scholarly journals Simian Virus 5 Is a Poor Inducer of Chemokine Secretion from Human Lung Epithelial Cells: Identification of Viral Mutants That Activate Interleukin-8 Secretion by Distinct Mechanisms

2003 ◽  
Vol 77 (12) ◽  
pp. 7124-7130 ◽  
Author(s):  
Virginia A. Young ◽  
Griffith D. Parks
Keyword(s):  
Gene Expression ◽  
Human Lung ◽  
A549 Cells ◽  
Viral Gene Expression ◽  
Simian Virus ◽  
Interleukin 8 ◽  
Parainfluenza Virus ◽  
Viral Gene ◽  
Simian Virus 5 ◽  
Infected Cells

ABSTRACT We have compared chemokine secretion from human lung A549 cells infected with simian virus 5 (SV5) with other members of the Rubulavirus genus of paramyxoviruses. High levels of the chemokines interleukin-8 (IL-8) and macrophage chemoattractant protein-1 (MCP-1) were secreted from A549 cells infected with Human parainfluenza virus type 2 (HPIV-2) but not from cells infected with wild-type (WT) SV5. The lack of IL-8 secretion from SV5-infected cells was not due to a global block in all signal transduction pathways leading to IL-8 secretion, since SV5-infected A549 cells secreted IL-8 after stimulation with exogenously added tumor necrosis factor alpha or by coinfection with HPIV-2. A previously described, recombinant SV5 containing substitutions in the shared region of the P/V gene (rSV5-P/V-CPI−) induced IL-8 secretion by a mechanism that was dependent on viral gene expression. By contrast, an SV5 variant isolated from persistently infected cells (Wake Forest strain of Canine parainfluenza virus) induced IL-8 secretion by a mechanism that was largely not affected by inhibitors of viral gene expression. Together, these data demonstrate that SV5 is unusual compared to other closely related paramyxoviruses, since SV5 is a very poor inducer of the cytokines IL-8 and MCP-1. The isolation of two recombinant SV5 mutants that are defective in preventing chemokine induction will allow an identification of mechanisms utilized by WT SV5 to avoid activation of host cell innate immune responses to infection.

Protein-RNA interactome analysis reveals wide association of KSHV ORF57 with host non-coding RNAs and polysomes

2021 ◽  
Author(s):  
Beatriz Alvarado-Hernandez ◽  
Yanping Ma ◽  
Nishi R. Sharma ◽  
Vladimir Majerciak ◽  
Alexei Lobanov ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Splicing ◽  
Rna Binding ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
28S Rrna ◽  
Protein Coding ◽  
Infected Cells ◽  
Non Coding Rnas ◽  
Rna Interaction

