Production Of Virus Particles Research Articles

Mouse Fibroblast L929 Cell Line as a Useful Tool for Replication and Adaptation of Infectious Bursal Disease Virus.

Infectious bursal disease virus (IBDV) causes a highly contagious disease associated with immunosuppression in young chickens. Production of either egg-based or primary cell-based high-quality vaccines requires time-consuming and costly procedures. To determine a suitable cell line for IBDV replication, L929 cell line was a candidate for the growth kinetics processing of the virus. The L929 cells were proliferated in monolayer, and doubling time was calculated. Replication kinetics an IBDV isolate at the multiplicity of infection 0.1 PFU/cell were determined using virus titration. To adapt IBDV on L929 cells, seven consecutive passages were performed. Virus titer and levels of apoptosis were quantitatively analyzed at each passage. The viral VP2 gene was amplified and sequenced in three passages. An average doubling time of 21 h was estimated for monolayers of L929 cells. Although during early passages, virus growth did not produce a clear cytopathic effect (CPE), an increase in IBDV titers was observed. Serial passages led to the evidence of marked CPEs and an increase in the virus titer in the third passage. During the fourth to seventh passages, consistent CPEs characterized by the formation of granulated and round cells were evident within 24 to 48 hours post-inoculation. The titer of the virus was increased in the third passage onwards to peak in the fourth and constant at 5.9 TCID50 until the end passage. The IBDV replication in connection with DNA fragmentation and FITC, revealed the characteristic picture of apoptosis in a time-dependent manner. We found that the IBDV could easily be adapted to L929 cells, increasing virus yields by about two orders of magnitude. These results indicated that the cell line may be useful in the production of efficient virus particles.

Favipiravir Suppresses Zika Virus (ZIKV) through Activity as a Mutagen.

In a companion paper, we demonstrated that the nucleoside analogue favipiravir (FAV) suppressed Zika virus (ZIKV) replication in three human-derived cell lines-HeLa, SK-N-MC, and HUH-7. Our results revealed that FAV's effect was most pronounced in HeLa cells. In this work, we aimed to explain variation in FAV activity, investigating its mechanism of action and characterizing host cell factors relevant to tissue-specific differences in drug effect. Using viral genome sequencing, we show that FAV therapy was associated with an increase in the number of mutations and promoted the production of defective viral particles in all three cell lines. Our findings demonstrate that defective viral particles made up a larger portion of the viral population released from HeLa cells both at increasing FAV concentrations and at increasing exposure times. Taken together, our companion papers show that FAV acts via lethal mutagenesis against ZIKV and highlight the host cell's influence on the activation and antiviral activity of nucleoside analogues. Furthermore, the information gleaned from these companion papers can be applied to gain a more comprehensive understanding of the activity of nucleoside analogues and the impact of host cell factors against other viral infections for which we currently have no approved antiviral therapies.

Experimental studies from shake flasks to 3 L stirred tank bioreactor of nutrients and oxygen supply conditions to improve the growth of the avian cell line DuckCelt®-T17

BackgroundTo produce viral vaccines, avian cell lines are interesting alternatives to replace the egg-derived processes for viruses that do not grow well on mammalian cells. The avian suspension cell line DuckCelt®-T17 was previously studied and investigated to produce a live attenuated metapneumovirus (hMPV)/respiratory syncytial virus (RSV) and influenza virus vaccines. However, a better understanding of its culture process is necessary for an efficient production of viral particles in bioreactors.ResultsThe growth and metabolic requirements of the avian cell line DuckCelt®-T17 were investigated to improve its cultivation parameters. Several nutrient supplementation strategies were studied in shake flasks highlighting the interest of (i) replacing L-glutamine by glutamax as main nutrient or (ii) adding these two nutrients in the serum-free growth medium in a fed-batch strategy. The scale-up in a 3 L bioreactor was successful for these types of strategies confirming their efficiencies in improving the cells’ growth and viability. Moreover, a perfusion feasibility test allowed to achieve up to ~ 3 times the maximum number of viable cells obtained with the batch or fed-batch strategies. Finally, a strong oxygen supply – 50% dO2 – had a deleterious effect on DuckCelt®-T17 viability, certainly because of the greater hydrodynamic stress imposed.ConclusionsThe culture process using glutamax supplementation with a batch or a fed-batch strategy was successfully scaled-up to 3 L bioreactor. In addition, perfusion appeared as a very promising culture process for subsequent continuous virus harvesting.

