scholarly journals Expressing the pro-apoptotic Reaper protein via insertion into the structural open reading frame of Sindbis virus reduces the ability to infect Aedes aegypti mosquitoes

2022 ◽  
Author(s):  
Alexis Carpenter ◽  
Rollie J Clem
Keyword(s):  
Aedes Aegypti ◽  
Sindbis Virus ◽  
Cultured Cells ◽  
Virus Titer ◽  
Open Reading Frame ◽  
Infection Prevalence ◽  
Reading Frame ◽  
Disseminated Infection ◽  
Control Virus ◽  
Induced Apoptosis

Arboviruses continue to threaten a significant portion of the human population, and a better understanding is needed of the determinants of successful arbovirus infection of arthropod vectors. Avoiding apoptosis has been shown to be one such determinant. Previous work showed that a Sindbis virus (SINV) construct called MRE/rpr that expresses the pro-apoptotic protein Reaper via a duplicated subgenomic promoter had a reduced ability to orally infect Aedes aegypti mosquitoes at 3 days post-blood meal (PBM), but this difference diminished over time as virus variants containing deletions in the inserted reaper gene rapidly predominated. The goal of this study was to generate a SINV construct that more stably expressed Reaper, in order to further clarify the effect of midgut apoptosis on disseminated infection in Ae. aegypti. We did this by inserting reaper as an in-frame fusion into the structural open reading frame (ORF) of SINV. This construct, MRE/rprORF, successfully expressed Reaper, replicated similarly to MRE/rpr in cell lines, and induced apoptosis in cultured cells and in mosquito midgut tissue. Mosquitoes that fed on blood containing MRE/rprORF developed less midgut and disseminated infection when compared to MRE/rpr or a control virus up to at least 7 days PBM, when less than 50% of mosquitoes that ingested MRE/rprORF had detectable disseminated infection, compared with around 80% or more of mosquitoes fed with MRE/rpr or control virus. However, virus titer in mosquitoes infected with MRE/rprORF was not significantly different from control virus, suggesting that induction of apoptosis by expression of Reaper by this method can reduce infection prevalence, but if infection is established then apoptosis induced by this method has limited ability to continue to suppress replication.

scholarly journals Infection of Aedes aegypti mosquitoes with midgut-attenuated Sindbis virus reduces, but does not eliminate, disseminated infection

2021 ◽  
Author(s):  
Alexis Carpenter ◽  
William Bart Bryant ◽  
Scott R. Santos ◽  
Rollie J. Clem
Keyword(s):  
Aedes Aegypti ◽  
Sindbis Virus ◽  
Midgut Epithelium ◽  
New Host ◽  
Reading Frame ◽  
Disseminated Infection ◽  
Control Virus ◽  
Almost All ◽  
Blood Meals ◽  
Mirna Targeting

Arboviruses are transmitted by specific vectors and the reasons for this specificity are not fully understood. One contributing factor is the existence of tissue barriers within the vector such as the midgut escape barrier. We used miRNA targeting of Sindbis virus (SINV) to study how replication in midgut cells contributes to overcoming this barrier in the mosquito Aedes aegypti. SINV constructs were designed to be attenuated specifically in midgut cells by inserting binding sites for midgut-specific miRNAs into either the 3′ untranslated region (MRE3′miRT) or the structural open reading frame (MRE-ORFmiRT) of the SINV genome. Both miRNA-targeted viruses replicated less efficiently than control viruses in the presence of these miRNAs. When mosquitoes were given infectious blood meals containing miRNA-targeted viruses, only around 20% (MRE3′miRT) or 40% (MRE-ORFmiRT) of mosquitoes developed disseminated infection. In contrast, dissemination occurred in almost all mosquitoes fed control viruses. Deep sequencing of virus populations from individual mosquitoes ruled out selection for mutations in the inserted target sequences as being the cause for dissemination in these mosquitoes. In mosquitoes that became infected with miRNA-targeted viruses, titers were equivalent to control virus in both the midgut and the carcass and there was no evidence of a threshold titer necessary for dissemination. Instead, it appeared that if infection was successfully established in the midgut, replication and dissemination were largely normal. Our results support the hypothesis that replication is an important factor in allowing SINV to overcome the midgut escape barrier, but hint that other factors are also likely involved. IMPORTANCE When a mosquito ingests an arbovirus during a blood meal, the arbovirus must escape from the midgut of the vector and infect the salivary glands in order to be transmitted to a new host. We used tissue-specific miRNA targeting to examine the requirement for Sindbis virus (SINV) to replicate in midgut epithelium in order to cause disseminated infection in the mosquito Aedes aegypti. Our results indicate that specifically reducing the ability of SINV to replicate in the mosquito midgut reduces its overall ability to establish infection in the mosquito, but if infection is established, replication and dissemination occur normally. These results are consistent with an importance for replication in the midgut epithelium in aiding arboviruses in crossing the midgut barrier.

