope-tagged L proteins

2009 ◽  
Vol 90 (2) ◽  
pp. 297-306 ◽  
Author(s):  
Xiaohong Shi ◽  
Richard M. Elliott
Keyword(s):  
Reverse Genetics ◽  
Immunofluorescent Staining ◽  
Cell Fractionation ◽  
Protein Insertion ◽  
Recombinant Viruses ◽  
L Protein ◽  
Size Growth ◽  
Infected Cells ◽  
L Proteins

The L protein of Bunyamwera virus (BUNV; family Bunyaviridae) is an RNA-dependent RNA polymerase, 2238 aa in length, that catalyses transcription and replication of the negative-sense, tripartite RNA genome. To learn more about the molecular interactions of the L protein and to monitor its intracellular distribution we inserted a 14 aa V5 epitope derived from parainfluenza virus type 5, against which high-affinity antibodies are available, into different regions of the protein. Insertion of the epitope at positions 1935 or 2046 resulted in recombinant L proteins that retained functionality in a minireplicon assay. Two viable recombinant viruses, rBUNL4V5 and rBUNL5V5, expressing the tagged L protein were rescued by reverse genetics, and characterized with respect to their plaque size, growth kinetics and protein synthesis profile. The recombinant viruses behaved similarly to wild-type (wt) BUNV in BHK-21 cells, but formed smaller plaques and grew to lower titres in Vero E6 cells compared with wt BUNV. Immunofluorescent staining of infected cells showed the L protein to have a punctate to reticular distribution in the cytoplasm, and cell fractionation studies indicated that the L protein was present in both soluble and microsomal fractions. Co-immunoprecipitation and confocal microscopic assays confirmed an interaction between BUNV L and N proteins. The recombinant viruses expressing tagged L protein will be highly valuable reagents for the detailed dissection of the role of the BUNV L protein in virus replication.

scholarly journals S-Adenosyl Homocysteine-Induced Hyperpolyadenylation of Vesicular Stomatitis Virus mRNA Requires the Methyltransferase Activity of L Protein

2008 ◽  
Vol 82 (24) ◽  
pp. 12280-12290 ◽  
Author(s):  
Summer E. Galloway ◽  
Gail W. Wertz
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Competitive Inhibitor ◽  
Recombinant Viruses ◽  
L Protein ◽  
Mrna Capping ◽  
Adenosyl Methionine ◽  
Infected Cells ◽  
Vesicular Stomatitis ◽  
L Proteins

ABSTRACT There are many unique aspects of vesicular stomatitis virus (VSV) transcription. In addition to its unusual mRNA capping and methyltransferase mechanisms, the addition of S-adenosyl homocysteine (SAH), which is the by-product and competitive inhibitor of S-adenosyl methionine (SAM)-mediated methyltransferase reactions, leads to synthesis of poly(A) tails on the 3′ end of VSV mRNAs that are 10- or 20-fold longer than normal. The mechanism by which this occurs is not understood, since it has been shown that productive transcription is not dependent on 5′ cap methylation and full-length VSV mRNAs can be synthesized in the absence of SAM. To investigate this unusual phenotype, we assayed the effects of SAH on transcription using a panel of recombinant viruses that contained mutations in domain VI of the VSV L protein. The L proteins we investigated displayed a range of 5′ cap methyltransferase activities. In the present study, we show that the ability of the VSV L protein to catalyze methyl transfer correlates with its sensitivity to SAH with respect to polyadenylation, thereby indicating an intriguing connection between 5′ and 3′ end mRNA modifications. We also identified an L protein mutant that hyperpolyadenylates mRNA irrespective of the presence or absence of exogenous SAH. Further, the data presented here show that the wild-type L protein hyperpolyadenylates a percentage of VSV mRNAs in infected cells as well as in vitro.

scholarly journals Tula hantavirus L protein is a 250 kDa perinuclear membrane-associated protein

2004 ◽  
Vol 85 (5) ◽  
pp. 1181-1189 ◽  
Author(s):  
Sami K. J. Kukkonen ◽  
Antti Vaheri ◽  
Alexander Plyusnin
Keyword(s):  
Matrix Protein ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
Expression System ◽  
Peak Level ◽  
Cell Fractionation ◽  
L Protein ◽  
System L ◽  
Infected Cells ◽  
In Cells