Cell-to-Cell Transmission of HIV-1 and HIV-2 from Infected Macrophages and Dendritic Cells to CD4+ T Lymphocytes.

Macrophages (Mø) and dendritic cells (DCs) are key players in human immunodeficiency virus (HIV) infection and pathogenesis. They are essential for the spread of HIV to CD4+ T lymphocytes (TCD4+) during acute infection. In addition, they constitute a persistently infected reservoir in which viral production is maintained for long periods of time during chronic infection. Defining how HIV interacts with these cells remains a critical area of research to elucidate the pathogenic mechanisms of acute spread and sustained chronic infection and transmission. To address this issue, we analyzed a panel of phenotypically distinct HIV-1 and HIV-2 primary isolates for the efficiency with which they are transferred from infected DCs or Mø to TCD4+. Our results show that infected Mø and DCs spread the virus to TCD4+ via cell-free viral particles in addition to other alternative pathways. We demonstrate that the production of infectious viral particles is induced by the co-culture of different cell populations, indicating that the contribution of cell signaling driven by cell-to-cell contact is a trigger for viral replication. The results obtained do not correlate with the phenotypic characteristics of the HIV isolates, namely their co-receptor usage, nor do we find significant differences between HIV-1 and HIV-2 in terms of cis- or trans-infection. The data presented here may help to further elucidate the cell-to-cell spread of HIV and its importance in HIV pathogenesis. Ultimately, this knowledge is critical for new therapeutic and vaccine approaches.

TDP-43 Controls HIV-1 Viral Production and Virus Infectiveness.

The transactive response DNA-binding protein (TARDBP/TDP-43) is known to stabilize the anti-HIV-1 factor, histone deacetylase 6 (HDAC6). TDP-43 has been reported to determine cell permissivity to HIV-1 fusion and infection acting on tubulin-deacetylase HDAC6. Here, we studied the functional involvement of TDP-43 in the late stages of the HIV-1 viral cycle. The overexpression of TDP-43, in virus-producing cells, stabilized HDAC6 (i.e., mRNA and protein) and triggered the autophagic clearance of HIV-1 Pr55Gag and Vif proteins. These events inhibited viral particle production and impaired virion infectiveness, observing a reduction in the amount of Pr55Gag and Vif proteins incorporated into virions. A nuclear localization signal (NLS)-TDP-43 mutant was not able to control HIV-1 viral production and infection. Likewise, specific TDP-43-knockdown reduced HDAC6 expression (i.e., mRNA and protein) and increased the expression level of HIV-1 Vif and Pr55Gag proteins and α-tubulin acetylation. Thus, TDP-43 silencing favored virion production and enhanced virus infectious capacity, thereby increasing the amount of Vif and Pr55Gag proteins incorporated into virions. Noteworthy, there was a direct relationship between the content of Vif and Pr55Gag proteins in virions and their infection capacity. Therefore, for TDP-43, the TDP-43/HDAC6 axis could be considered a key factor to control HIV-1 viral production and virus infectiveness.

Antiviral Activity of Quercetin Hydrate against Zika Virus.

