scholarly journals Immunological Effects and Viral Gene Expression Determine the Efficacy of Oncolytic Measles Vaccines Encoding IL-12 or IL-15 Agonists

Viruses ◽  
2019 ◽  
Vol 11 (10) ◽  
pp. 914 ◽  
Author(s):  
Backhaus ◽  
Veinalde ◽  
Hartmann ◽  
Dunder ◽  
Jeworowski ◽  
...  
Keyword(s):  
Gene Expression ◽  
Fusion Protein ◽  
Therapeutic Efficacy ◽  
Measles Virus ◽  
Nk Cell ◽  
Viral Gene Expression ◽  
Interleukin 12 ◽  
Viral Gene ◽  
Interleukin 15

Tumor-targeted immunomodulation using oncolytic viral vectors is currently being investigated as a promising strategy in cancer therapy. In a previous study, we showed that a measles virus Schwarz vaccine strain (MeVac) vector encoding an interleukin-12 fusion protein (FmIL-12) is an effective immunotherapy in the MC38cea murine colon adenocarcinoma model. We hypothesized that MeVac encoding interleukin-15 may mediate enhanced T and NK cell responses and thus increase the therapeutic efficacy, especially in NK cell-controlled tumors. Therefore, we generated MeVac vectors encoding an interleukin-15 superagonist, FmIL-15. Replication and oncolytic capacity, transgene expression, and functionality of MeVac FmIL-15 vectors were validated in vitro. Effects on the tumor immune landscape and therapeutic efficacy of both FmIL-12 and FmIL-15 vectors were studied in the MC38cea and B16hCD46 tumor models. Treatment with MeVac FmIL-15 increased T and NK cell infiltration in both models. However, MeVac FmIL-12 showed more robust viral gene expression and immune activation, resulting in superior anti-tumor efficacy. Based on these results, MeVac encoding a human IL-12 fusion protein was developed for future clinical translation.

scholarly journals HCMV exploits STING signaling and counteracts IFN and ISG induction to facilitate dendritic cell infection

2022 ◽  
Author(s):  
Bibiana Costa ◽  
Jennifer Becker ◽  
Tobias Krammer ◽  
Felix Mulenge ◽  
Verónica Durán ◽  
...  
Keyword(s):  
Gene Expression ◽  
Viral Gene Expression ◽  
Productive Infection ◽  
Viral Gene ◽  
Human Pathogen ◽  
Cell Infection ◽  
Complex Interactions ◽  
Life Threatening ◽  
Immunocompromised Hosts

Abstract Human cytomegalovirus (HCMV) is a widespread obligatory human pathogen causing life-threatening disease in immunocompromised hosts. Myeloid cells such as monocyte-derived dendritic cells (moDCs) are targets of HCMV. Here, we performed single-cell RNA sequencing, which revealed infection of most moDCs upon in vitro HCMV exposure, whereas only a fraction of them initiated viral gene expression. We identified three moDC subsets, of which CD1a−/CD86− cells showed the highest susceptibility. Upon HCMV entry, STING activation not only induced IFN-β, but also promoted viral gene expression. Upon progression of infection, IFN-β but not IFN-λ1 expression was inhibited. Similarly, ISG expression was initially induced and then shut off and thus allowed productive infection. Increased viral gene expression was associated with the induction of several pro- (RHOB, HSP1A1, DNAJB1) and anti-viral (RNF213, TNFSF10, IFI16) genes. Thus, moDC permissiveness to HCMV depends on complex interactions between virus sensing, regulation of IFNs/ISGs and viral gene expression.

scholarly journals Kaposi's Sarcoma-Associated Herpesvirus Induces Sustained NF-κB Activation during De Novo Infection of Primary Human Dermal Microvascular Endothelial Cells That Is Essential for Viral Gene Expression

2007 ◽  
Vol 81 (8) ◽  
pp. 3949-3968 ◽  
Author(s):  
Sathish Sadagopan ◽  
Neelam Sharma-Walia ◽  
Mohanan Valiya Veettil ◽  
Hari Raghu ◽  
Ramu Sivakumar ◽  
...  
Keyword(s):  
Gene Expression ◽  
Growth Factors ◽  
De Novo ◽  
Viral Gene Expression ◽  
Angiogenic Factors ◽  
Viral Gene ◽  
Kshv Infection ◽  
Time Points ◽  
Lytic Genes

