Production Of Virus Particles Research Articles

Cell culture\u2013based production of defective interfering influenza A virus particles in perfusion mode using an alternating tangential flow filtration system

Respiratory diseases including influenza A virus (IAV) infections represent a major threat to human health. While the development of a vaccine requires a lot of time, a fast countermeasure could be the use of defective interfering particles (DIPs) for antiviral therapy. IAV DIPs are usually characterized by a large internal deletion in one viral RNA segment. Consequentially, DIPs can only propagate in presence of infectious standard viruses (STVs), compensating the missing gene function. Here, they interfere with and suppress the STV replication and might act “universally” against many IAV subtypes. We recently reported a production system for purely clonal DIPs utilizing genetically modified cells. In the present study, we established an automated perfusion process for production of a DIP, called DI244, using an alternating tangential flow filtration (ATF) system for cell retention. Viable cell concentrations and DIP titers more than 10 times higher than for a previously reported batch cultivation were observed. Furthermore, we investigated a novel tubular cell retention device for its potential for continuous virus harvesting into the permeate. Very comparable performances to typically used hollow fiber membranes were found during the cell growth phase. During the virus replication phase, the tubular membrane, in contrast to the hollow fiber membrane, allowed 100% of the produced virus particles to pass through. To our knowledge, this is the first time a continuous virus harvest was shown for a membrane-based perfusion process. Overall, the process established offers interesting possibilities for advanced process integration strategies for next-generation virus particle and virus vector manufacturing.Key points• An automated perfusion process for production of IAV DIPs was established.• DIP titers of 7.40E + 9 plaque forming units per mL were reached.• A novel tubular cell retention device enabled continuous virus harvesting.

Interferon-Induced HERC5 Inhibits Ebola Virus Particle Production and Is Antagonized by Ebola Glycoprotein

Survival following Ebola virus (EBOV) infection correlates with the ability to mount an early and robust interferon (IFN) response. The host IFN-induced proteins that contribute to controlling EBOV replication are not fully known. Among the top genes with the strongest early increases in expression after infection in vivo is IFN-induced HERC5. Using a transcription- and replication-competent VLP system, we showed that HERC5 inhibits EBOV virus-like particle (VLP) replication by depleting EBOV mRNAs. The HERC5 RCC1-like domain was necessary and sufficient for this inhibition and did not require zinc finger antiviral protein (ZAP). Moreover, we showed that EBOV (Zaire) glycoprotein (GP) but not Marburg virus GP antagonized HERC5 early during infection. Our data identify a novel ‘protagonist–antagonistic’ relationship between HERC5 and GP in the early stages of EBOV infection that could be exploited for the development of novel antiviral therapeutics.

Mathematical modelling of SARS-CoV-2 infection of human and animal host cells reveals differences in the infection rates and delays in viral particle production by infected cells

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV -2), a causative agent of COVID-19 disease, poses a significant threat to public health. Since its outbreak in December 2019, Wuhan, China, extensive collection of diverse data from cell culture and animal infections as well as population level data from an ongoing pandemic, has been vital in assessing strategies to battle its spread. Mathematical modelling plays a key role in quantifying determinants that drive virus infection dynamics, especially those relevant for epidemiological investigations and predictions as well as for proposing efficient mitigation strategies. We utilized a simple mathematical model to describe and explain experimental results on viral replication cycle kinetics during SARS-CoV-2 infection of animal and human derived cell lines, green monkey kidney cells, Vero-E6, and human lung epithelium cells, A549-ACE2, respectively. We conducted cell infections using two distinct initial viral concentrations and quantified viral loads over time. We then fitted the model to our experimental data and quantified the viral parameters. We showed that such cellular tropism generates significant differences in the infection rates and incubation times of SARS-CoV-2, that is, the times to the first release of newly synthesised viral progeny by SARS-CoV-2-infected cells. Specifically, the rate at which A549-ACE2 cells were infected by SARS-CoV-2 was 15 times lower than that in the case of Vero-E6 cell infection and the duration of latent phase of A549-ACE2 cells was 1.6 times longer than that of Vero-E6 cells. On the other hand, we found no statistically significant differences in other viral parameters, such as viral production rate or infected cell death rate. Since in vitro infection assays represent the first stage in the development of antiviral treatments against SARS-CoV-2, discrepancies in the viral parameter values across different cell hosts have to be identified and quantified to better target vaccine and antiviral research.