Kaposi’s sarcoma-associated herpesvirus (KSHV) ORF57 is an RNA-binding post-transcriptional regulator. We recently applied an affinity-purified anti-ORF57 antibody to conduct ORF57-CLIP (Cross-linking Immunoprecipitation) in combination with RNA-sequencing (CLIP-seq) and analyzed the genome-wide host RNA transcripts in association with ORF57 in BCBL-1 cells with lytic KSHV infection. Mapping of the CLIPed RNA reads to the human genome (GRCh37) revealed that most of the ORF57-associated RNA reads were from rRNAs. The remaining RNA reads mapped to several classes of host non-coding and protein-coding mRNAs. We found ORF57 binds and regulates expression of a subset of host lncRNAs, including LINC00324, LINC00355, and LINC00839 which are involved in cell growth. ORF57 binds snoRNAs responsible for 18S and 28S rRNA modifications, but does not interact with fibrillarin and NOP58. We validated ORF57 interactions with 67 snoRNAs by ORF57-RNA immunoprecipitation (RIP)-snoRNA-array assays. Most of the identified ORF57 rRNA binding sites (BS) overlap with the sites binding snoRNAs. We confirmed ORF57-snoRA71B RNA interaction in BCBL-1 cells by ORF57-RIP and Northern blot analyses using a 32 P-labeled oligo probe from the 18S rRNA region complementary to snoRA71B. Using RNA oligos from the rRNA regions that ORF57 binds for oligo pulldown-Western blot assays, we selectively verified ORF57 interactions with 5.8S and 18S rRNAs. Polysome profiling revealed that ORF57 associates with both monosomes and polysomes and its association with polysomes increases PABPC1 binding to, but prevent Ago2 from polysomes. Our data indicate a functional correlation with ORF57 binding and suppression of Ago2 activities for ORF57 promotion of gene expression. Significance As an RNA-binding protein, KSHV ORF57 regulates RNA splicing, stability, and translation and inhibits host innate immunity by blocking the formation of RNA granules in virus infected cells. In this report, ORF57 was found to interact many host non-coding RNAs, including lncRNAs, snoRNAs and ribosomal RNAs to carry out additional unknown functions. ORF57 binds a group of lncRNAs via the identified RNA motifs by ORF57 CLIP-seq to regulate their expression. ORF57 associates with snoRNAs independently of fibrillarin and NOP58 proteins, and with ribosomal RNA in the regions that commonly bind snoRNAs. Knockdown of fibrillarin expression decreases the expression of snoRNAs and CDK4, but not affect viral gene expression. More importantly, we found that ORF57 binds translationally active polysomes and enhances PABPC-1 but prevents Ago2 association with polysomes. Data provide a compelling evidence on how ORF57 in KSHV infected cells might regulate protein synthesis by blocking Ago2’s hostile activities on translation.

scholarly journals Adenovirus Virus-Associated RNA Is Processed to Functional Interfering RNAs Involved in Virus Production

2006 ◽  
Vol 80 (3) ◽  
pp. 1376-1384 ◽  
Author(s):  
Oscar Aparicio ◽  
Nerea Razquin ◽  
Mikel Zaratiegui ◽  
Iñigo Narvaiza ◽  
Puri Fortes
Keyword(s):  
Gene Expression ◽  
Virus Production ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Small Interfering Rnas ◽  
Control Of Gene Expression ◽  
Posttranscriptional Gene Silencing ◽  
Argonaute 2 ◽  
Complementary Sequences ◽  
Infected Cells

ABSTRACT Posttranscriptional gene silencing allows sequence-specific control of gene expression. Specificity is guaranteed by small antisense RNAs such as microRNAs (miRNAs) or small interfering RNAs (siRNAs). Functional miRNAs derive from longer double-stranded RNA (dsRNA) molecules that are cleaved to pre-miRNAs in the nucleus and are transported by exportin 5 (Exp 5) to the cytoplasm. Adenovirus-infected cells express virus-associated (VA) RNAs, which are dsRNA molecules similar in structure to pre-miRNAs. VA RNAs are also transported by Exp 5 to the cytoplasm, where they accumulate. Here we show that small RNAs derived from VA RNAs (svaRNAs), similar to miRNAs, can be found in adenovirus-infected cells. VA RNA processing to svaRNAs requires neither viral replication nor viral protein expression, as evidenced by the fact that svaRNA accumulation can be detected in cells transfected with VA sequences. svaRNAs are efficiently bound by Argonaute 2, the endonuclease of the RNA-induced silencing complex, and behave as functional siRNAs, in that they inhibit the expression of reporter genes with complementary sequences. Blocking svaRNA-mediated inhibition affects efficient adenovirus production, indicating that svaRNAs are required for virus viability. Thus, svaRNA-mediated silencing could represent a novel mechanism used by adenoviruses to control cellular or viral gene expression.

scholarly journals The Immune Evasion Paradox: Immunoevasins of Murine Cytomegalovirus Enhance Priming of CD8 T Cells by Preventing Negative Feedback Regulation