ABSTRACT In vitro Kaposi's sarcoma-associated herpesvirus (KSHV) infection of primary human dermal microvascular endothelial (HMVEC-d) cells and human foreskin fibroblast (HFF) cells is characterized by the induction of preexisting host signal cascades, sustained expression of latency-associated genes, transient expression of a limited number of lytic genes, and induction of several cytokines, growth factors, and angiogenic factors. Since NF-κB is a key molecule involved in the regulation of several of these factors, here, we examined NF-κB induction during de novo infection of HMVEC-d and HFF cells. Activation of NF-κB was observed as early as 5 to 15 min postinfection by KSHV, and translocation of p65-NF-κB into nuclei was detected by immunofluorescence assay, electrophoretic mobility shift assay, and p65 enzyme-linked immunosorbent assay. IκB phosphorylation inhibitor (Bay11-7082) reduced this activation significantly. A sustained moderate level of NF-κB induction was seen during the observed 72 h of in vitro KSHV latency. In contrast, high levels of ERK1/2 activation at earlier time points and a moderate level of activation at later times were observed. p38 mitogen-activated protein kinase was activated only at later time points, and AKT was activated in a cyclic manner. Studies with UV-inactivated KSHV suggested a role for virus entry stages in NF-κB induction and a requirement for KSHV viral gene expression in sustained induction. Inhibition of NF-κB did not affect target cell entry by KSHV but significantly reduced the expression of viral latent open reading frame 73 and lytic genes. KSHV infection induced the activation of several host transcription factors, including AP-1 family members, as well as several cytokines, growth factors, and angiogenic factors, which were significantly affected by NF-κB inhibition. These results suggest that during de novo infection, KSHV induces sustained levels of NF-κB to regulate viral and host cell genes and thus possibly regulates the establishment of latent infection.

Cell-cycle-dependent localization of human cytomegalovirus UL83 phosphoprotein in the nucleolus and modulation of viral gene expression in human embryo fibroblasts in vitro

2011 ◽  
Vol 112 (1) ◽  
pp. 307-317 ◽  
Author(s):  
Maria-Cristina Arcangeletti ◽  
Isabella Rodighiero ◽  
Prisco Mirandola ◽  
Flora De Conto ◽  
Silvia Covan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Human Cytomegalovirus ◽  
Human Embryo ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Embryo Fibroblasts ◽  
Cycle Dependent

scholarly journals Expression of varicella-zoster virus VLT-ORF63 fusion transcript induces broad viral gene expression during reactivation from neuronal latency

2020 ◽  
Author(s):  
Werner J. D. Ouwendijk ◽  
Daniel P. Depledge ◽  
Labchan Rajbhandari ◽  
Tihana Lenac Rovis ◽  
Stipan Jonjic ◽  
...  
Keyword(s):  
Gene Expression ◽  
Varicella Zoster Virus ◽  
Viral Gene Expression ◽  
Fusion Transcript ◽  
Viral Gene ◽  
Reading Frame ◽  
Varicella Zoster ◽  
Rapid Amplification

SummaryVaricella-zoster virus (VZV) establishes lifelong neuronal latency in most humans world-wide, reactivating in one-third to cause herpes zoster and occasionally chronic pain. How VZV establishes, maintains and reactivates from latency is largely unknown. Latent VZV gene expression is restricted to VZV latency-associated transcript (VLT) and open reading frame 63 (ORF63) in naturally VZV-infected human trigeminal ganglia (TG). Notably, these transcript levels positively correlated suggesting co-regulated transcription during latency. Here, we used direct RNA-sequencing to identify fusion transcripts that combine VLT and ORF63 loci (VLT-ORF63) and are expressed during both lytic and latent VZV infections. Furthermore, real-time PCR, RNA in situ hybridization and 5’ rapid amplification of cDNA ends (RACE) all confirmed VLT-ORF63, but not canonical ORF63, expression in human TG. During lytic infection, one of the two major VLT-ORF63 isoforms encodes a novel fusion protein combining VLT and ORF63 proteins (pVLT-ORF63). In vitro, VLT is transcribed in latently VZV-infected human sensory neurons, whereas VLT-ORF63 expression is induced by reactivation stimuli. Moreover, the pVLT-ORF63-encoding VLT-ORF63 isoform induced transcription of lytic VZV genes. Collectively, our findings show that VZV expresses a unique set of VLT-ORF63 transcripts, potentially involved in the transition from latency to lytic VZV infection.

