scholarly journals Loquacious Modulates Flaviviral RNA Replication in Mosquito Cells

2021 ◽  
Author(s):  
Shwetha Shivaprasad ◽  
Kuo-Feng Weng ◽  
Yaw Shin Ooi ◽  
Julia Belk ◽  
Jan E. Carette ◽  
...  
Keyword(s):  
Gene Expression ◽  
Viral Genome ◽  
Viral Gene Expression ◽  
Rna Replication ◽  
Viral Rna ◽  
Infected Mosquito ◽  
Viral Gene ◽  
Host Interactions ◽  
Mosquito Cells ◽  
Detection Assay

AbstractArthropod-borne viruses infect both mosquito and mammalian hosts. While much is known about virus-host interactions that modulate viral gene expression in their mammalian host, much less is known about the interactions that involve inhibition, subversion or avoidance strategies in the mosquito host. A novel RNA-Protein interaction detection assay was used to detect proteins that directly or indirectly bind to dengue viral genomes in infected mosquito cells. Membrane-associated mosquito proteins SEC61A1 and Loquacious (Loqs) were found to be in complex with the viral RNA. Depletion analysis demonstrated that both SEC61A1 and Loqs have pro-viral functions in the dengue viral infectious cycle. Co-localization and pull-down assays showed that Loqs interacts with viral protein NS3 and both full-length and subgenomic viral RNAs. While Loqs coats the entire positive-stranded viral RNA, it binds selectively to the 3’ end of the negative-strand of the viral genome. In-depth analyses showed that the absence of Loqs did not affect translation or turnover of the viral RNA but modulated viral replication. Loqs also displayed pro-viral functions for several flaviviruses in infected mosquito cells, suggesting a conserved role for Loqs in flavivirus-infected mosquito cells.Author SummaryThere is a wealth of information that dictates virus-host interactions in flavivirus-infected mammalian cells, yet there is only sparse information on the mechanisms that modulate viral gene expression in the mosquito host. Using a novel RNA-protein detection assay, the interactions of SEC61A1 and Loqs with the dengue viral genome were found to have proviral functions in infected mosquito cells. In particular, Loqs forms complexes with the positive-strand of the viral RNA and the very 3’ end of the negative-strand viral RNA. Further analyses showed that Loqs modulates viral RNA replication of dengue virus and gene amplification of several other flaviviral genomes. These findings argue that Loqs is an essential proviral host factor in mosquitos.

scholarly journals The [KIL-d] Element Specifically Regulates Viral Gene Expression in Yeast

Genetics ◽  
2000 ◽  
Vol 155 (2) ◽  
pp. 601-609 ◽  
Author(s):  
Zsolt Tallóczy ◽  
Rebecca Mazar ◽  
Denise E Georgopoulos ◽  
Fausto Ramos ◽  
Michael J Leibowitz
Keyword(s):  
Gene Expression ◽  
Phenotypic Expression ◽  
Viral Gene Expression ◽  
Virus Genome ◽  
Viral Rna ◽  
High Rate ◽  
Viral Gene ◽  
Double Stranded Rna ◽  
Yeast Prions ◽  
Killer Virus

Abstract The cytoplasmically inherited [KIL-d] element epigenetically regulates killer virus gene expression in Saccharomyces cerevisiae. [KIL-d] results in variegated defects in expression of the M double-stranded RNA viral segment in haploid cells that are “healed” in diploids. We report that the [KIL-d] element is spontaneously lost with a frequency of 10−4–10−5 and reappears with variegated phenotypic expression with a frequency of ≥10−3. This high rate of loss and higher rate of reappearance is unlike any known nucleic acid replicon but resembles the behavior of yeast prions. However, [KIL-d] is distinct from the known yeast prions in its relative guanidinium hydrochloride incurability and independence of Hsp104 protein for its maintenance. Despite its transmissibility by successive cytoplasmic transfers, multiple cytoplasmic nucleic acids have been proven not to carry the [KIL-d] trait. [KIL-d] epigenetically regulates the expression of the M double-stranded RNA satellite virus genome, but fails to alter the expression of M cDNA. This specificity remained even after a cycle of mating and meiosis. Due to its unique genetic properties and viral RNA specificity, [KIL-d] represents a new type of genetic element that interacts with a viral RNA genome.

scholarly journals Herpes Simplex Virus Latency Is Noisier the Closer We Look

2019 ◽  
Vol 94 (4) ◽  
Author(s):  
Navneet Singh ◽  
David C. Tscharke
Keyword(s):  
Gene Expression ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Viral Genome ◽  
Infectious Virus ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Peripheral Neurons ◽  
Virus Latency ◽  
Simplex Virus