scholarly journals Evidence That RNA Silencing-Mediated Resistance to Beet necrotic yellow vein virus Is Less Effective in Roots Than in Leaves

2005 ◽  
Vol 18 (3) ◽  
pp. 194-204 ◽  
Author(s):  
Ida Bagus Andika ◽  
Hideki Kondo ◽  
Tetsuo Tamada
Keyword(s):  
Nicotiana Benthamiana ◽  
Rna Silencing ◽  
Defense Mechanism ◽  
Virus Titer ◽  
Virus Multiplication ◽  
Open Reading Frame ◽  
Yellow Vein ◽  
Polymyxa Betae ◽  
Small Interfering Rnas ◽  
Reading Frame

In plants, RNA silencing is part of a defense mechanism against virus infection but there is little information as to whether RNA silencing-mediated resistance functions similarly in roots and leaves. We have obtained transgenic Nicotiana benthamiana plants encoding the coat protein readthrough domain open reading frame (54 kDa) of Beet necrotic yellow vein virus (BNYVV), which either showed a highly resistant or a recovery phenotype following foliar rub-inoculation with BNYVV. These phenotypes were associated with an RNA silencing mechanism. Roots of the resistant plants that were immune to foliar rub-inoculation with BNYVV could be infected by viruliferous zoospores of the vector fungus Polymyxa betae, although virus multiplication was greatly limited. In addition, virus titer was reduced in symptomless leaves of the plants showing the recovery phenotype, but it was high in roots of the same plants. Compared with leaves of silenced plants, higher levels of transgene mRNAs and lower levels of transgene-derived small interfering RNAs (siRNAs) accumulated in roots. Similarly, in nontransgenic plants inoculated with BNYVV, accumulation level of viral RNA-derived siRNAs in roots was lower than in leaves. These results indicate that the RNA silencing-mediated resistance to BNYVV is less effective in roots than in leaves.

Open reading frame 5 of porcine reproductive and respiratory syndrome virus as a cause of virus-induced apoptosis.

1996 ◽  
Vol 70 (5) ◽  
pp. 2876-2882 ◽  
Author(s):  
P Suárez ◽  
M Díaz-Guerra ◽  
C Prieto ◽  
M Esteban ◽  
J M Castro ◽  
...  
Keyword(s):  
Open Reading Frame ◽  
Respiratory Syndrome Virus ◽  
Reading Frame ◽  
Induced Apoptosis

scholarly journals Open reading frame Bm21 of Bombyx mori nucleopolyhedrovirus is not essential for virus replication in vitro, but its deletion extends the median survival time of infected larvae

2008 ◽  
Vol 89 (4) ◽  
pp. 922-930 ◽  
Author(s):  
Jinshan Huang ◽  
Bifang Hao ◽  
Fei Deng ◽  
Xiulian Sun ◽  
Hualin Wang ◽  
...  
Keyword(s):  
Survival Time ◽  
Bombyx Mori ◽  
Median Survival ◽  
Virus Replication ◽  
Median Survival Time ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Group I ◽  
Control Virus