High-Throughput NanoBiT-Based Screening for Inhibitors of HIV-1 Vpu and Host BST-2 Protein Interaction.

Bone marrow stromal cell antigen 2 (BST-2), also known as CD317 or tetherin, has been identified as a host restriction factor that suppresses the release of enveloped viruses from host cells by physically tethering viral particles to the cell surface; however, this host defense can be subverted by multiple viruses. For example, human immunodeficiency virus (HIV)-1 encodes a specific accessory protein, viral protein U (Vpu), to counteract BST-2 by binding to it and directing its lysosomal degradation. Thus, blocking the interaction between Vpu and BST-2 will provide a promising strategy for anti-HIV therapy. Here, we report a NanoLuc Binary Technology (NanoBiT)-based high-throughput screening assay to detect inhibitors that disrupt the Vpu-BST-2 interaction. Out of more than 1000 compounds screened, four inhibitors were identified with strong activity at nontoxic concentrations. In subsequent cell-based BST-2 degradation assays, inhibitor Y-39983 HCl restored the cell-surface and total cellular level of BST-2 in the presence of Vpu. Furthermore, the Vpu-mediated enhancement of pesudotyped viral particle production was inhibited by Y-39983 HCl. Our findings indicate that our newly developed assay can be used for the discovery of potential antiviral molecules with novel mechanisms of action.

Newly identified proviruses in Thermotogota suggest that viruses are the vehicles on the highways of interphylum gene sharing.

Phylogenomic analyses of bacteria from the phylum Thermotogota have shown extensive lateral gene transfer with distantly related organisms, particularly with Firmicutes. One likely mechanism of such DNA transfer is viruses. However, to date, only three temperate viruses have been characterized in this phylum, all infecting bacteria from the Marinitoga genus. Here we report 17 proviruses integrated into genomes of bacteria belonging to eight Thermotogota genera and induce viral particle production from one of the proviruses. All except an incomplete provirus from Mesotoga fall into two groups based on sequence similarity, gene synteny and taxonomic classification. Proviruses of Group 1 are found in the genera Geotoga, Kosmotoga, Marinitoga, Thermosipho and Mesoaciditoga and are similar to the previously characterized Marinitoga viruses, while proviruses from Group 2 are distantly related to the Group 1 proviruses, have different genome organization and are found in Petrotoga and Defluviitoga. Genes carried by both groups are closely related to Firmicutes and Firmicutes (pro)viruses in phylogenetic analyses. Moreover, one of the groups show evidence of recent gene exchange and may be capable of infecting cells from both phyla. We hypothesize that viruses are responsible for a large portion of the observed gene flow between Firmicutes and Thermotogota.

The impact of local assembly rules on RNA packaging in a T = 1 satellite plant virus.

The vast majority of viruses consist of a nucleic acid surrounded by a protective icosahedral protein shell called the capsid. During viral infection of a host cell, the timing and efficiency of the assembly process is important for ensuring the production of infectious new progeny virus particles. In the class of single-stranded RNA (ssRNA) viruses, the assembly of the capsid takes place in tandem with packaging of the ssRNA genome in a highly cooperative co-assembly process. In simple ssRNA viruses such as the bacteriophage MS2 and small RNA plant viruses such as STNV, this cooperative process results from multiple interactions between the protein shell and sites in the RNA genome which have been termed packaging signals. Using a stochastic assembly algorithm which includes cooperative interactions between the protein shell and packaging signals in the RNA genome, we demonstrate that highly efficient assembly of STNV capsids arises from a set of simple local rules. Altering the local assembly rules results in different nucleation scenarios with varying assembly efficiencies, which in some cases depend strongly on interactions with RNA packaging signals. Our results provide a potential simple explanation based on local assembly rules for the ability of some ssRNA viruses to spontaneously assemble around charged polymers and other non-viral RNAs in vitro.