The complete open reading frame of Tula hantavirus (TULV) L RNA was cloned in three parts. The middle third (nt 2191–4344) could be expressed in E. coli and was used to immunize rabbits. The resultant antiserum was then used to immunoblot concentrated TULV and infected Vero E6 cells. The L protein of a hantavirus was detected, for the first time, in infected cells and was found to be expressed as a single protein with an apparent molecular mass of 250 kDa in both virions and infected cells. Using the antiserum, the expression level of the L protein was followed and image analysis of immunoblots indicated that there were 104 copies per cell at the peak level of expression. The antiserum was also used to detect the L protein in cell fractionation studies. In cells infected with TULV and cells expressing recombinant L, the protein pelleted with the microsomal membrane fraction. The membrane association was confirmed with membrane flotation assays. To visualize L protein localization in cells, a fusion protein of L and enhanced green fluorescent protein, L–EGFP, was expressed in Vero E6 cells with a plasmid-driven T7 expression system. L–EGFP localized in the perinuclear region where it had partial co-localization with the Golgi matrix protein GM130 and the TULV nucleocapsid protein.

scholarly journals N-glycosylation of infectious bronchitis virus M41 spike determines receptor specificity

2020 ◽  
Vol 101 (6) ◽  
pp. 599-608
Author(s):  
K. M. Bouwman ◽  
N. Habraeken ◽  
A. Laconi ◽  
A. J. Berends ◽  
L. Groenewoud ◽  
...  
Keyword(s):  
Infectious Bronchitis Virus ◽  
Reverse Genetics ◽  
Glycosylation Site ◽  
Host Cells ◽  
Receptor Specificity ◽  
Knock Out ◽  
Recombinant Viruses ◽  
Infectious Bronchitis

Infection of chicken coronavirus infectious bronchitis virus (IBV) is initiated by binding of the viral heavily N-glycosylated attachment protein spike to the alpha-2,3-linked sialic acid receptor Neu5Ac. Previously, we have shown that N-glycosylation of recombinantly expressed receptor binding domain (RBD) of the spike of IBV-M41 is of critical importance for binding to chicken trachea tissue. Here we investigated the role of N-glycosylation of the RBD on receptor specificity and virus replication in the context of the virus particle. Using our reverse genetics system we were able to generate recombinant IBVs for nine-out-of-ten individual N-glycosylation mutants. In vitro growth kinetics of these viruses were comparable to the virus containing the wild-type M41-S1. Furthermore, Neu5Ac binding by the recombinant viruses containing single N-glycosylation site knock-out mutations matched the Neu5Ac binding observed with the recombinant RBDs. Five N-glycosylation mutants lost the ability to bind Neu5Ac and gained binding to a different, yet unknown, sialylated glycan receptor on host cells. These results demonstrate that N-glycosylation of IBV is a determinant for receptor specificity.

scholarly journals The K1 Protein of Kaposi's Sarcoma-Associated Herpesvirus Augments Viral Lytic Replication

2016 ◽  
Vol 90 (17) ◽  
pp. 7657-7666 ◽  
Author(s):  
Zhigang Zhang ◽  
Wuguo Chen ◽  
Marcia K. Sanders ◽  
Kevin F. Brulois ◽  
Dirk P. Dittmer ◽  
...  
Keyword(s):  
Life Cycle ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Akt Kinase ◽  
Recombinant Viruses ◽  
Infected Cells ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACTThe K1 gene product of Kaposi's sarcoma-associated herpesvirus (KSHV) is encoded by the first open reading frame (ORF) of the viral genome. To investigate the role of the K1 gene during the KSHV life cycle, we constructed a set of recombinant viruses that contained either wild-type (WT) K1, a deleted K1 ORF (KSHVΔK1), stop codons within the K1 ORF (KSHV-K15×STOP), or a revertant K1 virus (KSHV-K1REV). We report that the recombinant viruses KSHVΔK1 and KSHV-K15×STOPdisplayed significantly reduced lytic replication compared to WT KSHV and KSHV-K1REVupon reactivation from latency. Additionally, cells infected with the recombinant viruses KSHVΔK1 and KSHV-K15×STOPalso yielded smaller amounts of infectious progeny upon reactivation than did WT KSHV- and KSHV-K1REV-infected cells. Upon reactivation from latency, WT KSHV- and KSHV-K1REV-infected cells displayed activated Akt kinase, as evidenced by its phosphorylation, while cells infected with viruses deleted for K1 showed reduced phosphorylation and activation of Akt kinase. Overall, our results suggest that K1 plays an important role during the KSHV life cycle.IMPORTANCEKaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of three human malignancies, and KSHV K1 is a signaling protein that has been shown to be involved in cellular transformation and to activate the phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR pathway. In order to investigate the role of the K1 protein in the life cycle of KSHV, we constructed recombinant viruses that were deficient for K1. We found that K1 deletion viruses displayed reduced lytic replication compared to the WT virus and also yielded smaller numbers of infectious progeny. We report that K1 plays an important role in the life cycle of KSHV.