In this report, the open reading frame 21 (Bm21) of Bombyx mori nucleopolyhedrovirus (BmNPV), one of the unique genes of group I NPVs, was characterized. Bm21 is predicted to encode a protein of 55.8 kDa and was found to contain imperfectly conserved leucine-rich repeats. 3′ Rapid amplification of cDNA ends (3′RACE) showed that the transcript of Bm21 was first detected from 6 h post-infection and that it also encompassed the complete Bm20. 5′RACE revealed three transcription initiation sites, one of which mapped to the baculovirus early transcription motifs CGTGC and CAGT. Transient-expression and superinfection assays indicated that BM21 localized in the nucleus of infected BmN cells. To study the function of BM21, a Bm21-null virus was constructed using bacmid technology. Viral one-step growth curve analyses showed that the Bm21-null virus had similar budded virus production kinetics to those of the parental virus. Bioassay analyses showed that the median lethal concentration (LC50) of the Bm21-null virus was similar to that of the control virus; however, the median survival time (ST50) of the knockout virus was significantly longer than the control virus. These results indicate that BM21 is not essential for virus replication in vitro, but that deletion of the gene delays the killing of the infected larvae.

scholarly journals Investigation of Biological Factors Contributing to Individual Variation in Viral Titer after Oral Infection of Aedes aegypti Mosquitoes by Sindbis Virus

Viruses ◽  
2022 ◽  
Vol 14 (1) ◽  
pp. 131
Author(s):  
Peter Hodoameda ◽  
Linus Addae ◽  
Rollie J. Clem
Keyword(s):  
Aedes Aegypti ◽  
Sindbis Virus ◽  
Vector Competence ◽  
High Titer ◽  
Mosquito Species ◽  
Virus Titer ◽  
Laboratory Strain ◽  
Virus Concentration ◽  
Biological Factors ◽  
Single Experiment

The mechanisms involved in determining arbovirus vector competence, or the ability of an arbovirus to infect and be transmitted by an arthropod vector, are still incompletely understood. It is well known that vector competence for a particular arbovirus can vary widely among different populations of a mosquito species, which is generally attributed to genetic differences between populations. What is less understood is the considerable variability (up to several logs) that is routinely observed in the virus titer between individual mosquitoes in a single experiment, even in mosquitoes from highly inbred lines. This extreme degree of variation in the virus titer between individual mosquitoes has been largely ignored in past studies. We investigated which biological factors can affect titer variation between individual mosquitoes of a laboratory strain of Aedes aegypti, the Orlando strain, after Sindbis virus infection. Greater titer variation was observed after oral versus intrathoracic infection, suggesting that the midgut barrier contributes to titer variability. Among the other factors tested, only the length of the incubation period affected the degree of titer variability, while virus strain, mosquito strain, mosquito age, mosquito weight, amount of blood ingested, and virus concentration in the blood meal had no discernible effect. We also observed differences in culture adaptability and in the ability to orally infect mosquitoes between virus populations obtained from low and high titer mosquitoes, suggesting that founder effects may affect the virus titer in individual mosquitoes, although other explanations also remain possible.

scholarly journals Release of Genotype 1 Hepatitis E Virus from Cultured Hepatoma and Polarized Intestinal Cells Depends on Open Reading Frame 3 Protein and Requires an Intact PXXP Motif

2010 ◽  
Vol 84 (18) ◽  
pp. 9059-9069 ◽  
Author(s):  
Suzanne U. Emerson ◽  
Hanh T. Nguyen ◽  
Udana Torian ◽  
Danielle Burke ◽  
Ronald Engle ◽  
...  
Keyword(s):  
Hepatitis E Virus ◽  
Cultured Cells ◽  
Hepatitis E ◽  
Open Reading Frame ◽  
Intestinal Cells ◽  
Protein Accumulation ◽  
Genotype 1 ◽  
Virus Genotype ◽  
Reading Frame ◽  
Orf3 Protein