Zika virus (ZIKV) has re-emerged in recent decades, leading to outbreaks of Zika fever in Africa, Asia, and Central and South America. Despite its drastic re-emergence and clinical impact, no vaccines or antiviral compounds are available to prevent or control ZIKV infection. This study evaluated the potential antiviral activity of quercetin hydrate against ZIKV infection and demonstrated that this substance inhibits virus particle production in A549 and Vero cells under different treatment conditions. In vitro antiviral activity was long-lasting (still observed 72 h post-infection), suggesting that quercetin hydrate affects multiple rounds of ZIKV replication. Molecular docking indicates that quercetin hydrate can efficiently interact with the specific allosteric binding site cavity of the NS2B-NS3 proteases and NS1-dimer. These results identify quercetin as a potential compound to combat ZIKV infection in vitro.

Amino acid variation at VP1-145 of enterovirus A71 determines the viral infectivity and receptor usage in a primary human intestinal model.

With the near eradication of polio worldwide, polio-like illness (as is increasingly caused by EV-A71 infections) is of emerging concern. EV-A71 is indeed the most neurotropic enterovirus that poses a major threat globally to public health and specifically in infants and young children. Our findings will contribute to the understanding of the virulence and the pathogenicity of this virus. Further, our data also supports the identification of potential therapeutic targets against severe EV-A71 infection especially among infants and young children. Furthermore, our work highlights the key role of HSPG-binding mutations in the disease outcome of EV-A71. Additionally, EV-A71 is not able to infect the gut (the primary replication site in humans) in traditionally used animal models. Thus, our research highlights the need for human-based models to study human viral infections.Graphical Abstract.

Mathematical modeling of plus-strand RNA virus replication to identify broad-spectrum antiviral treatment strategies.

Plus-strand RNA viruses are the largest group of viruses. Many are human pathogens that inflict a socio-economic burden. Interestingly, plus-strand RNA viruses share remarkable similarities in their replication. A hallmark of plus-strand RNA viruses is the remodeling of intracellular membranes to establish replication organelles (so-called "replication factories"), which provide a protected environment for the replicase complex, consisting of the viral genome and proteins necessary for viral RNA synthesis. In the current study, we investigate pan-viral similarities and virus-specific differences in the life cycle of this highly relevant group of viruses. We first measured the kinetics of viral RNA, viral protein, and infectious virus particle production of hepatitis C virus (HCV), dengue virus (DENV), and coxsackievirus B3 (CVB3) in the immuno-compromised Huh7 cell line and thus without perturbations by an intrinsic immune response. Based on these measurements, we developed a detailed mathematical model of the replication of HCV, DENV, and CVB3 and showed that only small virus-specific changes in the model were necessary to describe the in vitro dynamics of the different viruses. Our model correctly predicted virus-specific mechanisms such as host cell translation shut off and different kinetics of replication organelles. Further, our model suggests that the ability to suppress or shut down host cell mRNA translation may be a key factor for in vitro replication efficiency, which may determine acute self-limited or chronic infection. We further analyzed potential broad-spectrum antiviral treatment options in silico and found that targeting viral RNA translation, such as polyprotein cleavage and viral RNA synthesis, may be the most promising drug targets for all plus-strand RNA viruses. Moreover, we found that targeting only the formation of replicase complexes did not stop the in vitro viral replication early in infection, while inhibiting intracellular trafficking processes may even lead to amplified viral growth.

Packaging and Uncoating of CRISPR/Cas Ribonucleoproteins for Efficient Gene Editing with Viral and Non-Viral Extracellular Nanoparticles

Rapid progress in gene editing based on clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) has revolutionized functional genomic studies and genetic disease correction. While numerous gene editing applications have been easily adapted by experimental science, the clinical utility of CRISPR/Cas remains very limited due to difficulty in delivery to primary cells and possible off-target effects. The use of CRISPR in the form of a ribonucleoprotein (RNP) complex substantially reduces the time of DNA exposure to the effector nuclease and minimizes its off-target activity. The traditional electroporation and lipofection methods lack the cell-type specificity of RNP delivery, can be toxic for cells, and are less efficient when compared to nanoparticle transporters. This review focuses on CRISPR/Cas RNP packaging and delivery using retro/lentiviral particles and exosomes. First, we briefly describe the natural stages of viral and exosomal particle formation, release and entry into the target cells. This helps us understand the mechanisms of CRISPR/Cas RNP packaging and uncoating utilized by the current delivery systems, which we discuss afterward. Much attention is given to the exosomes released during viral particle production that can be passively loaded with RNPs as well as the mechanisms necessary for particle fusion, RNP release, and transportation inside the target cells. Collectively, together with specific packaging mechanisms, all these factors can substantially influence the editing efficiency of the system. Finally, we discuss ways to improve CRISPR/Cas RNP delivery using extracellular nanoparticles.