ABSTRACT During herpes simplex virus (HSV) latency, the viral genome is harbored in peripheral neurons in the absence of infectious virus but with the potential to restart infection. Advances in epigenetics have helped explain how viral gene expression is largely inhibited during latency. Paradoxically, at the same time, the view that latency is entirely silent has been eroding. This low-level noise has implications for our understanding of HSV latency and should not be ignored.

scholarly journals A Single Amino Acid Residue Change in the P Protein of Parainfluenza Virus 5 Elevates Viral Gene Expression

2008 ◽  
Vol 82 (18) ◽  
pp. 9123-9133 ◽  
Author(s):  
Khalid A. Timani ◽  
Dengyun Sun ◽  
Minghao Sun ◽  
Celia Keim ◽  
Yuan Lin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Amino Acid ◽  
Amino Acid Residue ◽  
Viral Gene Expression ◽  
Viral Rna ◽  
Single Amino Acid ◽  
Viral Gene ◽  
Wild Type ◽  
V Protein ◽  
P Protein

ABSTRACT Parainfluenza virus 5 (PIV5) is a prototypical paramyxovirus. The V/P gene of PIV5 encodes two mRNA species through a process of pseudotemplated insertion of two G residues at a specific site during transcription, resulting in two viral proteins, V and P, whose N termini of 164 amino acid residues are identical. Previously it was reported that mutating six amino acid residues within this identical region results in a recombinant PIV5 (rPIV5-CPI−) that exhibits elevated viral protein expression and induces production of cytokines, such as beta interferon and interleukin 6. Because the six mutations correspond to the shared region of the V protein and the P protein, it is not clear whether the phenotypes associated with rPIV5-CPI− are due to mutations in the P protein and/or mutations in the V protein. To address this question, we used a minigenome system and recombinant viruses to study the effects of mutations on the functions of the P and V proteins. We found that the P protein with six amino acid residue changes (Pcpi−) was more efficient than wild-type P in facilitating replication of viral RNA, while the V protein with six amino acid residue changes (Vcpi−) still inhibits minigenome replication as does the wild-type V protein. These results indicate that elevated viral gene expression in rPIV5-CPI− virus-infected cells can be attributed to a P protein with an increased ability to facilitate viral RNA synthesis. Furthermore, we found that a single amino acid residue change at position 157 of the P protein from Ser (the residue in the wild-type P protein) to Phe (the residue in Pcpi−) is sufficient for elevated viral gene expression. Using mass spectrometry and 33P labeling, we found that residue S157 of the P protein is phosphorylated. Based on these results, we propose that phosphorylation of the P protein at residue 157 plays an important role in regulating viral RNA replication.

scholarly journals Herpes Simplex Virus VP16, but Not ICP0, Is Required To Reduce Histone Occupancy and Enhance Histone Acetylation on Viral Genomes in U2OS Osteosarcoma Cells

2009 ◽  
Vol 84 (3) ◽  
pp. 1366-1375 ◽  
Author(s):  
Meaghan H. Hancock ◽  
Anna R. Cliffe ◽  
David M. Knipe ◽  
James R. Smiley
Keyword(s):  
Gene Expression ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Chromatin Structure ◽  
Viral Genome ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Osteosarcoma Cell ◽  
U2os Cells ◽  
Simplex Virus

ABSTRACT The herpes simplex virus (HSV) genome rapidly becomes associated with histones after injection into the host cell nucleus. The viral proteins ICP0 and VP16 are required for efficient viral gene expression and have been implicated in reducing the levels of underacetylated histones on the viral genome, raising the possibility that high levels of underacetylated histones inhibit viral gene expression. The U2OS osteosarcoma cell line is permissive for replication of ICP0 and VP16 mutants and appears to lack an innate antiviral repression mechanism present in other cell types. We therefore used chromatin immunoprecipitation to determine whether U2OS cells are competent to load histones onto HSV DNA and, if so, whether ICP0 and/or VP16 are required to reduce histone occupancy and enhance acetylation in this cell type. High levels of underacetylated histone H3 accumulated at several locations on the viral genome in the absence of VP16 activation function; in contrast, an ICP0 mutant displayed markedly reduced histone levels and enhanced acetylation, similar to wild-type HSV. These results demonstrate that U2OS cells are competent to load underacetylated histones onto HSV DNA and uncover an unexpected role for VP16 in modulating chromatin structure at viral early and late loci. One interpretation of these findings is that ICP0 and VP16 affect viral chromatin structure through separate pathways, and the pathway targeted by ICP0 is defective in U2OS cells. We also show that HSV infection results in decreased histone levels on some actively transcribed genes within the cellular genome, demonstrating that viral infection alters cellular chromatin structure.