ABSTRACT Hepatitis E virus genotype 1 strain Sar55 replicated in subcloned Caco-2 intestinal cells and Huh7 hepatoma cells that had been transfected with in vitro transcribed viral genomes, and hepatitis E virions were released into the culture medium of both cell lines. Virus egress from cells depended on open reading frame 3 (ORF3) protein, and a proline-rich sequence in ORF3 was important for egress from cultured cells and for infection of macaques. Both intracellular ORF3 protein accumulation and virus release occurred at the apical membrane of polarized Caco-2 cells. ORF3 protein and lipids were intimately associated with virus particles produced in either cell line; ORF2 epitopes were masked in these particles and could not be immunoprecipitated with anti-ORF2.

scholarly journals Sindbis Virus Induces Apoptosis through a Caspase-Dependent, CrmA-Sensitive Pathway

1998 ◽  
Vol 72 (1) ◽  
pp. 452-459 ◽  
Author(s):  
Victor E. Nava ◽  
Antony Rosen ◽  
Michael A. Veliuona ◽  
Rollie J. Clem ◽  
Beth Levine ◽  
...  
Keyword(s):  
Cell Death ◽  
Sindbis Virus ◽  
Cultured Cells ◽  
Cowpox Virus ◽  
Necrosis Factor Alpha ◽  
Viral Proteases ◽  
Factor Alpha ◽  
Induced Apoptosis ◽  
Cell Suicide

ABSTRACT Sindbis virus infection of cultured cells and of neurons in mouse brains leads to programmed cell death exhibiting the classical characteristics of apoptosis. Although the mechanism by which Sindbis virus activates the cell suicide program is not known, we demonstrate here that Sindbis virus activates caspases, a family of death-inducing proteases, resulting in cleavage of several cellular substrates. To study the role of caspases in virus-induced apoptosis, we determined the effects of specific caspase inhibitors on Sindbis virus-induced cell death. CrmA (a serpin from cowpox virus) and zVAD-FMK (N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone) inhibited Sindbis virus-induced cell death, suggesting that cellular caspases facilitate apoptosis induced by Sindbis virus. Furthermore, CrmA significantly increased the rate of survival of infected mice. These inhibitors appear to protect cells by inhibiting the cellular death pathway rather than impairing virus replication or by inhibiting the nsP2 and capsid viral proteases. The specificity of CrmA indicates that the Sindbis virus-induced death pathway is similar to that induced by Fas or tumor necrosis factor alpha rather than being like the death pathway induced by DNA damage. Taken together, these data suggest a central role for caspases in Sindbis virus-induced apoptosis.

scholarly journals Human Polyomavirus-Encoded Circular RNAs

2020 ◽  
Author(s):  
Rong Yang ◽  
Eunice E. Lee ◽  
Jiwoong Kim ◽  
Joon H. Choi ◽  
Yating Chen ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Cultured Cells ◽  
Reporter Genes ◽  
T Antigen ◽  
Open Reading Frame ◽  
Human Polyomavirus ◽  
Circular Rnas ◽  
Large T Antigen ◽  
Reading Frame ◽  
Human Polyomaviruses

ABSTRACTCircular RNAs (circRNAs) are a conserved class of RNAs with diverse functions. A subset of circRNAs are translated into peptides. Here we describe circular RNAs encoded by human polyomaviruses (HPyVs), including circular forms of RNAs encoding variants of the previously described alternative large T antigen open reading frame (ALTO) gene. Circular ALTO RNAs (circALTOs) can be detected in virus positive Merkel cell carcinoma (VP-MCC) cell lines and tumor samples. CircALTOs are stable, predominantly located in the cytoplasm, and N6-methyladenosine (m6A) modified. MCPyV circALTOs produce ALTO protein in cultured cells. MCPyV ALTO promotes the transcription of co-transfected reporter genes. MCPyV circALTOs are enriched in exosomes derived from VP-MCC lines and circALTO-transfected 293T cells, and purified exosomes can mediate ALTO expression and transcriptional activation. The related trichodysplasia spinulosa polyomavirus (TSPyV) also expresses a circALTO that can be detected in infected tissues and produces ALTO protein in cultured cells. Thus, human polyomavirus circRNAs are expressed in human tumors and tissues, encode for proteins, and may contribute to the infectious and tumorigenic properties of these viruses.