scholarly journals hsp72, a Host Determinant of Measles Virus Neurovirulence

2006 ◽  
Vol 80 (22) ◽  
pp. 11031-11039 ◽  
Author(s):  
Thomas Carsillo ◽  
Zachary Traylor ◽  
Changsun Choi ◽  
Stefan Niewiesk ◽  
Michael Oglesbee
Keyword(s):  
Gene Expression ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Protein Interactions ◽  
Measles Virus ◽  
Viral Rna ◽  
Viral Gene ◽  
Febrile Episodes

ABSTRACT Transient hyperthermia such as that experienced during febrile episodes increases expression of the major inducible 70-kDa heat shock protein (hsp72). Despite the relevance of febrile episodes to viral pathogenesis and the multiple in vitro roles of heat shock proteins in viral replication and gene expression, the in vivo significance of virus-heat shock protein interactions is unknown. The present work determined the in vivo relationship between hsp72 levels and neurovirulence of an hsp72-responsive virus using the mouse model of measles virus (MV) encephalitis. Transgenic C57BL/6 mice were created to constitutively overexpress hsp72 in neurons, and these mice were inoculated intracranially with Edmonston MV (Ed MV) at 42 h of age. The mean viral RNA burden in brain was approximately 2 orders of magnitude higher in transgenic animals than in nontransgenic animals 2 to 4 weeks postinfection, and this increased burden was associated with a fivefold increase in mortality. Mice were also challenged with an Ed MV variant exhibiting an attenuated in vitro response to hsp72-dependent stimulation of viral transcription (Ed N-522D). This virus exhibited an attenuated neuropathogenicity in transgenic mice, where mortality and viral RNA burdens were not significantly different from nontransgenic mice infected with either Ed N-522D or parent Ed MV. Collectively, these results indicate that hsp72 levels can serve as a host determinant of viral neurovirulence in C57BL/6 mice, reflecting the direct influence of hsp72 on viral gene expression.

scholarly journals Genome-Wide Analysis of Heterogeneous Nuclear Ribonucleoprotein (hnRNP) Binding to HIV-1 RNA Reveals a Key Role for hnRNP H1 in Alternative Viral mRNA Splicing

2019 ◽  
Vol 93 (21) ◽  
Author(s):  
Sebla B. Kutluay ◽  
Ann Emery ◽  
Srinivasa R. Penumutchu ◽  
Dana Townsend ◽  
Kasyap Tenneti ◽  
...  
Keyword(s):  
Gene Expression ◽  
Alternative Splicing ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Hnrnp A1 ◽  
Viral Mrnas ◽  
Temporal Regulation ◽  
Coding Potential ◽  
Hiv 1

ABSTRACT Alternative splicing of HIV-1 mRNAs increases viral coding potential and controls the levels and timing of gene expression. HIV-1 splicing is regulated in part by heterogeneous nuclear ribonucleoproteins (hnRNPs) and their viral target sequences, which typically repress splicing when studied outside their native viral context. Here, we determined the location and extent of hnRNP binding to HIV-1 mRNAs and their impact on splicing in a native viral context. Notably, hnRNP A1, hnRNP A2, and hnRNP B1 bound to many dispersed sites across viral mRNAs. Conversely, hnRNP H1 bound to a few discrete purine-rich sequences, a finding that was mirrored in vitro. hnRNP H1 depletion and mutation of a prominent viral RNA hnRNP H1 binding site decreased the use of splice acceptor A1, causing a deficit in Vif expression and replicative fitness. This quantitative framework for determining the regulatory inputs governing alternative HIV-1 splicing revealed an unexpected splicing enhancer role for hnRNP H1 through binding to its target element. IMPORTANCE Alternative splicing of HIV-1 mRNAs is an essential yet quite poorly understood step of virus replication that enhances the coding potential of the viral genome and allows the temporal regulation of viral gene expression. Although HIV-1 constitutes an important model system for general studies of the regulation of alternative splicing, the inputs that determine the efficiency with which splice sites are utilized remain poorly defined. Our studies provide an experimental framework to study an essential step of HIV-1 replication more comprehensively and in much greater detail than was previously possible and reveal novel cis-acting elements regulating HIV-1 splicing.

scholarly journals Effects of Altering the Transcription Termination Signals of Respiratory Syncytial Virus on Viral Gene Expression and Growth In Vitro and In Vivo