Early antitermination in the atypical coliphage mEp021 mediated by the Gp17 protein

The coliphage mEp021 belongs to a phage group with a unique immunity repressor, and its life cycle requires the host factor Nus. mEp021 has been classified as non-lambdoid based on its specific characteristics. The mEp021 genome carries a gene encoding an Nλ-like antiterminator protein, termed Gp17, and three nut sites (nutL, nutR1, and nutR2). Analysis of plasmid constructs containing these nut sites, a transcription terminator, and a GFP reporter gene showed high levels of fluorescence when Gp17 was expressed, but not in its absence. Like lambdoid N proteins, Gp17 has an arginine-rich motif (ARM), and mutations in its arginine codons inhibit its function. In infection assays using the mutant phage mEp021ΔGp17::Kan (where gp17 has been deleted), gene transcripts located downstream of transcription terminators were obtainedonly when Gp17 was expressed. In contrast to phage lambda, mEp021 virus particle production was partially restored (>1/3 relative to wild type) when nus mutants (nusA1, nusB5, nusC60, and nusE71) were infected with mEp021 and Gp17 was overexpressed. Our results suggest that RNA polymerase reads through the third nut site (nutR2), which is more than 7.9 kbp downstream of nutR1.

Targeting first trimester trophoblast cell metabolism modulates its susceptibility to Zika virus infection.

In the last 15 years Zika virus (ZIKV) caused several outbreaks of increasing scale in Micronesia, South Pacific islands, and more recently in the Caribbean and South America. The severity of the clinical presentation in neonates from pregnant women infected with ZIKV during the last outbreak supports the relevance of unraveling the mechanism of infection and viral persistence in the placenta with local viral isolates. Here, we investigated the relevance of trophoblast metabolic rewiring for viral multiplication and the role of the vasoactive intestinal peptide (VIP) as an endogenous factor associated with placental restriction to ZIKV infection at early pregnancy. Our in vitro model demonstrated that ZIKV triggers metabolic rewiring in first trimester cytotrophoblast-derived cells by increasing glucose utilization as fuel to sustain its replication, decreasing long-chain polyunsaturated fatty acid uptake, and promoting lipid droplets accumulation to favor its multiplication. Of note, variations in nutrient availability modulated viral spread in trophoblast cultures. The presence of VIP during trophoblast infection impaired ZIKV infective particle production and viral replication, restoring cell migration and metabolism. Moreover, the blockade of endogenous VIP signaling increased viral particle production and the viral entry receptor AXL expression. These results highlight the potential role of VIP as an endogenous antiviral factor related to trophoblast cell permissiveness to ZIKV infection at early pregnancy.

Complementary Cell Lines for Protease Gene-Deleted Single-Cycle Adenovirus Vectors.

