scholarly journals Characterization of Adenovirus 5 E1A Exon 1 Deletion Mutants in the Viral Replicative Cycle

Viruses ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 213
Author(s):  
Rita Costa ◽  
Nikolas Akkerman ◽  
Drayson Graves ◽  
Leandro Crisostomo ◽  
Scott Bachus ◽  
...  
Keyword(s):  
Viral Replication ◽  
Virus Replication ◽  
Cellular Localization ◽  
Negative Impact ◽  
S Phase ◽  
Viral Fitness ◽  
Deletion Mutants ◽  
Gene And Protein Expression ◽  
Exon 1 ◽  
Replicative Cycle

Human adenovirus infection is driven by Early region 1A (E1A) proteins, which are the first proteins expressed following the delivery of the viral genome to the cellular nucleus. E1A is responsible for reprogramming the infected cell to support virus replication alongside the activation of expression of all viral transcriptional units during the course of the infection. Although E1A has been extensively studied, most of these studies have focused on understanding the conserved region functions outside of a full infection. Here, we investigated the effects of small deletions in E1A exon 1 on the viral replicative cycle. Almost all deletions were found to have a negative impact on viral replication with the exception of one deletion found in the mutant dl1106, which replicated better than the wild-type E1A expressing dl309. In addition to growth, we assessed the virus mutants for genome replication, induction of the cytopathic effect, gene and protein expression, sub-cellular localization of E1A mutant proteins, induction of cellular S-phase, and activation of S-phase specific cellular genes. Importantly, our study found that virus replication is likely limited by host-specific factors, rather than specific viral aspects such as the ability to replicate genomes or express late proteins, after a certain level of these has been expressed. Furthermore, we show that mutants outside of the conserved regions have significant influence on viral fitness. Overall, our study is the first comprehensive evaluation of the dl1100 series of exon 1 E1A deletion mutants in viral fitness and provides important insights into the contribution that E1A makes to viral replication in normal human cells.

scholarly journals Dominant-Negative FADD Rescues the In Vivo Fitness of a Cytomegalovirus Lacking an Antiapoptotic Viral Gene

2007 ◽  
Vol 82 (5) ◽  
pp. 2056-2064 ◽  
Author(s):  
Luka Čičin-Šain ◽  
Zsolt Ruzsics ◽  
Juergen Podlech ◽  
Ivan Bubić ◽  
Carine Menard ◽  
...  
Keyword(s):  
Virus Replication ◽  
Control Cell ◽  
Death Receptor ◽  
Formal Proof ◽  
Dominant Negative ◽  
Viral Fitness ◽  
Viral Gene ◽  
Apoptosis Inhibition

ABSTRACT Genes that inhibit apoptosis have been described for many DNA viruses. Herpesviruses often contain even more than one gene to control cell death. Apoptosis inhibition by viral genes is postulated to contribute to viral fitness, although a formal proof is pending. To address this question, we studied the mouse cytomegalovirus (MCMV) protein M36, which binds to caspase-8 and blocks death receptor-induced apoptosis. The growth of MCMV recombinants lacking M36 (ΔM36) was attenuated in vitro and in vivo. In vitro, caspase inhibition by zVAD-fmk blocked apoptosis in ΔM36-infected macrophages and rescued the growth of the mutant. In vivo, ΔM36 infection foci in liver tissue contained significantly more apoptotic hepatocytes and Kupffer cells than did revertant virus foci, and apoptosis occurred during the early phase of virus replication prior to virion assembly. To further delineate the mode of M36 function, we replaced the M36 gene with a dominant-negative FADD (FADDDN) in an MCMV recombinant. FADDDN was expressed in cells infected with the recombinant and blocked the death-receptor pathway, replacing the antiapoptotic function of M36. Most importantly, FADDDN rescued ΔM36 virus replication, both in vitro and in vivo. These findings have identified the biological role of M36 and define apoptosis inhibition as a key determinant of viral fitness.

scholarly journals Network-Guided Discovery of Influenza Virus Replication Host Factors

mBio ◽  
2018 ◽  
Vol 9 (6) ◽  
Author(s):  
Emily E. Ackerman ◽  
Eiryo Kawakami ◽  
Manami Katoh ◽  
Tokiko Watanabe ◽  
Shinji Watanabe ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Viral Replication ◽  
Virus Replication ◽  
Drug Targets ◽  
Antiviral Drug ◽  
Viral Proteins ◽  
Host Factors ◽  
Ppi Network ◽  
Host Proteins ◽  
Influenza Virus Replication