Viral gene expression during the establishment of human cytomegalovirus latent infection in myeloid progenitor cells

Blood ◽  
2006 ◽  
Vol 108 (12) ◽  
pp. 3691-3699 ◽  
Author(s):  
Allen K. L. Cheung ◽  
Allison Abendroth ◽  
Anthony L. Cunningham ◽  
Barry Slobedman
Keyword(s):  
Gene Expression ◽  
Viral Genome ◽  
Latent Infection ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Time Points ◽  
Myeloid Progenitor ◽  
Viral Genes ◽  
Myeloid Progenitor Cells ◽  
Establishment Phase

AbstractHuman cytomegalovirus (HCMV) establishes and maintains a latent infection in myeloid cells and can reactivate to cause serious disease in allograft recipients. To better understand the molecular events associated with the establishment of latency, we tracked the virus following infection of primary human myeloid progenitor cells at days 1, 2, 3, 5, and 11. At all time points, the viral genome was maintained in most cells at approximately 10 copies. Infectious virus was not detected, but virus could be reactivated by extended fibroblast coculture. In contrast to wild-type HCMV, the viral genome was rapidly lost from myeloid progenitors infected with ultraviolet (UV)–inactivated virus, suggesting viral gene expression was required for efficient establishment of latency. To identify viral genes associated with the establishment phase, RNA from each time point was interrogated using custom-made HCMV gene microarrays. Using this approach, we detected expression of viral RNAs at all time points. The pattern of expression differed from that which occurs during productive infection, and decreased over time. This study provides evidence that a molecular pathway into latency is associated with expression of a unique subset of viral transcripts. Viral genes expressed during the establishment phase may serve as targets for therapies to interrupt this process.

scholarly journals Inhibition of Viral Gene Expression and Replication in Mosquito Cells by dsRNA-Triggered RNA Interference

2002 ◽  
Vol 6 (2) ◽  
pp. 243-251 ◽  
Author(s):  
Natasha J Caplen ◽  
Zhili Zheng ◽  
Barry Falgout ◽  
Richard A Morgan
Keyword(s):  
Gene Expression ◽  
Rna Interference ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Mosquito Cells

scholarly journals A Sensitive Yellow Fever Virus Entry Reporter Identifies Valosin-Containing Protein (VCP/p97) as an Essential Host Factor for Flavivirus Uncoating

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 147
Author(s):  
Shuo Zhang ◽  
Harish N. Ramanathan ◽  
Florian Douam ◽  
Katrina B. Mar ◽  
Jinhong Chang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Yellow Fever ◽  
Yellow Fever Virus ◽  
Virus Entry ◽  
Critical Role ◽  
Viral Gene Expression ◽  
Rna Replication ◽  
Fever Virus ◽  
Viral Gene ◽  
Positive Strand Rna

Flaviviruses are enveloped, arthropod-borne, positive-strand RNA viruses that cause significant human disease. While the basic mechanisms of flavivirus entry and fusion are understood, little is known about the postfusion events that precede RNA replication, such as nucleocapsid disassembly. We recently developed a sensitive, conditionally replication-defective yellow fever virus (YFV) entry reporter to quantitively monitor the translation of incoming virus particle-delivered genomes. We validated that viral gene expression can be neutralized by YFV-specific antisera and requires known pathways of flavivirus entry; however, as expected, gene expression from the defective reporter virus was insensitive to a small molecule inhibitor of YFV RNA replication. The initial round of viral gene expression was also shown to require: (i) cellular ubiquitylation, consistent with recent findings that dengue virus capsid protein must be ubiquitylated in order for nucleocapsid uncoating to occur, and (ii) valosin-containing protein (VCP)/p97, a cellular ATPase that unfolds and extracts ubiquitylated client proteins from large macromolecular complexes. RNA transfection and washout experiments showed that VCP/p97 functions at a postfusion, pretranslation step in YFV entry. Together, these data support a critical role for VCP/p97 in the disassembly of incoming flavivirus nucleocapsids during a postfusion step in virus entry.

scholarly journals Compensatory Point Mutations in the Human Immunodeficiency Virus Type 1 Gag Region That Are Distal from Deletion Mutations in the Dimerization Initiation Site Can Restore Viral Replication