scholarly journals A recombinant rotavirus harboring a spike protein with a heterologous peptide reveals a novel role of VP4 in viroplasm stability

2021 ◽  
Author(s):  
Guido Papa ◽  
Janine Vetter ◽  
Michael Seyffert ◽  
Kapila Gunasekera ◽  
Giuditta De Lorenzo ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
Similar Distribution ◽  
Cell Tropism ◽  
Wild Type ◽  
Lectin Domain ◽  
Infected Cells ◽  
Sirna Silencing ◽  
Genetically Manipulated ◽  
Replication Fitness

The rotavirus (RV) VP4 spike protrudes as a trimeric structure from the five-fold axes of the virion triple-layer. Infectious RV particles need to be proteolytically cleaved in VP4 into two subunits, VP8* and VP5*, constituting both the distal part and central body of the virus spike. Modification of VP4 has been challenging as it is involved in biological processes such as the interaction with sialic acid and integrins, cell tropism and hemagglutinin activity. Using RV reverse genetics, four loops in the lectin domain of the VP8* subunit were engineered independently to harbor a small biotin acceptor peptide (BAP) tag and then tested for their ability to rescue virus. Only a single recombinant virus, rRV/VP4-BAP, harboring VP4 with a modified loop at position K145-G150 was rescued. This rRV/VP4-BAP internalizes, replicates, and generates virus progeny, demonstrating that the VP4 spike of RV particles can be genetically manipulated by the incorporation of at least 15 exogenous amino acids. VP4-BAP had a similar distribution as VP4 in infected cells by localizing in the cytoskeleton and surrounding viroplasms. However, compared to wild-type RV, rRV/VP4-BAP featured a reduced replication fitness and impaired viroplasm stability. Upon treatment with 1,6-hexanediol, a drug disrupting liquid-liquid phase-separated condensates, the kinetic of rRV/VP4-BAP viroplasm recovery was delayed, and their size and numbers reduced when compared to viroplasms of wild type RV. Moreover, siRNA silencing of VP4 expression in RV strain SA11 showed similar recovery patterns as rRV/VP4-BAP, revealing a novel function of VP4 in viroplasm stability.

scholarly journals Structural basis for recognition and regulation of arenavirus polymerase L by Z protein

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Huiling Kang ◽  
Jingyuan Cong ◽  
Chenlong Wang ◽  
Wenxin Ji ◽  
Yuhui Xin ◽  
...  
Keyword(s):  
Rational Design ◽  
Viral Infections ◽  
Hemorrhagic Fever ◽  
Structural Basis ◽  
Exit Site ◽  
L Protein ◽  
Argentine Hemorrhagic Fever ◽  
L Proteins ◽  
Z Protein

AbstractJunin virus (JUNV) causes Argentine hemorrhagic fever, a debilitating human disease of high mortality rates and a great risk to public health worldwide. Studying the L protein that replicates and transcribes the genome of JUNV, and its regulator Z protein should provide critical clues to identify therapeutic targets for disrupting the life cycle of JUNV. Here we report the 3.54 Å cryo-EM structure of the JUNV L protein complexed with regulator Z protein. JUNV L structure reveals a conserved architecture containing signature motifs found in other L proteins. Structural analysis shows that L protein is regulated by binding of Z protein at the RNA product exit site. Based on these findings, we propose a model for the role of Z protein as a switch to turn on/off the viral RNA synthesis via its interaction with L protein. Our work unveils the mechanism of JUNV transcription, replication and regulation, which provides a framework for the rational design of antivirals for combating viral infections.

scholarly journals Role of Virally-Encoded Deubiquitinating Enzymes in Regulation of the Virus Life Cycle

2021 ◽  
Vol 22 (9) ◽  
pp. 4438
Author(s):  
Jessica Proulx ◽  
Kathleen Borgmann ◽  
In-Woo Park
Keyword(s):  
Immune Response ◽  
Life Cycle ◽  
Deubiquitinating Enzyme ◽  
Deubiquitinating Enzymes ◽  
Antiviral Immune Response ◽  
Cellular Processes ◽  
Virus Life Cycle ◽  
Infected Cells ◽  
Dependent Processes

The ubiquitin (Ub) proteasome system (UPS) plays a pivotal role in regulation of numerous cellular processes, including innate and adaptive immune responses that are essential for restriction of the virus life cycle in the infected cells. Deubiquitination by the deubiquitinating enzyme, deubiquitinase (DUB), is a reversible molecular process to remove Ub or Ub chains from the target proteins. Deubiquitination is an integral strategy within the UPS in regulating survival and proliferation of the infecting virus and the virus-invaded cells. Many viruses in the infected cells are reported to encode viral DUB, and these vial DUBs actively disrupt cellular Ub-dependent processes to suppress host antiviral immune response, enhancing virus replication and thus proliferation. This review surveys the types of DUBs encoded by different viruses and their molecular processes for how the infecting viruses take advantage of the DUB system to evade the host immune response and expedite their replication.