ABSTRACTThe positions of host factors required for viral replication within a human protein-protein interaction (PPI) network can be exploited to identify drug targets that are robust to drug-mediated selective pressure. Host factors can physically interact with viral proteins, be a component of virus-regulated pathways (where proteins do not interact with viral proteins), or be required for viral replication but unregulated by viruses. Here, we demonstrate a method of combining human PPI networks with virus-host PPI data to improve antiviral drug discovery for influenza viruses by identifying target host proteins. Analysis shows that influenza virus proteins physically interact with host proteins in network positions significant for information flow, even after the removal of known abundance-degree bias within PPI data. We have isolated a subnetwork of the human PPI network that connects virus-interacting host proteins to host factors that are important for influenza virus replication without physically interacting with viral proteins. The subnetwork is enriched for signaling and immune processes distinct from those associated with virus-interacting proteins. Selecting proteins based on subnetwork topology, we performed an siRNA screen to determine whether the subnetwork was enriched for virus replication host factors and whether network position within the subnetwork offers an advantage in prioritization of drug targets to control influenza virus replication. We found that the subnetwork is highly enriched for target host proteins—more so than the set of host factors that physically interact with viral proteins. Our findings demonstrate that network positions are a powerful predictor to guide antiviral drug candidate prioritization.IMPORTANCEIntegrating virus-host interactions with host protein-protein interactions, we have created a method using these established network practices to identify host factors (i.e., proteins) that are likely candidates for antiviral drug targeting. We demonstrate that interaction cascades between host proteins that directly interact with viral proteins and host factors that are important to influenza virus replication are enriched for signaling and immune processes. Additionally, we show that host proteins that interact with viral proteins are in network locations of power. Finally, we demonstrate a new network methodology to predict novel host factors and validate predictions with an siRNA screen. Our results show that integrating virus-host proteins interactions is useful in the identification of antiviral drug target candidates.

scholarly journals Murine Norovirus Replication Induces G0/G1Cell Cycle Arrest in Asynchronously Growing Cells

2015 ◽  
Vol 89 (11) ◽  
pp. 6057-6066 ◽  
Author(s):  
Colin Davies ◽  
Chris M. Brown ◽  
Dana Westphal ◽  
Joanna M. Ward ◽  
Vernon K. Ward
Keyword(s):  
Cell Cycle ◽  
Viral Replication ◽  
Host Cell ◽  
Virus Replication ◽  
Cell Cycle Progression ◽  
Cycle Phase ◽  
Murine Norovirus ◽  
Cycle Progression ◽  
Infected Cells ◽  
Favorable Conditions

ABSTRACTMany viruses replicate most efficiently in specific phases of the cell cycle, establishing or exploiting favorable conditions for viral replication, although little is known about the relationship between caliciviruses and the cell cycle. Microarray and Western blot analysis of murine norovirus 1 (MNV-1)-infected cells showed changes in cyclin transcript and protein levels indicative of a G1phase arrest. Cell cycle analysis confirmed that MNV-1 infection caused a prolonging of the G1phase and an accumulation of cells in the G0/G1phase. The accumulation in G0/G1phase was caused by a reduction in cell cycle progression through the G1/S restriction point, with MNV-1-infected cells released from a G1arrest showing reduced cell cycle progression compared to mock-infected cells. MNV-1 replication was compared in populations of cells synchronized into specific cell cycle phases and in asynchronously growing cells. Cells actively progressing through the G1phase had a 2-fold or higher increase in virus progeny and capsid protein expression over cells in other phases of the cell cycle or in unsynchronized populations. These findings suggest that MNV-1 infection leads to prolonging of the G1phase and a reduction in S phase entry in host cells, establishing favorable conditions for viral protein production and viral replication. There is limited information on the interactions between noroviruses and the cell cycle, and this observation of increased replication in the G1phase may be representative of other members of theCaliciviridae.IMPORTANCENoroviruses have proven recalcitrant to growth in cell culture, limiting our understanding of the interaction between these viruses and the infected cell. In this study, we used the cell-culturable MNV-1 to show that infection of murine macrophages affects the G1/S cell cycle phase transition, leading to an arrest in cell cycle progression and an accumulation of cells in the G0/G1phase. Furthermore, we show that MNV replication is enhanced in the G1phase compared to other stages of the cell cycle. Manipulating the cell cycle or adapting to cell cycle responses of the host cell is a mechanism to enhance virus replication. To the best of our knowledge, this is the first report of a norovirus interacting with the host cell cycle and exploiting the favorable conditions of the G0/G1phase for RNA virus replication.

scholarly journals Sequences in the human c-myc P2 promoter affect the elongation and premature termination of transcripts initiated from the upstream P1 promoter.

