Production Of Virus Particles Research Articles

Adeno-Associated Virus Production, Purification, and Titering.

Adeno-associated virus (AAV) vectors are exemplary tools for studying gene function in vivo and are particularly favorable for transferring genes of interest into brain tissues. They have shown great promise as a gene therapy vector for preclinical and clinical applications. However, the ability to use this tool is often hampered because the viruses themselves are not readily available. Many methods have been developed for AAV production. Here, we describe a simple method for small- to medium-scale (1012 -1013 viral particles) production of AAV based on Polyethylenimine Max (PEI Max)-mediated triple transfection of HEK 293 cells and purification with iodixanol gradient ultracentrifugation. These methods will provide users with ample material of sufficient quality for performing in vivo gene transfer. © 2018 by John Wiley & Sons, Inc.

Tomatidine, a novel antiviral compound towards dengue virus

Dengue is the most common arboviral disease worldwide with 96 million symptomatic cases annually. Despite its major impact on global human health and huge economic burden there is no antiviral drug available to treat the disease. The first tetravalent dengue virus vaccine was licensed in 2015 for individuals aged 9 to 45, however, most cases are reported in infants and young children. This, together with the limited efficacy of the vaccine to dengue virus (DENV) serotype 2, stresses the need to continue the search for compounds with antiviral activity to DENV. In this report, we describe tomatidine as a novel compound with potent antiviral properties towards all DENV serotypes and the related Zika virus. The strongest effect was observed for DENV-2 with an EC50 and EC90 value of 0.82 and 1.61 μM, respectively, following infection of Huh7 cells at multiplicity of infection of 1. The selectivity index is 97.7. Time-of-drug-addition experiments revealed that tomatidine inhibits virus particle production when added pre, during and up to 12 h post-infection. Subsequent experiments show that tomatidine predominantly acts at a step after virus-cell binding and membrane fusion but prior to the secretion of progeny virions. Tomatidine was found to control the expression of the cellular protein activating transcription factor 4 (ATF4), yet, this protein is not solely responsible for the observed antiviral effect. Here, we propose tomatidine as a candidate for the treatment of dengue given its potent antiviral activity.

Nucleolar Localization of HIV-1 Rev Is Required, Yet Insufficient for Production of Infectious Viral Particles.

Combination antiretroviral therapy fails in complete suppression of HIV-1 due to drug resistance and persistent latency. Novel therapeutic intervention requires knowledge of intracellular pathways responsible for viral replication, specifically those untargeted by antiretroviral drugs. An understudied phenomenon is the nucleolar localization of Rev phosphoprotein, which completes nucleocytoplasmic transport of unspliced/partially spliced HIV mRNA through multimerization with intronic cis-acting targets—the Rev-response element (RRE). Rev contains a nucleolar localization signal (NoLS) comprising the COOH terminus of the arginine-rich motif for accumulation within nucleoli—speculated as the interaction ground for Rev with cellular proteins mediating mRNA-independent nuclear export and splicing. Functionality of Rev nucleolar access during HIV-1 production and infection was investigated in the context of deletion and single-point mutations within Rev-NoLS. Mutations induced upon Rev-NoLS are hypothesized to inactivate the HIV-1 infectious cycle. HIV-1HXB2 replication ceased with Rev mutations lacking nucleolar access due to loss or replacement of multiple arginine residues. Rev mutations missing single arginine residues remained strictly nucleolar in pattern and participated in proviral production, however, with reduced efficiency. Viral RNA packaging also decreased in efficiency after expression of nucleolar-localizing mutations. These results were observed during propagation of variant HIV-1NL4-3 containing nucleolar-localizing mutations within the viral backbone (M4, M5, and M6). Lentiviral particles produced with Rev single-point mutations were transducible at extremely low frequency. Similarly, HIV-1NL4-3 Rev-NoLS variants lost infectivity, unlike virulent WT (wild type) HIV-1NL4-3. HIV-1NL4-3 variants were capable of CD4+ host entry and reverse transcription as WT HIV-1NL4-3, but lacked ability to complete a full infectious cycle. We currently reveal that viral integration is deregulated in the presence of Rev-NoLS mutations.

Metagenomic Sequencing From Mosquitoes in China Reveals a Variety of Insect and Human Viruses.

We collected 8,700 mosquitoes in three sites in China, which belonged to seven species. Their viromes were tested using metagenomic sequencing and bioinformatic analysis. The abundant viral sequences were detected and annotated belonging to more than 50 viral taxonomic families. The results were verified by PCR, followed by phylogenetic analysis. In the present study, we identified partial viral genes of dengue virus (DENV), a novel circovirus (CCV), densovirus (DNV), Japanese encephalitis virus (JEV), and Wuhan mosquito virus (WMV) in mosquitoes. Metagenomic analysis and PCR amplification revealed three DENV sequences, which were as homologous to the NS3 gene of DENV from Singapore isolated in 2005, with at least 91% nucleotide (nt) identity. Seven fragments of JEV encoding structural proteins were identified belonging to genotype I. They all shared high homology with structural protein genes of JEV isolated from Laos in 2009. The production of infectious virus particles of the newly isolated virus YunnanJEV2017-4 increased after passage from the BHK-21 cell line to the Vero cell line. Novel circovirus-related genes were identified and as being related to an unnamed gene of a mosquito circovirus (MCCV) sequence from the USA isolated in 2011, with at least 41% nt identity: this distant relationship suggests that the parent virus might belong to a novel circovirus genus. Additionally, numerous known viruses and some unknown viruses were also detected in mosquitoes from Yunnan province, China, which will be tested for propagation.

