scholarly journals The Reovirus μ2 C-Terminal Loop Inversely Regulates NTPase and Transcription Functions versus Binding to Factory-Forming μNS and Promotes Replication in Tumorigenic Cells

2021 ◽  
Vol 95 (10) ◽  
Author(s):  
Wan Kong (Wynton) Yip ◽  
Francisca Cristi ◽  
Georgi Trifonov ◽  
Nashae Narayan ◽  
Mark Kubanski ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Virus Replication ◽  
Rna Synthesis ◽  
Burst Size ◽  
Terminal Region ◽  
Virus Protein ◽  
Antiviral Immune Response ◽  
Reovirus Infection ◽  
Terminal Loop ◽  
Tumorigenic Cells

ABSTRACT The wild-type reovirus serotype 3 Dearing PL strain (T3wt) is being heavily evaluated as an oncolytic and immunotherapeutic treatment for cancers. Mutations that promote reovirus entry into tumor cells were previously reported to enhance oncolysis; here, we aimed to discover mutations that enhance the postentry steps of reovirus infection in tumor cells. Using directed evolution, we found that reovirus variant T3v10M1 exhibited enhanced replication relative to that of T3wt on a panel of cancer cells. T3v10M1 contains an alanine-to-valine substitution (A612V) in the core-associated μ2, which was previously found to have nucleoside-triphosphatase (NTPase) activities in virions and to facilitate virus factory formation by association with μNS. Paradoxically, the A612V mutation in μ2 from T3v10M1 was discovered to impair NTPase activities and RNA synthesis, leading to a 5-fold higher probability of abortive infection for T3v10M1 relative to that with T3wt. The A612V mutation resides in a previously uncharacterized C-terminal region that juxtaposes the template entry site of the polymerase μ2; our findings thus support an important role for this domain during virus transcription. Despite crippled onset of infection, T3v10M1 exhibited greater accumulation of viral proteins and progeny during replication, leading to increased overall virus burst size. Both far-Western blotting and coimmunoprecipitation (Co-IP) approaches corroborated that the A612V mutation in μ2 increased association with the nonstructural virus protein μNS and enhances burst size. Together, the data show that mutations in the C-terminal loop domain of μ2 inversely regulate NTPase and RNA synthesis versus interactions with μNS, but with a net gain of replication in tumorigenic cells. IMPORTANCE Reovirus is a model system for understanding virus replication and also a clinically relevant virus for cancer therapy. We identified the first mutation that increases reovirus infection in tumorigenic cells by enhancing postentry stages of reovirus replication. The mutation is in a previously uncharacterized C-terminal region of the M1-derived μ2 protein, which we demonstrated affects multiple functions of μ2, namely, NTPase, RNA synthesis, inhibition of antiviral immune response, and association with the virus replication factory-forming μNS protein. These findings promote a mechanistic understanding of viral protein functions. In the future, the benefits of μ2 mutations may be useful for enhancing reovirus potency in tumors.

scholarly journals Inhibition of Indoleamine-2,3-dioxygenase (IDO) in Glioblastoma Cells by Oncolytic Herpes Simplex Virus

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Bonnie Reinhart ◽  
Lucia Mazzacurati ◽  
Adriana Forero ◽  
Chang-Sook Hong ◽  
Junichi Eguchi ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Virus Replication ◽  
Immune Escape ◽  
Immune Surveillance ◽  
Innate Immune ◽  
Glioblastoma Cells ◽  
Indoleamine 2,3 Dioxygenase ◽  
Oncolytic Herpes Simplex Virus ◽  
Hel Cells ◽  
Simplex Virus

Successful oncolytic virus treatment of malignant glioblastoma multiforme depends on widespread tumor-specific lytic virus replication and escape from mitigating innate immune responses to infection. Here we characterize a new HSV vector, JD0G, that is deleted for ICP0 and the joint sequences separating the unique long and short elements of the viral genome. We observed that JD0G replication was enhanced in certain glioblastoma cell lines compared to HEL cells, suggesting that a vector backbone deleted for ICP0 may be useful for treatment of glioblastoma. The innate immune response to virus infection can potentially impede oncolytic vector replication in human tumors. Indoleamine-2,3-dioxygenase (IDO) is expressed in response to interferonγ(IFNγ) and has been linked to both antiviral functions and to the immune escape of tumor cells. We observed that IFNγtreatment of human glioblastoma cells induced the expression of IDO and that this expression was quelled by infection with both wild-type and JD0G viruses. The role of IDO in inhibiting virus replication and the connection of this protein to the escape of tumor cells from immune surveillance suggest that IDO downregulation by HSV infection may enhance the oncolytic activity of vectors such as JD0G.