Increased capsid oligomerization is deleterious to dengue virus particle production.

The capsid protein (C) of dengue virus is required for viral infectivity as it packages viral RNA genome into infectious particles. C exists as a homodimer that forms via hydrophobic interactions between the α2 and α4 helices of monomers. To identify C region(s) important for virus particle production, a complementation system was employed in which single-round infectious particles are generated by trans-encapsidation of a viral C-deleted genome by recombinant C expressed in mosquito cells. Mutants harbouring a complete α3 deletion, or a dual Ile65-/Trp69-to-Ala substitution in the α3 helix, exhibited reduced production of infectious virus. Unexpectedly, higher proportions of oligomeric C were detected in cells expressing both mutated forms as compared with the wild-type counterpart, indicating that the α3 helix, through its internal hydrophobic residues, may down-modulate oligomerization of C during particle formation. Compared with wild-type C, the double Ile65-/Trp69 to Ala mutations appeared to hamper viral infectivity but not C and genomic RNA incorporation into the pseudo-infectious virus particles, suggesting that increased C oligomerization may impair DENV replication at the cell entry step.

Hostile Takeover: How Viruses Reprogram Prokaryotic Metabolism.

To reproduce, prokaryotic viruses must hijack the cellular machinery of their hosts and redirect it toward the production of viral particles. While takeover of the host replication and protein synthesis apparatus has long been considered an essential feature of infection, recent studies indicate that extensive reprogramming of host primary metabolism is a widespread phenomenon among prokaryotic viruses that is required to fulfill the biosynthetic needs of virion production. In this review we provide an overview of the most significant recent findings regarding virus-induced reprogramming of prokaryotic metabolism and suggest how quantitative systems biology approaches may be used to provide a holistic understanding of metabolic remodeling during lytic viral infection.

Multiple Roles of Brd4 in the Infectious Cycle of Human Papillomaviruses.

Human Papillomaviruses (HPV) reproduce in stratified epithelia by establishing a reservoir of low- level infection in the dividing basal cells and restricting the production of viral particles to terminally differentiated cells. These small DNA viruses hijack pivotal cellular processes and pathways to support the persistent infectious cycle. One cellular factor that is key to multiple stages of viral replication and transcription is the BET (bromodomain and extra-terminal domain) protein, Brd4 (Bromodomain containing protein 4). Here we provide an overview of the multiple interactions of Brd4 that occur throughout the HPV infectious cycle.

Characterisation of Antiviral Activity of Cathelicidins from Naked Mole Rat and Python bivittatus on Human Herpes Simplex Virus 1.

Hg-CATH and Pb-CATH4 are cathelicidins from Heterocephalus glaber and Python bivittatus that have been previously identified as potent antibacterial peptides. However, their antiviral properties were not previously investigated. In this study, their activity against the herpes simplex virus (HSV)-1 was evaluated during primary human keratinocyte infection. Both of them significantly reduced HSV-1 DNA replication and production of infectious viral particles in keratinocytes at noncytotoxic concentrations, with the stronger activity of Pb-CATH4. These peptides did not show direct virucidal activity and did not exhibit significant immunomodulatory properties, except for Pb-CATH4, which exerted a moderate proinflammatory action. All in all, our results suggest that Hg-CATH and Pb-CATH4 could be potent candidates for the development of new therapies against HSV-1.

Repurposing nonnucleoside antivirals against SARS-CoV2 NSP12 (RNA dependent RNA polymerase): In silico-molecular insight

The pandemic of SARS-CoV-2 has necessitated expedited research efforts towards finding potential antiviral targets and drug development measures. While new drug discovery is time consuming, drug repurposing has been a promising area for elaborate virtual screening and identification of existing FDA approved drugs that could possibly be used for targeting against functions of various proteins of SARS-CoV-2 virus. RNA dependent RNA polymerase (RdRp) is an important enzyme for the virus that mediates replication of the viral RNA. Inhibition of RdRp could inhibit viral RNA replication and thus new virus particle production. Here, we screened non-nucleoside antivirals and found three out of them to be strongest in binding to RdRp out of which two retained binding even using molecular dynamic simulations. We propose these two drugs as potential RdRp inhibitors which need further in-depth testing.