1992 ◽  
Vol 12 (10) ◽  
pp. 4590-4600 ◽  
Author(s):  
T Meulia ◽  
A Krumm ◽  
C Spencer ◽  
M Groudine
Keyword(s):  
Mammalian Cells ◽  
Xenopus Oocyte ◽  
Xenopus Oocytes ◽  
Premature Termination ◽  
Deletion Mutants ◽  
Potential Contribution ◽  
Germinal Vesicles ◽  
Exon 1 ◽  
P2 Promoter ◽  
Steady State Levels

A conditional block to transcription elongation provides one mechanism for controlling the steady-state levels of c-myc RNA in mammalian cells. Although prematurely terminated c-myc RNAs are not detectable in mammalian cells, truncated c-myc RNAs with 3' ends that map near the end of the first exon are transcribed from human c-myc templates injected into Xenopus oocytes germinal vesicles. A series of linker scanner and deletion mutants within the c-myc P2 promoter was tested in the Xenopus oocyte injection assay to determine the potential contribution of promoter elements to the elongation or premature termination of c-myc transcription. Although this analysis failed to identify sequences in the P2 promoter that significantly affect the elongation or termination of P2-initiated transcripts, our results suggest that sequences within the P2 promoter contribute to the premature termination of transcripts initiated at the upstream P1 promoter. A subset of these sequences is essential for the efficient elongation of P1-initiated transcripts through intrinsic sites of termination at the end of exon 1. These sequences affect P1 elongation when they are downstream of the site of initiation, and we hypothesize that they may be analogous to a class of prokaryotic elements required for antitermination.

scholarly journals Viral Characteristics Associated with the Clinical Nonprogressor Phenotype Are Inherited by Viruses from a Cluster of HIV-1 Elite Controllers

mBio ◽  
2018 ◽  
Vol 9 (2) ◽  
Author(s):  
Concepción Casado ◽  
Sara Marrero-Hernández ◽  
Daniel Márquez-Arce ◽  
María Pernas ◽  
Sílvia Marfil ◽  
...  
Keyword(s):  
Viral Replication ◽  
Clinical Phenotype ◽  
Functional Characterization ◽  
Viral Fitness ◽  
Elite Controller ◽  
Replication Capacity ◽  
Viral Fusion ◽  
Elite Controllers ◽  
Hiv 1

ABSTRACTA small group of HIV-1-infected individuals, called long-term nonprogressors (LTNPs), and in particular a subgroup of LTNPs, elite controllers (LTNP-ECs), display permanent control of viral replication and lack of clinical progression. This control is the result of a complex interaction of host, immune, and viral factors. We identified, by phylogenetic analysis, a cluster of LTNP-ECs infected with very similar low-replication HIV-1 viruses, suggesting the contribution of common viral features to the clinical LTNP-EC phenotype. HIV-1 envelope (Env) glycoprotein mediates signaling and promotes HIV-1 fusion, entry, and infection, being a key factor of viral fitnessin vitro, cytopathicity, and infection progressionin vivo. Therefore, we isolated full-lengthenvgenes from viruses of these patients and from chronically infected control individuals. Functional characterization of the initial events of the viral infection showed that Envs from the LTNP-ECs were ineffective in the binding to CD4 and in the key triggering of actin/tubulin-cytoskeleton modifications compared to Envs from chronic patients. The viral properties of the cluster viruses result in a defective viral fusion, entry, and infection, and these properties were inherited by every virus of the cluster. Therefore, inefficient HIV-1 Env functions and signaling defects may contribute to the low viral replication capacity and transmissibility of the cluster viruses, suggesting a direct role in the LTNP-EC phenotype of these individuals. These results highlight the important role of viral characteristics in the LTNP-EC clinical phenotype. These Env viral properties were common to all the cluster viruses and thus support the heritability of the viral characteristics.IMPORTANCEHIV-1 long-term nonprogressor elite controller patients, due to their permanent control of viral replication, have been the object of numerous studies to identify the factors responsible for this clinical phenotype. In this work, we analyzed the viral characteristics of the envelopes of viruses from a phylogenetic cluster of LTNP-EC patients. These envelopes showed ineffective binding to CD4 and the subsequent signaling activity to modify actin/tubulin cytoskeletons, which result in low fusion and deficient entry and infection capacities. These Env viral characteristics could explain the nonprogressor clinical phenotype of these patients. In addition, these inefficientenvviral properties were present in all viruses of the cluster, supporting the heritability of the viral phenotype.