2004 ◽  
Vol 78 (2) ◽  
pp. 692-699 ◽  
Author(s):  
Kim C. Tran ◽  
Peter L. Collins ◽  
Michael N. Teng
Keyword(s):  
Gene Expression ◽  
Respiratory Syncytial Virus ◽  
Respiratory Tract ◽  
Transcription Termination ◽  
Regulation Of Gene Expression ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Mrna Levels ◽  
Syncytial Virus

ABSTRACT Nonsegmented negative-sense RNA viruses (mononegaviruses) control viral gene expression largely through a transcription gradient such that promoter-proximal genes are transcribed more abundantly than downstream genes. For some paramyxoviruses, naturally occurring differences in the levels of efficiency of transcription termination by various gene end (GE) signals provide an additional level of regulation of gene expression. The first two genes (NS1 and NS2) of respiratory syncytial virus (RSV) are particularly inefficient in termination. We investigated whether altering the termination efficiency (TE) of these two genes in infectious recombinant virus would affect transcription of promoter-proximal and promoter-distal genes, production of viral proteins, and viral replication in cell culture and in the respiratory tract of mice. Recombinant RSVs were constructed with mutations that increased or decreased the TE of the NS1 GE signal, increased that of the NS2 GE signal, or increased that of both signals. Increasing the TE of either or both GE signals resulted in decreased production of the related polycistronic readthrough mRNAs, which normally arise due to the failure of the viral polymerase to recognize the GE signal. This was accompanied by a small increase in the levels of monocistronic NS1 and NS2 mRNAs. Conversely, decreasing the TE of the NS1 GE increased the production of readthrough mRNAs concomitant with a decrease of monocistronic NS1 and NS2 mRNA levels. These changes were reflected in the levels of NS1 and NS2 protein. All of the mutant viruses displayed growth kinetics and virus yields similar to wild-type recombinant RSV (rA2) in both HEp-2 and Vero cells. In addition, all mutants grew similarly to rA2 in the upper- and lower-respiratory tract of BALB/c mice, though some of the mutants displayed slightly decreased replication. These data suggest that the natural inefficiencies of transcription termination by the NS1 and NS2 GE signals do not play important roles in controlling the magnitude of RSV gene expression or the efficiency of virus replication. Furthermore, while changes in the TE of a GE signal clearly can affect the transcription of its gene as well as that of the one immediately downstream, these changes did not have a significant effect on the overall transcriptional gradient.

scholarly journals HTLV-1 Rex is required for viral spread and persistence in vivo but is dispensable for cellular immortalization in vitro

Blood ◽  
2003 ◽  
Vol 102 (12) ◽  
pp. 3963-3969 ◽  
Author(s):  
Jianxin Ye ◽  
Lee Silverman ◽  
Michael D. Lairmore ◽  
Patrick L. Green
Keyword(s):  
Gene Expression ◽  
Interleukin 2 ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Viral Oncoprotein ◽  
Cell Leukemia Virus Type ◽  
Viral Spread ◽  
Human T Cell

Abstract Human T-cell leukemia virus type 1 (HTLV-1) is associated with leukemia/lymphoma and neurologic disorders. Although the viral transcriptional activator Tax is the critical viral oncoprotein, Rex, which regulates the expression of the viral structural and enzymatic genes, is essential for efficient viral replication. Herein, we investigate the contribution of Rex in HTLV-1 immortalization of primary T cells in vitro and viral survival in an infectious rabbit animal model. A Rex-deficient HTLV-1 (HTLVRex-) was constructed and characterized for viral gene expression, protein production, and immortalization capacity. Cells transiently transfected with the HTLVRex- proviral clone produced low detectable levels of p19 Gag. 729HTLVRex- stable transfectants produced functional Tax, but undetectable levels of Rex or p19 Gag. Coculture of irradiated 729HTLVRex- cells with peripheral blood mononuclear cells (PBMCs) resulted in sustained interleukin-2 (IL-2)-dependent growth of primary T lymphocytes. These cells carried the HTLVRex- genome and expressed tax/rex mRNA but produced no detectable Rex or p19 Gag. Rabbits inoculated with irradiated 729HTLVRex- cells or 729HTLVRex- cells transiently transfected with a Rex cDNA expression plasmid failed to become persistently infected or mount a detectable antibody response to the viral gene products. Together, our results provide the first direct evidence that Rex and its function to modulate viral gene expression and virion production is not required for in vitro immortalization by HTLV-1. However, Rex is critical for efficient infection of cells and persistence in vivo.

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