Viperin, an IFN-Stimulated Protein, Delays Rotavirus Release by Inhibiting Non-Structural Protein 4 (NSP4)-Induced Intrinsic Apoptosis.

Viral infections lead to expeditious activation of the host’s innate immune responses, most importantly the interferon (IFN) response, which manifests a network of interferon-stimulated genes (ISGs) that constrain escalating virus replication by fashioning an ill-disposed environment. Interestingly, most viruses, including rotavirus, have evolved numerous strategies to evade or subvert host immune responses to establish successful infection. Several studies have documented the induction of ISGs during rotavirus infection. In this study, we evaluated the induction and antiviral potential of viperin, an ISG, during rotavirus infection. We observed that rotavirus infection, in a stain independent manner, resulted in progressive upregulation of viperin at increasing time points post-infection. Knockdown of viperin had no significant consequence on the production of total infectious virus particles. Interestingly, substantial escalation in progeny virus release was observed upon viperin knockdown, suggesting the antagonistic role of viperin in rotavirus release. Subsequent studies unveiled that RV-NSP4 triggered relocalization of viperin from the ER, the normal residence of viperin, to mitochondria during infection. Furthermore, mitochondrial translocation of NSP4 was found to be impeded by viperin, leading to abridged cytosolic release of Cyt c and subsequent inhibition of intrinsic apoptosis. Additionally, co-immunoprecipitation studies revealed that viperin associated with NSP4 through regions including both its radical SAM domain and its C-terminal domain. Collectively, the present study demonstrated the role of viperin in restricting rotavirus egress from infected host cells by modulating NSP4 mediated apoptosis, highlighting a novel mechanism behind viperin’s antiviral action in addition to the intricacy of viperin–virus interaction.

HIV Viral Dynamic under Treatment with Intracellular Delay and Virus Decay as Interactive Parameters

Treatment with antiviral drugs for human immunodeficiency virus type 1 (HIV-1) infection causes a rapid reduction in plasma viral load. Viral decline occurs in several stages and provides information on important kinetic constants of virus replication in vivo and pharmacodynamic properties. We present a mathematical model that not only considers the intracellular phase of the viral life cycle, defined as the time between the infection of a cell and the production of new viral particles, but we also consider that this parameter together with the virus decay are interactive fuzzy numbers.

Full assembly of HIV-1 particles requires assistance of the membrane curvature factor IRSp53.

During HIV-1 particle formation, the requisite plasma membrane curvature is thought to be solely driven by the retroviral Gag protein. Here, we reveal that the cellular I-BAR protein IRSp53 is required for the progression of HIV-1 membrane curvature to complete particle assembly. siRNA-mediated knockdown of IRSp53 gene expression induces a decrease in viral particle production and a viral bud arrest at half completion. Single-molecule localization microscopy at the cell plasma membrane shows a preferential localization of IRSp53 around HIV-1 Gag assembly sites. In addition, we observe the presence of IRSp53 in purified HIV-1 particles. Finally, HIV-1 Gag protein preferentially localizes to curved membranes induced by IRSp53 I-BAR domain on giant unilamellar vesicles. Overall, our data reveal a strong interplay between IRSp53 I-BAR and Gag at membranes during virus assembly. This highlights IRSp53 as a crucial host factor in HIV-1 membrane curvature and its requirement for full HIV-1 particle assembly.

HIV Replication Is Increased by RNA Methylation METTL3/METTL14/WTAP Complex Activators.