scholarly journals Kaposi's Sarcoma-Associated Herpesvirus Open Reading Frame 50 Represses p53-Induced Transcriptional Activity and Apoptosis

2001 ◽  
Vol 75 (13) ◽  
pp. 6245-6248 ◽  
Author(s):  
Yousang Gwack ◽  
Seungmin Hwang ◽  
Hyewon Byun ◽  
Chunghun Lim ◽  
Jin Woo Kim ◽  
...  
Keyword(s):  
Kaposi's Sarcoma ◽  
Transcriptional Activity ◽  
Kaposi’S Sarcoma ◽  
Inhibitory Effect ◽  
Open Reading Frame ◽  
Creb Binding Protein ◽  
Reading Frame ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Induced Apoptosis ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) open reading frame 50 (ORF50) encodes a viral transcriptional activator which stimulates the transcription of viral early and late genes of KSHV. Here we show that ORF50 represses transcriptional activity of p53 and p53-induced apoptosis through interaction with CREB binding protein (CBP). This inhibitory effect of ORF50 on the transcriptional activity of p53 was relieved by the addition of CBP. ORF50 mutants, which are defective in interaction with CBP, lost the inhibitory effects on p53. Our data provide a framework for delineating the regulatory mechanisms used by KSHV to modulate cellular transcription and the cell cycle.

scholarly journals Vaccinia Virus Encodes I5, a Small Hydrophobic Virion Membrane Protein That Enhances Replication and Virulence in Mice

2008 ◽  
Vol 82 (20) ◽  
pp. 10071-10078 ◽  
Author(s):  
Cindy L. Sood ◽  
Jerrold M. Ward ◽  
Bernard Moss
Keyword(s):  
Vaccinia Virus ◽  
Stop Codon ◽  
Open Reading Frame ◽  
Wild Type Virus ◽  
Epidermal Keratinocytes ◽  
In Vitro Cultivation ◽  
Human Epidermal Keratinocytes ◽  
Reading Frame ◽  
Control Virus

ABSTRACT The vaccinia virus I5L open reading frame encodes a 79-amino-acid protein, with two predicted transmembrane domains, that is conserved among all sequenced members of the chordopoxvirus subfamily. No nonpoxvirus homologs or functional motifs have been recognized, and the role of the I5 protein remains unknown. We found that synthesis of I5 was dependent on viral DNA replication and occurred exclusively at late times, consistent with a consensus late promoter motif adjacent to the start of the open reading frame. I5 was present in preparations of purified virions and could be extracted with nonionic detergent, suggesting membrane insertion. Transmission electron microscopy of immunogold-labeled thawed cryosections of infected cells revealed the association of an epitope-tagged I5 with the membranes of immature and mature virions. Viable I5L deletion and frameshift mutants were constructed and found to replicate like wild-type virus in a variety of cell lines and primary human epidermal keratinocytes, indicating that the protein was dispensable for in vitro cultivation. However, mouse intranasal challenge experiments indicated that a mutant virus with a frameshift resulting in a stop codon near the N terminus of I5 was attenuated compared to control virus. The attenuation was correlated with clearance of mutant viruses from the respiratory tract and with less progression and earlier resolution of pathological changes. We suggest that I5 is involved in an aspect of host defense that is evolutionarily conserved although a role in cell tropism should also be considered.

Sign in / Sign up

Export Citation Format

Share Document