scholarly journals Enhanced virus translation enables miR-122-independent Hepatitis C Virus propagation

2021 ◽  
Author(s):  
Mamata Panigrahi ◽  
Michael Palmer ◽  
Joyce A Wilson
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Replication ◽  
Rna Structure ◽  
Genome Stability ◽  
Internal Ribosomal Entry Site ◽  
Terminal Region ◽  
Translation Regulation ◽  
Virus Propagation ◽  
Entry Site

The 5’UTR of the Hepatitis C Virus genome forms RNA structures that regulate virus replication and translation. The region contains a viral internal ribosomal entry site and a 5’ terminal region. Binding of the liver specific miRNA, miR-122, to two conserved binding sites in the 5’ terminal region regulates viral replication, translation, and genome stability, and is essential for efficient virus replication, but its precise mechanism of its action is still under debate. A current hypothesis is that miR-122 binding stimulates viral translation by facilitating the viral 5’ UTR to form the translationally active HCV IRES RNA structure. While miR-122 is essential for detectable virus replication in cell culture, several viral variants with 5’ UTR mutations exhibit low level replication in the absence of miR-122. We show that HCV mutants capable of replicating independently of miR-122 also replicate independently of other microRNAs generated by the canonical miRNA synthesis pathway. Further, we also show that the mutant genomes display an enhanced translation phenotype that correlates with their ability to replicate independently of miR-122. Finally, we provide evidence that translation regulation is the major role for miR-122, and show that miR-122-independent HCV replication can be rescued to miR-122-dependent levels by the combined impacts of 5’ UTR mutations that stimulate translation, and by stabilizing the viral genome by knockdown of host exonucleases and phosphatases that degrade the genome. Thus, we provide a model suggesting that translation stimulation and genome stabilization are the primary roles for miR-122 in the virus life cycle.

scholarly journals Herpes simplex virus 1 evades cellular antiviral response by inducing microRNA-24, which attenuates STING synthesis

2021 ◽  
Vol 17 (9) ◽  
pp. e1009950
Author(s):  
Nikhil Sharma ◽  
Chenyao Wang ◽  
Patricia Kessler ◽  
Ganes C. Sen
Keyword(s):  
Immune Response ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Replication ◽  
Map Kinases ◽  
Nodal Point ◽  
Type I ◽  
Herpes Simplex Virus 1 ◽  
Antiviral Immune Response ◽  
Simplex Virus

STING is a nodal point for cellular innate immune response to microbial infections, autoimmunity and cancer; it triggers the synthesis of the antiviral proteins, type I interferons. Many DNA viruses, including Herpes Simplex Virus 1 (HSV1), trigger STING signaling causing inhibition of virus replication. Here, we report that HSV1 evades this antiviral immune response by inducing a cellular microRNA, miR-24, which binds to the 3’ untranslated region of STING mRNA and inhibits its translation. Expression of the gene encoding miR-24 is induced by the transcription factor AP1 and activated by MAP kinases in HSV1-infected cells. Introduction of exogenous miR-24 or prior activation of MAPKs, causes further enhancement of HSV1 replication in STING-expressing cells. Conversely, transfection of antimiR-24 inhibits virus replication in those cells. HSV1 infection of mice causes neuropathy and death; using two routes of infection, we demonstrated that intracranial injection of antimiR-24 alleviates both morbidity and mortality of the infected mice. Our studies reveal a new immune evasion strategy adopted by HSV1 through the regulation of STING and demonstrates that it can be exploited to enhance STING’s antiviral action.

scholarly journals High-Resolution 3D Imaging of Virus Infections in Solvent-Cleared Organs: Novel Insights into Virus Replication and Tropism In Vivo