2008 ◽  
Vol 82 (23) ◽  
pp. 11637-11650 ◽  
Author(s):  
Verena Böhm ◽  
Christian O. Simon ◽  
Jürgen Podlech ◽  
Christof K. Seckert ◽  
Dorothea Gendig ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
Negative Feedback ◽  
Cd8 T Cells ◽  
Viral Gene Expression ◽  
Cd8 T Cell ◽  
Murine Cytomegalovirus ◽  
Viral Gene ◽  
Infected Cells

ABSTRACT Cytomegaloviruses express glycoproteins that interfere with antigen presentation to CD8 T cells. Although the molecular modes of action of these “immunoevasins” differ between cytomegalovirus species, the convergent biological outcome is an inhibition of the recognition of infected cells. In murine cytomegalovirus, m152/gp40 retains peptide-loaded major histocompatibility complex class I molecules in a cis-Golgi compartment, m06/gp48 mediates their vesicular sorting for lysosomal degradation, and m04/gp34, although not an immunoevasin in its own right, appears to assist in the concerted action of all three molecules. Using the Ld-restricted IE1 epitope YPHFMPTNL in the BALB/c mouse model as a paradigm, we provide here an explanation for the paradox that immunoevasins enhance CD8 T-cell priming although they inhibit peptide presentation in infected cells. Adaptive immune responses are initiated in the regional lymph node (RLN) draining the site of pathogen exposure. In particular for antigens that are not virion components, the magnitude of viral gene expression providing the antigens is likely a critical parameter in priming efficacy. We have therefore focused on the events in the RLN and have related priming to intranodal viral gene expression. We show that immunoevasins enhance priming by downmodulating an early CD8 T-cell-mediated “negative feedback” control of the infection in the cortical region of the RLN, thus supporting the model that immunoevasins improve antigen supply for indirect priming by uninfected antigen-presenting cells. As an important consequence, these findings predict that deletion of immunoevasin genes in a replicative vaccine virus is not a favorable option but may, rather, be counterproductive.

scholarly journals Multiple RSV strains infecting HEp-2 and A549 cells reveal cell line-dependent differences in resistance to RSV infection.

2021 ◽  
Author(s):  
Anubama Rajan ◽  
Felipe-Andres Piedra ◽  
Letisha Aideyan ◽  
Trevor McBride ◽  
Matthew J Robertson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Cell Lines ◽  
A549 Cells ◽  
Viral Gene Expression ◽  
Model Systems ◽  
Viral Gene ◽  
Transformed Cell ◽  
Syncytial Virus ◽  
Transformed Cell Lines

Respiratory syncytial virus (RSV) is a leading cause of pediatric acute respiratory infection worldwide. There are currently no approved vaccines or antivirals to combat RSV disease. A few transformed cell lines and two historic strains have been extensively used to study RSV. Here we report a thorough molecular and cell biological characterization of HEp-2 and A549 cells infected with four strains of RSV representing both major subgroups as well as historic and more contemporaneous genotypes -- [RSV/A/Tracy (GA1), RSV/A/Ontario (ON), RSV/B/18537 (GB1), RSV/B/Buenos Aires (BA)] -- via measurements of viral replication kinetics and viral gene expression, immunofluorescence-based imaging of gross cellular morphology and cell-associated RSV, and measurements of host response including transcriptional changes and levels of secreted cytokines and growth factors. Our findings strongly suggest 1) the existence of a conserved difference in gene expression between RSV subgroups A and B; 2) the A549 cell line is a more stringent and natural host of replicating RSV than the HEp-2 cell line; and 3) consistent with previous studies, determining the full effects of viral genetic variation in RSV pathogenesis requires model systems as tractable as transformed cell lines but better representative of the human host.

scholarly journals Cell Cycle-Independent Expression of Immediate-Early Gene 3 Results in G1 and G2 Arrest in Murine Cytomegalovirus-Infected Cells