To increase the safety of adenovirus vector (AdV)-based therapy without reducing its efficacy, a single-cycle adenovirus vector (SC-AdV) with a deletion in the protease gene (PS) was developed in order to be used as a substitute for the replication-competent adenovirus (RC-AdV). Since no infectious viral particles are assembled, there is no risk of viral shedding. The complementary cell lines for this developed AdV proved to be suboptimal for the production of viral particles and require the presence of fetal bovine serum (FBS) to grow. In the current study, we produced both stable pools and clones using adherent and suspension cells expressing the PS gene. The best adherent cell pool can be used in the early stages for the generation of protease-deleted adenovirus, plaque purification, and titration. Using this, we produced over 3400 infectious viral particles per cell. Additionally, the best suspension subclone that was cultured in the absence of FBS yielded over 4000 infectious viral particles per cell. Harvesting time, culture media, and concentration of the inducer for the best suspension subclone were further characterized. With these two types of stable cells (pool and subclone), we successfully improved the titer of protease-deleted adenovirus in adherent and suspension cultures and eliminated the need for FBS during the scale-up production. Eight lots of SC-AdV were produced in the best suspension subclone at a scale of 2 to 8.2 L. The viral and infectious particle titers were influenced by the virus backbone and expressed transgene.

Mathematical Modeling and Analysis of the Dynamics of RNA Viruses in Presence of Immunity and Treatment: A Case Study of SARS-CoV-2.

The emergence of novel RNA viruses like SARS-CoV-2 poses a greater threat to human health. Thus, the main objective of this article is to develop a new mathematical model with a view to better understand the evolutionary behavior of such viruses inside the human body and to determine control strategies to deal with this type of threat. The developed model takes into account two modes of transmission and both classes of infected cells that are latently infected cells and actively infected cells that produce virus particles. The cure of infected cells in latent period as well as the lytic and non-lytic immune response are considered into the model. We first show that the developed model is well-posed from the biological point of view by proving the non-negativity and boundedness of model's solutions. Our analytical results show that the dynamical behavior of the model is fully determined by two threshold parameters one for viral infection and the other for humoral immunity. The effect of antiviral treatment is also investigated. Furthermore, numerical simulations are presented in order to illustrate our analytical results.

Protein post-translational modification in SARS-CoV-2 and host interaction.

SARS-CoV-2 can cause lung diseases, such as pneumonia and acute respiratory distress syndrome, and multi-system dysfunction. Post-translational modifications (PTMs) related to SARS-CoV-2 are conservative and pathogenic, and the common PTMs are glycosylation, phosphorylation, and acylation. The glycosylation of SARS-CoV-2 mainly occurs on spike (S) protein, which mediates the entry of the virus into cells through interaction with angiotensin-converting enzyme 2. SARS-CoV-2 utilizes glycans to cover its epitopes and evade the immune response through glycosylation of S protein. Phosphorylation of SARS-CoV-2 nucleocapsid (N) protein improves its selective binding to viral RNA and promotes viral replication and transcription, thereby increasing the load of the virus in the host. Succinylated N and membrane(M) proteins of SARS-CoV-2 synergistically affect virus particle assembly. N protein regulates its affinity for other proteins and the viral genome through acetylation. The acetylated envelope (E) protein of SARS-CoV-2 interacts with bromodomain-containing protein 2/4 to influence the host immune response. Both palmitoylation and myristoylation sites on S protein can affect the virus infectivity. Papain-like protease is a domain of NSP3 that dysregulates host inflammation by deubiquitination and impinges host IFN-I antiviral immune responses by deISGylation. Ubiquitination of ORF7a inhibits host IFN-α signaling by blocking STAT2 phosphorylation. The methylation of N protein can inhibit the formation of host stress granules and promote the binding of N protein to viral RNA, thereby promoting the production of virus particles. NSP3 macrodomain can reverse the ADP-ribosylation of host proteins, and inhibit the cascade immune response with IFN as the core, thereby promoting the intracellular replication of SARS-CoV-2. On the whole, PTMs have fundamental roles in virus entry, replication, particle assembly, and host immune response. Mutations in various SARS-CoV-2 variants, which lead to changes in PTMs at corresponding sites, cause different biological effects. In this paper, we mainly reviewed the effects of PTMs on SARS-CoV-2 and host cells, whose application is to inform the strategies for inhibiting viral infection and facilitating antiviral treatment and vaccine development for COVID-19.

An optimized HEK293T cell expansion protocol using a hollow-fiber bioreactor system.