scholarly journals An MDM2 inhibitor achieves synergistic cytotoxic effects with adenoviruses lacking E1B55kDa gene on mesothelioma with the wild-type p53 through augmenting NFI expression

2021 ◽  
Vol 12 (7) ◽  
Author(s):  
Thao Thi Thanh Nguyen ◽  
Masato Shingyoji ◽  
Michiko Hanazono ◽  
Boya Zhong ◽  
Takao Morinaga ◽  
...  
Keyword(s):  
Wild Type ◽  
Inhibitory Effects ◽  
Cytotoxic Effects ◽  
P53 Expression ◽  
Nuclear Factor I ◽  
Infected Cells ◽  
Wild Type P53 ◽  
Mdm2 Inhibitor ◽  
P16 Expression

AbstractA majority of mesothelioma specimens were defective of p14 and p16 expression due to deletion of the INK4A/ARF region, and the p53 pathway was consequently inactivated by elevated MDM2 functions which facilitated p53 degradaton. We investigated a role of p53 elevation by MDM2 inhibitors, nutlin-3a and RG7112, in cytotoxicity of replication-competent adenoviruses (Ad) lacking the p53-binding E1B55kDa gene (Ad-delE1B). We found that a growth inhibition by p53-activating Ad-delE1B was irrelevant to p53 expression in the infected cells, but combination of Ad-delE1B and the MDM2 inhibitor produced synergistic inhibitory effects on mesothelioma with the wild-type but not mutated p53 genotype. The combination augmented p53 phosphorylation, activated apoptotic but not autophagic pathway, and enhanced DNA damage signals through ATM-Chk2 phosphorylation. The MDM2 inhibitors facilitated production of the Ad progenies through augmented expression of nuclear factor I (NFI), one of the transcriptional factors involved in Ad replications. Knocking down of p53 with siRNA did not increase the progeny production or the NFI expression. We also demonstrated anti-tumor effects by the combination of Ad-delE1B and the MDM2 inhibitors in an orthotopic animal model. These data collectively indicated that upregulation of wild-type p53 expression contributed to cytotoxicity by E1B55kDa-defective replicative Ad through NFI induction and suggested that replication-competent Ad together with augmented p53 levels was a therapeutic strategy for p53 wild-type mesothelioma.

scholarly journals Genome-Wide Expression Patterns of Rhoptry Kinases during the Eimeria tenella Life-Cycle

2021 ◽  
Vol 9 (8) ◽  
pp. 1621
Author(s):  
Adeline Ribeiro E Silva ◽  
Alix Sausset ◽  
Françoise I. Bussière ◽  
Fabrice Laurent ◽  
Sonia Lacroix-Lamandé ◽  
...  
Keyword(s):  
Life Cycle ◽  
Expression Patterns ◽  
Housekeeping Genes ◽  
Catalytic Triad ◽  
Eimeria Tenella ◽  
Apicomplexan Parasites ◽  
Genome Wide ◽  
Infected Cells ◽  
First And Second Generation

Kinome from apicomplexan parasites is composed of eukaryotic protein kinases and Apicomplexa specific kinases, such as rhoptry kinases (ROPK). Ropk is a gene family that is known to play important roles in host–pathogen interaction in Toxoplasma gondii but is still poorly described in Eimeria tenella, the parasite responsible for avian coccidiosis worldwide. In the E. tenella genome, 28 ropk genes are predicted and could be classified as active (n = 7), inactive (incomplete catalytic triad, n = 12), and non-canonical kinases (active kinase with a modified catalytic triad, n = 9). We characterized the ropk gene expression patterns by real-time quantitative RT-PCR, normalized by parasite housekeeping genes, during the E. tenella life-cycle. Analyzed stages were: non-sporulated oocysts, sporulated oocysts, extracellular and intracellular sporozoites, immature and mature schizonts I, first- and second-generation merozoites, and gametes. Transcription of all those predicted ropk was confirmed. The mean intensity of transcription was higher in extracellular stages and 7–9 ropk were specifically transcribed in merozoites in comparison with sporozoites. Transcriptional profiles of intracellular stages were closely related to each other, suggesting a probable common role of ROPKs in hijacking signaling pathways and immune responses in infected cells. These results provide a solid basis for future functional analysis of ROPK from E. tenella.

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