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 75
Author(s):  
Luca Zaeck ◽  
Madlin Potratz ◽  
Antonia Klein ◽  
Conrad M. Freuling ◽  
Thomas Müller ◽  
...  
Keyword(s):  
Rabies Virus ◽  
Glial Cells ◽  
Virus Replication ◽  
3D Visualization ◽  
Virus Protein ◽  
3D Analysis ◽  
Virus Infections ◽  
Thin Sections ◽  
Cellular Environment

The visualization of infection processes in relevant tissues and organs using microscopy methods reveals a unique link between the distribution, tropism, and abundance of pathogens and the physiological structure of the respective organ. To dissect virus replication and the host reaction in vivo at both a global and a single-cell level, conventional 2D imaging approaches can only provide limited insight. However, pathological studies of infected organ material are still mostly restricted to the immunostaining of thin sections from paraffin-embedded or frozen samples. While the 3D analysis of large tissue volumes is possible via laborious serial sectioning, a variety of problems and artifacts remain. Modern immunostaining-compatible tissue clearing techniques allow for the seamless 3D visualization of infection sites in optically cleared thick tissues sections or even entire organs. Benefiting from pure optical slicing, this approach enables the acquisition of multicolor high-volume 3D image stacks for coherent qualitative and quantitative analyses of the infection and its surrounding cellular environment. Here, we demonstrate the utility and power of this methodology by visualizing virus infections in different target tissues. For instance, we reconstructed the cellular context of rabies virus infection sites in mouse brain tissue, allowing a thorough investigation and quantitative analysis of rabies virus cell tropism. The systematic comparison of different rabies viruses with varying pathogenicity revealed a remarkable difference for highly virulent street rabies viruses and attenuated lab strains. While the virus protein expression was readily detectable at a comparable level in both neurons and non-neuronal glial cells from brains of mice infected with street rabies viruses, it was virtually absent in glial cells of lab strain-infected mice. These data provide novel and detailed insights into the pathogenesis of virus infections and substantially contribute to an improved understanding of virus–host interactions in vivo.

scholarly journals Distinct sites for myotoxic and membrane-damaging activities in the C-terminal region of a Lys49-phospholipase A2

2002 ◽  
Vol 366 (3) ◽  
pp. 971-976 ◽  
Author(s):  
Lucimara CHIOATO ◽  
Arthur H.C. de OLIVEIRA ◽  
Roberto RULLER ◽  
Juliana M. SÁ ◽  
Richard J. WARD
Keyword(s):  
Phospholipase A2 ◽  
Lipid Membrane ◽  
Terminal Region ◽  
Site Directed Mutagenesis ◽  
Phospholipid Bilayer ◽  
Directed Mutagenesis ◽  
Aromatic Residues ◽  
Loop Region ◽  
Terminal Loop ◽  
Arginine Residues

Bothropstoxin-I (BthTx-I) is a Lys49-phospholipase A2 from the venom of Bothrops jararacussu which demonstrates both myotoxic and Ca2+-independent membrane-damaging activities. The structural determinants of these activities are poorly defined, therefore site-directed mutagenesis has been used to substitute all cationic and aromatic residues between positions 115 and 129 in the C-terminal loop region of the protein. Substitution of lysine and arginine residues with alanine in the region 117—122 resulted in a significant reduction of myotoxic activity of the recombinant BthTx-I. With the exception of Lys122, these same substitutions did not significantly alter the Ca2+-independent membrane-damaging activity. In contrast, substitution of the positively-charged residues at positions 115, 116 and 122 resulted in reduced Ca2+-independent membrane-damaging activity but, with the exception of Lys122, had no effect on myotoxicity. These results indicate that the two activities are independent and are determined by discrete yet partially overlapping motifs in the C-terminal loop. Results from site-directed mutagenesis of the aromatic residues in the same part of the protein suggest that a region including residues 115—119 interacts superficially with the membrane interface and that the residues around position 125 partially insert into the lipid membrane. These results represent the first detailed mapping of a myotoxic site in a phospholipase A2, and support a model of a Ca2+-independent membrane-damaging mechanism in which the C-terminal region of BthTx-I interacts with and contributes to the perturbation of the phospholipid bilayer.

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