scholarly journals Cooperative nature of viral replication

2020 ◽  
Vol 6 (49) ◽  
pp. eabd4942
Author(s):  
Iván Andreu-Moreno ◽  
Juan-Vicente Bou ◽  
Rafael Sanjuán
Keyword(s):  
Viral Replication ◽  
Viral Fitness ◽  
Progeny Production ◽  
Short Term ◽  
Viral Particles ◽  
Viral Progeny ◽  
Cooperative Process ◽  
Complex Models ◽  
Rapid Dissemination ◽  
Human Viruses

The ability of viruses to infect their hosts depends on rapid dissemination following transmission. The notion that viral particles function as independent propagules has been challenged by recent observations suggesting that viral aggregates show enhanced infectivity and faster spread. However, these observations remain poorly understood. Here, we show that viral replication is a cooperative process, such that entry of multiple viral genome copies into the same cell disproportionately increases short-term viral progeny production. This cooperativity arises from the positive feedback established between replication templates and virus-encoded products involved in replication and should be a general feature of viruses. We develop a simple model that captures this effect, verify that cooperativity also emerges in more complex models for specific human viruses, validate our predictions experimentally using different mammalian viruses, and discuss the implications of cooperative replication for viral fitness.

scholarly journals Thrips as the Transmission Bottleneck for Mixed Infection of Two Orthotospoviruses

Plants ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 509
Author(s):  
Kaixi Zhao ◽  
Cristina Rosa
Keyword(s):  
Genetic Diversity ◽  
Virus Replication ◽  
Mixed Infection ◽  
Viral Fitness ◽  
Mixed Infections ◽  
Virus Species ◽  
Necrotic Spot ◽  
Tomato Spotted Wilt ◽  
Transmission Bottleneck

Mixed infections provide opportunities for viruses to increase genetic diversity by facilitating genomic reassortment or recombination, and they may lead to the emergence of new virus species. Mixed infections of two economically important orthotospoviruses, Tomato spotted wilt orthotospovirus (TSWV) and Impatiens necrotic spot orthotospovirus (INSV), were found in recent years, but no natural reassortants between INSV and TSWV were ever reported. The goal of this study was to establish how vector preferences and the ability to transmit INSV and TSWV influence transmission and establishment of mixed infections. Our results demonstrate that thrips prefer to oviposit on TSWV and INSV mixed-infected plants over singly infected or healthy plants, providing young nymphs with the opportunity to acquire both viruses. Conversely, we observed that thrips served as a bottleneck during transmission and favored transmission of one of the two viruses over the second one, or over transmission of both viruses simultaneously. This constraint was relaxed in plants, when transmission of TSWV and INSV occurred sequentially, demonstrating that plants serve as orthotospovirus permissive hosts, while thrips serve as a bottleneck. Viral fitness, as measured by virus replication, transmission, and competition with other viral strains, is not well studied in mixed infection. Our study looks at the success of transmission during mixed infection of orthotopoviruses, enhancing the understanding of orthotospovirus epidemiology and evolution.

scholarly journals Marek's Disease Virus Disables the ATR-Chk1 Pathway by Activating STAT3

2019 ◽  
Vol 93 (9) ◽  
Author(s):  
Xue Lian ◽  
Chenyi Bao ◽  
Xueqi Li ◽  
Xunhai Zhang ◽  
Hongjun Chen ◽  
...  
Keyword(s):  
Dna Damage ◽  
Viral Replication ◽  
Disease Virus ◽  
Virus Replication ◽  
Specific Inhibitor ◽  
Marek's Disease Virus ◽  
Marek's Disease ◽  
Marek’S Disease Virus ◽  
Marek’S Disease ◽  
Stat3 Phosphorylation