The N6-methyladenosine (m6A) modifications in both viral and host cell RNAs play an important role in HIV-1 virus genome transcription and virus replication. We demonstrate here that activators of the METTL3/METTL14/WTAP RNA methyltransferase complex enhance the production of virus particles in cells harboring HIV-1 provirus. In parallel, the amount of m6A residues in the host cell mRNA was increased in the presence of these activator compounds. Importantly, the m6A methylation of the HIV-1 RNA was also enhanced significantly (about 18%). The increase of virus replication by the small-molecule activators of the METTL3/METTL14/WTAP complex excludes them as potential anti-HIV-1 drug candidates. However, the compounds may be of large interest as activators for the latent HIV-1 provirus copies deposited in host cells’ genome and the subsequent virus eradication by an antiviral compound.

In Vivo Generation of BK and JC Polyomavirus Defective Viral Genomes in Human Urine Samples Associated with Higher Viral Loads.

ABSTRACTDefective viral genomes (DVGs) are parasitic viral sequences containing point mutations, deletions, or duplications that might interfere with replication. DVGs are often associated with viral passage at high multiplicities of infection in culture systems but have been increasingly reported in clinical specimens. To date however, only RNA viruses have been shown to contain DVGs in clinical specimens. Here, using direct deep sequencing with multiple library preparation strategies and confirmatory digital droplet PCR (ddPCR) of urine samples taken from immunosuppressed individuals, we show that clinical BK polyomavirus (BKPyV) and JC polyomavirus (JCPyV) strains contain widespread genomic rearrangements across multiple loci that likely interfere with viral replication. BKPyV DVGs were derived from BKPyV genotypes Ia, Ib-1, and Ic. The presence of DVGs was associated with specimens containing higher viral loads but never reached clonality, consistent with a model of parasitized replication. These DVGs persisted during clinical infection as evidenced in two separate pairs of samples containing BK virus collected from the same individual up to 302 days apart. In a separate individual, we observed the generation of DVGs after a 57.5-fold increase in viral load. In summary, by extending the presence of DVGs in clinical specimens to DNA viruses, we demonstrate the ubiquity of DVGs in clinical virology.IMPORTANCE Defective viral genomes (DVGs) can have a significant impact on the production of infectious virus particles. DVGs have only been identified in cultured viruses passaged at high multiplicities of infection and RNA viruses collected from clinical specimens; no DNA virus in the wild has been shown to contain DVGs. Here, we identified BK and JC polyomavirus DVGs in clinical urine specimens and demonstrated that these DVGs are more frequently identified in samples with higher viral loads. The strains containing DVGs had rearrangements throughout their genomes, with the majority affecting genes required for viral replication. Longitudinal analysis showed that these DVGs can persist during an infection but do not reach clonality within the chronically infected host. Our identification of polyomavirus DVGs suggests that these parasitic sequences exist across the many classes of viruses capable of causing human disease.

Heterogeneity of SARS-CoV-2 virus produced in cell culture revealed by shotgun proteomics and supported by genome sequencing

COVID-19 is the most disturbing pandemic of the past hundred years. Its causative agent, the SARS-CoV-2 virus, has been the subject of an unprecedented investigation to characterize its molecular structure and intimate functioning. While markers for its detection have been proposed and several diagnostic methodologies developed, its propensity to evolve and evade diagnostic tools and the immune response is of great concern. The recent spread of new variants with increased infectivity requires even more attention. Here, we document how shotgun proteomics can be useful for rapidly monitoring the evolution of the SARS-CoV-2 virus. We evaluated the heterogeneity of purified SARS-CoV-2 virus obtained after culturing in the Vero E6 cell line. We found that cell culture induces significant changes that are translated at the protein level, such changes being detectable by tandem mass spectrometry. Production of viral particles requires careful quality control which can be easily performed by shotgun proteomics. Although considered relatively stable so far, the SARS-CoV-2 genome turns out to be prone to frequent variations. Therefore, the sequencing of SARS-CoV-2 variants from patients reporting only the consensus genome after its amplification would deserve more attention and could benefit from more in-depth analysis of low level but crystal-clear signals, as well as complementary and rapid analysis by shotgun proteomics.Graphical abstract

A Thioester-Containing Protein Controls Dengue Virus Infection in Aedes aegypti Through Modulating Immune Response.