Viral vectors are commonly used to introduce genetic material into cells to modify cell function for a variety of purposes. Manufacture of those modified viruses may use a variety of cell types to generate high titers of viral particles; one of the most common being HEK293 cells. These cells have been modified into different lines aimed at satisfying specific use cases. HEK293T cells, for example, have been modified to include the SV40 large T antigen. Efficient viral particle production by HEK293T cells requires the maintenance of favorable cell culture conditions during expansion and transfection. This protocol describes the use of the Quantum® hollow-fiber bioreactor (HFB) system for the automated expansion of HEK293T cells, and the results derived using the protocol described herein were not compared with those from tissue culture flasks or other expansion platforms, as the parameters described are unique to Quantum's hollow fiber cell expansion environment. The purpose of this protocol is to help users of Quantum to focus on relevant parameters of expansion in the HFB milieu and to provide guidelines for a successful expansion of HEK293T cells in the Quantum system. The steps provided have been optimized to reliably control environmental factors related to glucose, lactate, and pH. Data reflecting this consistency are provided along with procedural time points reflected in text and figure formats.

Kaposi’s Sarcoma-Associated Herpesvirus ORF21 Enhances the Phosphorylation of MEK and the Infectivity of Progeny Virus

Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus-8, is the causative agent of Kaposi's sarcoma, Castleman's disease, and primary effusion lymphoma. Although the functions of the viral thymidine kinases (vTK) of herpes simplex virus-1/2 are well understood, that of KSHV ORF21 (an ortholog of vTK) is largely unknown. Here, we investigated the role of ORF21 in lytic replication and infection by generating two ORF21-mutated KSHV BAC clones: ORF21-kinase activity deficient KSHV (21KD) and stop codon-induced ORF21-deleted KSHV (21del). The results showed that both ORF21 mutations did not affect viral genome replication, lytic gene transcription, or the production of viral genome-encapsidated particles. The ORF21 molecule-dependent function, other than the kinase function of ORF21, was involved in the infectivity of the progeny virus. ORF21 was expressed 36 h after the induction of lytic replication, and endogenously expressed ORF21 was localized in the whole cytoplasm. Moreover, ORF21 upregulated the MEK phosphorylation and anchorage-independent cell growth. The inhibition of MEK signaling by U0126 in recipient target cells suppressed the number of progeny virus-infected cells. These suggest that ORF21 transmitted as a tegument protein in the progeny virus enhances the new infection through MEK up-regulation in the recipient cell. Our findings indicate that ORF21 plays key roles in the infection of KSHV through the manipulation of the cellular function.

Determining the Importance of Peroxisomal Proteins for Viral Infections in Cultured Mammalian Cells.

Peroxisomes have recently been shown to play important roles in the context of viral infections. However, further and more detailed studies should be performed to unravel the specific mechanisms involved. The analysis of the relevance of particular peroxisomal components, such as peroxisomal proteins, for viral infections can be performed by comparing the production of new virus particles in the absence and presence of those specific components. Different methodologies are used to quantify the production of infectious virus particles, depending on the virus, cell type, and the specific characteristics of the viral infection to be analyzed. Here we provide a detailed protocol to study the importance of a putative peroxisomal protein on infection by viruses that induce the death of their host cells. We use the influenza A virus (IAV) infection in A549 cells as a model, and the quantification of the newly produced infectious virus particles is performed by a plaque assay.