ABSTRACT Oncogenic virus replication often leads to genomic instability, causing DNA damage and inducing the DNA damage response (DDR) pathway. The DDR pathway is a cellular pathway that senses DNA damage and regulates the cell cycle to maintain genomic stability. Therefore, the DDR pathway is critical for the viral lifecycle and tumorigenesis. Marek’s disease virus (MDV), an alphaherpesvirus that causes lymphoma in chickens, has been shown to induce DNA damage in infected cells. However, the interaction between MDV and the host DDR is unclear. In this study, we observed that MDV infection causes DNA strand breakage in chicken fibroblast (CEF) cells along with an increase in the DNA damage markers p53 and p21. Interestingly, we showed that phosphorylation of STAT3 was increased during MDV infection, concomitantly with a decrease of Chk1 phosphorylation. In addition, we found that MDV infection was enhanced by VE-821, an ATR-specific inhibitor, but attenuated by hydroxyurea, an ATR activator. Moreover, inhibition of STAT3 phosphorylation by Stattic eliminates the ability of MDV to inhibit Chk1 phosphorylation. Finally, we showed that MDV replication was decreased by Stattic treatment. Taken together, these results suggest that MDV disables the ATR-Chk1 pathway through STAT3 activation to benefit its replication. IMPORTANCE MDV is used as a biomedical model to study virus-induced lymphoma due to the similar genomic structures and physiological characteristics of MDV and human herpesviruses. Upon infection, MDV induces DNA damage, which may activate the DDR pathway. The DDR pathway has a dual impact on viruses because it manipulates repair and recombination factors to facilitate viral replication and also initiates antiviral action by regulating other signaling pathways. Many DNA viruses evolve to manipulate the DDR pathway to promote virus replication. In this study, we identified a mechanism used by MDV to inhibit ATR-Chk1 pathways. ATR is a cellular kinase that responds to broken single-stranded DNA, which has been less studied in MDV infection. Our results suggest that MDV infection activates STAT3 to disable the ATR-Chk1 pathway, which is conducive to viral replication. This finding provides new insight into the role of STAT3 in interrupting the ATR-Chk1 pathway during MDV replication.

scholarly journals HLA-B57/B*5801 Human Immunodeficiency Virus Type 1 Elite Controllers Select for Rare Gag Variants Associated with Reduced Viral Replication Capacity and Strong Cytotoxic T-Lymphotye Recognition

2008 ◽  
Vol 83 (6) ◽  
pp. 2743-2755 ◽  
Author(s):  
Toshiyuki Miura ◽  
Mark A. Brockman ◽  
Arne Schneidewind ◽  
Michael Lobritz ◽  
Florencia Pereyra ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Viral Replication ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Viral Fitness ◽  
Replication Capacity ◽  
Elite Controllers ◽  
Immunodeficiency Virus ◽  
Viral Replication Capacity

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) elite controllers (EC) maintain viremia below the limit of commercial assay detection (<50 RNA copies/ml) in the absence of antiviral therapy, but the mechanisms of control remain unclear. HLA-B57 and the closely related allele B*5801 are particularly associated with enhanced control and recognize the same Gag240-249 TW10 epitope. The typical escape mutation (T242N) within this epitope diminishes viral replication capacity in chronically infected persons; however, little is known about TW10 epitope sequences in residual replicating viruses in B57/B*5801 EC and the extent to which mutations within this epitope may influence steady-state viremia. Here we analyzed TW10 in a total of 50 B57/B*5801-positive subjects (23 EC and 27 viremic subjects). Autologous plasma viral sequences from both EC and viremic subjects frequently harbored the typical cytotoxic T-lymphocyte (CTL)-selected mutation T242N (15/23 sequences [65.2%] versus 23/27 sequences [85.1%], respectively; P = 0.18). However, other unique mutants were identified in HIV controllers, both within and flanking TW10, that were associated with an even greater reduction in viral replication capacity in vitro. In addition, strong CTL responses to many of these unique TW10 variants were detected by gamma interferon-specific enzyme-linked immunospot assay. These data suggest a dual mechanism for durable control of HIV replication, consisting of viral fitness loss resulting from CTL escape mutations together with strong CD8 T-cell immune responses to the arising variant epitopes.

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