2008 ◽  
Vol 82 (20) ◽  
pp. 10188-10198 ◽  
Author(s):  
Lüder Wiebusch ◽  
Anke Neuwirth ◽  
Linus Grabenhenrich ◽  
Sebastian Voigt ◽  
Christian Hagemeier
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Viral Gene Expression ◽  
Immediate Early Gene ◽  
Early Gene ◽  
Immediate Early ◽  
Viral Gene ◽  
Cycle Arrest ◽  
Infected Cells

ABSTRACT The infectious cycle of human cytomegalovirus (HCMV) is intricately linked to the host's cell cycle. Viral gene expression can be initiated only in G0/G1 phase. Once expressed, the immediate-early gene product IE2 prevents cellular DNA synthesis, arresting infected cells with a G1 DNA content. This function is required for efficient viral replication in vitro. A prerequisite for addressing its in vivo relevance is the characterization of cell cycle-regulatory activities of CMV species for which animal models have been established. Here, we show that murine CMV (MCMV), like HCMV, has a strong antiproliferative capacity and arrests cells in G1. Unexpectedly, and in contrast to HCMV, MCMV can also block cells that have passed through S phase by arresting them in G2. Moreover, MCMV can also replicate in G2 cells. This is made possible by the cell cycle-independent expression of MCMV immediate-early genes. Transfection experiments show that of several MCMV candidate genes, only immediate-early gene 3 (ie3), the homologue of HCMV IE2, exhibits cell cycle arrest activity. Accordingly, an MCMV ie3 deletion mutant has lost the ability to arrest cells in either G1 or G2. Thus, despite interspecies variations in the cell cycle dependence of viral gene expression, the central theme of HCMV IE2-induced cell cycle arrest is conserved in the murine counterpart, raising the possibility of studying its physiological relevance at the level of the whole organism.

scholarly journals Nuclear Localization of Tegument-Delivered pp71 in Human Cytomegalovirus-Infected Cells Is Facilitated by One or More Factors Present in Terminally Differentiated Fibroblasts

2010 ◽  
Vol 84 (19) ◽  
pp. 9853-9863 ◽  
Author(s):  
Rhiannon R. Penkert ◽  
Robert F. Kalejta
Keyword(s):  
Gene Expression ◽  
Human Cytomegalovirus ◽  
Viral Entry ◽  
Viral Gene Expression ◽  
Cell Types ◽  
Lytic Replication ◽  
Viral Gene ◽  
Differentiated Cells ◽  
Virion Envelope ◽  
Infected Cells

ABSTRACT Herpesviral virions contain a tegument layer that consists primarily of viral proteins. The delivery of fully functional proteins to infected cells upon virion envelope fusion to the plasma membrane allows herpesviruses to modulate cellular activities prior to viral gene expression. Certain tegument proteins can also regulate viral processes. For example, the pp71 tegument protein encoded by the UL82 gene of human cytomegalovirus (HCMV) stimulates viral immediate early (IE) gene expression and thus acts to initiate the productive lytic infectious cycle. In terminally differentiated fibroblasts infected with HCMV, tegument-delivered pp71 traffics to the nucleus and degrades the cellular transcriptional corepressor Daxx to initiate viral IE gene expression and lytic replication. However, when HCMV infects incompletely differentiated cells, tegument-delivered pp71 remains in the cytoplasm, allowing the nucleus-localized Daxx protein to silence viral IE gene expression and promote the establishment of a latent infection in certain cell types. We sought to determine whether undifferentiated cells block the trafficking of tegument-delivered pp71 to the nucleus or whether differentiated cells facilitate the nuclear transport of tegument-delivered pp71. Heterogenous cell fusion experiments demonstrated that tegument-delivered pp71 found in the cytoplasm of undifferentiated NT2 cells could be driven into the nucleus by one or more factors provided by fully differentiated fibroblasts. Our data raise the intriguing possibility that latency is the default program launched by HCMV upon viral entry into cells and that lytic infection is initiated only in certain (differentiated) cells that can facilitate the delivery of incoming pp71 to the nucleus.

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