1998 ◽  
Vol 72 (8) ◽  
pp. 6629-6636 ◽  
Author(s):  
Chen Liang ◽  
Liwei Rong ◽  
Michael Laughrea ◽  
Lawrence Kleiman ◽  
Mark A. Wainberg
Keyword(s):  
Gene Expression ◽  
Point Mutations ◽  
Viral Gene Expression ◽  
Initiation Site ◽  
Viral Rna ◽  
Viral Gene ◽  
Rna Packaging ◽  
Immunodeficiency Virus ◽  
Dimerization Initiation Site

ABSTRACT The dimerization initiation site (DIS), downstream of the long terminal repeat within the human immunodeficiency virus type 1 (HIV-1) genome, can form a stem-loop structure (SL1) that has been shown to be involved in the packaging of viral RNA. In order to further determine the role of this region in the virus life cycle, we deleted the 16 nucleotides (nt) at positions +238 to +253 within SL1 to generate a construct termed BH10-LD3 and showed that this virus was impaired in viral RNA packaging, viral gene expression, and viral replication. Long-term culture of these mutated viruses in MT-2 cells, i.e., 18 passages, yielded revertant viruses that possessed infectivities similar to that of the wild type. Cloning and sequencing showed that these viruses retained the original 16-nt deletion but possessed two additional point mutations, which were located within the p2 and NC regions of the Gag coding region, respectively, and which were therefore named MP2 and MNC. Site-directed mutagenesis studies revealed that both of these point mutations were necessary to compensate for the 16-nt deletion in BH10-LD3. A construct with both the 16-nt deletion and the MP2 mutation, i.e., LD3-MP2, produced approximately five times more viral protein than BH10-LD3, while the MNC mutation, i.e., construct LD3-MNC, reversed the defects in viral RNA packaging. We also deleted nt +261 to +274 within the 3′ end of SL1 and showed that the diminished infectivity of the mutated virus, termed BH10-LD4, could also be restored by the MP2 and MNC point mutations. Therefore, compensatory mutations within the p2 and NC proteins, distal from deletions within the DIS region of the HIV genome, can restore HIV replication, viral gene expression, and viral RNA packaging to control levels.

scholarly journals New insights into internal ribosome entry site elements relevant for viral gene expression

2008 ◽  
Vol 89 (3) ◽  
pp. 611-626 ◽  
Author(s):  
Encarnación Martínez-Salas ◽  
Almudena Pacheco ◽  
Paula Serrano ◽  
Noemi Fernandez
Keyword(s):  
Gene Expression ◽  
Rna Binding ◽  
Viral Gene Expression ◽  
Rna Replication ◽  
Host Factors ◽  
Viral Gene ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Positive Strand ◽  
Positive Strand Rna

A distinctive feature of positive-strand RNA viruses is the presence of high-order structural elements at the untranslated regions (UTR) of the genome that are essential for viral RNA replication. The RNA of all members of the family Picornaviridae initiate translation internally, via an internal ribosome entry site (IRES) element present in the 5′ UTR. IRES elements consist of cis-acting RNA structures that usually require specific RNA-binding proteins for translational machinery recruitment. This specialized mechanism of translation initiation is shared with other viral RNAs, e.g. from hepatitis C virus and pestivirus, and represents an alternative to the cap-dependent mechanism. In cells infected with many picornaviruses, proteolysis or changes in phosphorylation of key host factors induces shut off of cellular protein synthesis. This event occurs simultaneously with the synthesis of viral gene products since IRES activity is resistant to the modifications of the host factors. Viral gene expression and RNA replication in positive-strand viruses is further stimulated by viral RNA circularization, involving direct RNA–RNA contacts between the 5′ and 3′ ends as well as RNA-binding protein bridges. In this review, we discuss novel insights into the mechanisms that control picornavirus gene expression and compare them to those operating in other positive-strand RNA viruses.

scholarly journals Human Cytomegalovirus pUL93 Is Required for Viral Genome Cleavage and Packaging

2015 ◽  
Vol 89 (23) ◽  
pp. 12221-12225 ◽  
Author(s):  
Bernadette M. DeRussy ◽  
Ritesh Tandon
Keyword(s):  
Gene Expression ◽  
Human Cytomegalovirus ◽  
Viral Genome ◽  
Unit Length ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Genome Packaging ◽  
Viral Dna ◽  
Virus Growth ◽  
Virus Life Cycle

Human cytomegalovirus (HCMV) pUL93 is essential for virus growth, but its precise function in the virus life cycle is unknown. Here, we characterize a UL93 stop mutant virus (UL93st-TB40/E-BAC) to demonstrate that the absence of this protein does not restrict viral gene expression; however, cleavage of viral DNA into unit-length genomes as well as genome packaging is abolished. Thus, pUL93 is required for viral genome cleavage and packaging.

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