Complement-like proteins in arthropods defend against invading pathogens in the early phases of infection. Thioester-containing proteins (TEPs), which exhibit high similarity to mammalian complement C3, are thought to play a key role in the innate immunity of arthropods. We identified and characterized anti-dengue virus (DENV) host factors, in particular complement-like proteins, in the mosquito Aedes aegypti. Our results indicate that TEP1 limits DENV infection in Ae. aegypti. We showed that TEP1 transcription is highly induced in mosquitoes following DENV infection. Silencing TEP1 resulted in the up-regulation of viral RNA and proteins. In addition, the production of infectious virus particles increased in the absence of TEP1. We generated a transgenic mosquito line with a TEP1 loss-of-function phenotype under a blood meal-inducible promoter. We showed that viral protein and titers increased in transgenic mosquitoes after an infectious blood meal. Interestingly, expression of transcription factor Rel2 and certain anti-microbial peptides (AMPs) were inhibited in transgenic mosquitoes. Overall, our results suggest that TEP1 regulates the immune response and consequently controls the replication of dengue virus in mosquitoes. This finding provides new insight into the molecular mechanisms of mosquito host factors in the regulation of DENV replication.

Characterizing infection of B lymphocytes with vaccine and wild-type strains of measles virus

Abstract Wild-type (w.t.) measles virus (MeV) infection can result in immunosuppression that does not occur following vaccination. A rhesus macaque model indicated that B cells are a target of infection. The role of B cell infection in MeV-induced immune suppression is not fully understood. We characterize the differences in viral replication in human B cells infected with vaccine (Edmonston Zagreb [EZ]) or w.t. virus. B cells from healthy donor blood were infected (MOI 1.0) with a GFP-expressing EZ virus or a w.t. virus to evaluate viral replication after 24 and 48 hours. The number of GFP+ cells (EZ infected) or hemagglutinin (H)+ cells (w.t. infected) was measured via flow cytometry. Transcription of the viral nucleoprotein (N) gene was measured by mRNA-specific cDNA generation and N gene specific qPCR. Production of infectious viral particles was measured via endpoint dilution. In B cells infected with EZ, 30% of cells expressed GFP at 24 hours and 45% of cells expressed GFP at 48 hours. There was a 4.4-fold increase in N gene transcription from 0 to 48 hours. There was no detectable increase in viral titer over input after 48 hours. In B cells infected with w.t. virus, 10% of cells expressed viral H at 24 hours with no increase in H expression at 48 hours. The w.t. virus had a 2.4-fold increase in viral transcription from 0 to 48 hours. There was no detectable increase in viral titer over input after 48 hours. MeV-infected B cells show viral protein production and N gene transcription at 24 and 48 hours. However, w.t. virus replication may be less efficient as shown by lower percentages of H+ cells at 24 and 48 hours and lower levels of N gene transcription at 48 hours. There is no evidence of infectious virus production by either strain of MeV despite evidence of replication.

The Epstein-Barr Virus Lytic Protein BMLF1 Induces Upregulation of GRP78 Expression through ATF6 Activation.

The unfolded protein response (UPR) is an intracellular signaling pathway essential for alleviating the endoplasmic reticulum (ER) stress. To support the productive infection, many viruses are known to use different strategies to manipulate the UPR signaling network. However, it remains largely unclear whether the UPR signaling pathways are modulated in the lytic cycle of Epstein-Barr virus (EBV), a widely distributed human pathogen. Herein, we show that the expression of GRP78, a central UPR regulator, is up-regulated during the EBV lytic cycle. Our data further revealed that knockdown of GRP78 in EBV-infected cell lines did not substantially affect lytic gene expression; however, GRP78 knockdown in these cells markedly reduced the production of virus particles. Importantly, we identified that the early lytic protein BMLF1 is the key regulator critically contributing to the activation of the grp78 gene promoter. Mechanistically, we found that BMLF1 can trigger the proteolytic cleavage and activation of the UPR senor ATF6, which then transcriptionally activates the grp78 promoter through the ER stress response elements. Our findings therefore provide evidence for the connection between the EBV lytic cycle and the UPR, and implicate that the BMLF1-mediated ATF6 activation may play critical roles in EBV lytic replication.