Characterization and Analysis of HEK293/Adenovirus Type 5 Cell Cultures under Simulated Microgravity Using Differential Neural Network Modeling

HEK293 cells are the common system to produce adenoviral vectors for vectorized vaccines and gene therapy products. Therefore, the improvement of this type of cell culture is highly relevant nowadays. Furthermore, the high aspect ratio vessel (HARV) bioreactor provides a simulated microgravity condition (SMGC) which allows the establishment of in vitro models of mammalian cells. The present work is aimed at showing the characterization of HEK293 cell cultures in serum‐free medium under SMGC and to identify a set of adaptable time differential equations from experimental data through a nonparametric mathematical modeling using differential neural networks (DNN). Results showed that the HARV bioreactor enables to obtain a gravity percentage equal to 〖%g〗_maximum = −0.053% (where minus sign implies against the gravity direction). In addition, the characterization of HEK293 culture under SMGC revealed that such condition favored the production of infective viral particles as well as the uptake of basic nutrients such as glucose and glutamine, but the cell concentration was not significantly affected. As shown, these results contribute to improve the adenoviral vector production, and the DNN analysis is useful to model the kinetic changes and to build the foundations for control strategies and optimization of adenoviral production in further studies.

Mechanistic modeling of viral particle production.

Viral systems such as wild-type viruses, viral vectors, and virus-like particles are essential components of modern biotechnology and medicine. Despite their importance, the commercial-scale production of viral systems remains highly inefficient for multiple reasons. Computational strategies are a promising avenue for improving process development, optimization, and control, but require a mathematical description of the system. This article reviews mechanistic modeling strategies for the production of viral particles, both at the cellular and bioreactor scales. In many cases, techniques and models from adjacent fields such as epidemiology and wild-type viral infection kinetics can be adapted to construct a suitable process model. These process models can then be employed for various purposes such as in-silico testing of novel process operating strategies and/or advanced process control.

Enterovirus-Cardiomyocyte Interactions: Impact of Terminally Deleted Genomic RNAs on Viral and Host Functions.

Group B enteroviruses, including coxsackievirus B3 (CVB3), can persistently infect cardiac tissue and cause dilated cardiomyopathy. Persistence is linked to 5' terminal deletions of viral genomic RNAs that have been detected together with minor populations of full-length genomes in human infections. In this study, we explored the functions and interactions of the different viral RNA forms found in persistently infected patients and their putative role(s) in pathogenesis. Since enterovirus cardiac pathogenesis is linked to the viral proteinase 2A, we investigated the effect of different terminal genomic RNA deletions on 2A activity. We discovered that 5' terminal deletions in CVB3 genomic RNAs decreased the levels of 2A proteinase activity but could not abrogate it. Using newly generated viral reporters encoding nano-luciferase, we found that 5' terminal deletions resulted in decreased levels of viral protein and RNA synthesis in singly transfected cardiomyocyte cultures. Unexpectedly, when full-length and terminally deleted forms were cotransfected into cardiomyocytes, a cooperative interaction was observed, leading to increased viral RNA and protein production. However, when viral infections were carried out in cells harboring 5' terminally deleted CVB3 RNAs, a decrease in infectious particle production was observed. Our results provide a possible explanation for the necessity of full-length viral genomes during persistent infection, as they would stimulate efficient viral replication compared to that of the deleted genomes alone. To avoid high levels of viral particle production that would trigger cellular immune activation and host cell death, the terminally deleted RNA forms act to limit the production of viral particles, possibly as trans-dominant inhibitors. IMPORTANCE Enteroviruses like coxsackievirus B3 are able to initiate acute infections of cardiac tissue and, in some cases, to establish a long-term persistent infection that can lead to serious disease sequelae, including dilated cardiomyopathy. Previous studies have demonstrated the presence of 5' terminally deleted forms of enterovirus RNAs in heart tissues derived from patients with dilated cardiomyopathy. These deleted RNAs are found in association with very low levels of full-length enterovirus genomic RNAs, an interaction that may facilitate continued persistence while limiting virus particle production. Even in the absence of detectable infectious virus particle production, these deleted viral RNA forms express viral proteinases at levels capable of causing viral pathology. Our studies provide mechanistic insights into how full-length and deleted forms of enterovirus RNA cooperate to stimulate viral protein and RNA synthesis without stimulating infectious viral particle production. They also highlight the importance of targeting enteroviral proteinases to inhibit viral replication while at the same time limiting the long-term pathologies they trigger.