The Endonucleolytic RNA Cleavage Function of nsp1 of Middle East Respiratory Syndrome Coronavirus Promotes the Production of Infectious Virus Particles in Specific Human Cell Lines.

Middle East respiratory syndrome coronavirus (MERS-CoV) nsp1 suppresses host gene expression in expressed cells by inhibiting translation and inducing endonucleolytic cleavage of host mRNAs, the latter of which leads to mRNA decay. We examined the biological functions of nsp1 in infected cells and its role in virus replication by using wild-type MERS-CoV and two mutant viruses with specific mutations in the nsp1; one mutant lacked both biological functions, while the other lacked the RNA cleavage function but retained the translation inhibition function. In Vero cells, all three viruses replicated efficiently with similar replication kinetics, while wild-type virus induced stronger host translational suppression and host mRNA degradation than the mutants, demonstrating that nsp1 suppressed host gene expression in infected cells. The mutant viruses replicated less efficiently than wild-type virus in Huh-7 cells, HeLa-derived cells, and 293-derived cells, the latter two of which stably expressed a viral receptor protein. In 293-derived cells, the three viruses accumulated similar levels of nsp1 and major viral structural proteins and did not induce IFN-β and IFN-λ mRNAs; however, both mutants were unable to generate intracellular virus particles as efficiently as wild-type virus, leading to inefficient production of infectious viruses. These data strongly suggest that the endonucleolytic RNA cleavage function of the nsp1 promoted MERS-CoV assembly and/or budding in a 293-derived cell line. MERS-CoV nsp1 represents the first CoV gene 1 protein that plays an important role in virus assembly/budding and is the first identified viral protein whose RNA cleavage-inducing function promotes virus assembly/budding.IMPORTANCE MERS-CoV represents a high public health threat. Because CoV nsp1 is a major viral virulence factor, uncovering the biological functions of MERS-CoV nsp1 could contribute to our understanding of MERS-CoV pathogenicity and spur development of medical countermeasures. Expressed MERS-CoV nsp1 suppresses host gene expression, but its biological functions for virus replication and effects on host gene expression in infected cells are largely unexplored. We found that nsp1 suppressed host gene expression in infected cells. Our data further demonstrated that nsp1, which was not detected in virus particles, promoted virus assembly or budding in a 293-derived cell line, leading to efficient virus replication. These data suggest that nsp1 plays an important role in MERS-CoV replication and possibly affects virus-induced diseases by promoting virus particle production in infected hosts. Our data, which uncovered an unexpected novel biological function of nsp1 in virus replication, contribute to further understanding of the MERS-CoV replication strategies.

Progress in gene therapy using oncolytic vaccinia virus as vectors.

Vaccinia virus was widely used in the World Health Organization's smallpox eradication campaign and is currently a promising vector for gene therapy owing to its unique characteristics. Vaccinia virus can selectively replicate and propagate productively in tumor cells, resulting in oncolysis. In addition, rapid viral particle production, wide host range, large genome size (approximately 200 kb), and safe handling render vaccinia virus a suitable vector for gene therapy. Cancer vaccines and gene therapy are being studied in clinical trials and experiment researches. However, we put forward unique challenges of optimal selection of foreign genes, administration and modification of VACV, personalized medicine, and other existing problems, based on current researches and our own experiments. This review presents an overview of the vaccinia virus from its mechanisms to medical researches and clinical trials. We believe that the solution to these problems will contribute to understanding mechanisms of VACV and provide a theoretical basis for clinical treatment.

Impact of immune suppressive agents on the BK-Polyomavirus non coding control region

BackgroundReactivation of the BK-Polyomavirus (BKPyV) can cause a polyomavirus associated nephropathy in approx. 10% of kidney transplant recipients. In these cases, current therapy is based on the reduction of immunosuppression. Since BKPyV-transcription is driven by the Non-Coding-Control-Region (NCCR) we were interested whether NCCR-activity is affected by immunosuppressive agents. MethodsPlasma samples from 45 BKPyV-positive patients after renal transplantation were subjected to PCR-analysis. NCCR-amplicons were cloned into a plasmid that allows the quantification of early and late NCCR-activity by tdTomato and eGFP expression, respectively. HEK293T-cells were transfected with the reporter-plasmids, treated with immunosuppressive agents, and subjected to FACS-analysis. In addition, H727-cells were infected with patient derived BKPyV, treated with mTOR-inhibitors, and NCCR activity was analysed using qRT-PCR. ResultsWhile tacrolimus and cyclosporine-A did not affect NCCR-promoter-activity, treatment with mTOR1-inhibitor rapamycin resulted in the reduction of early, but not late-NCCR-promoter-activity. Treatment with dual mTOR1/2 inhibitors (INK128 or pp242) led to significant inhibition of early, however, concomitantly enhanced late-promoter-activity. In BKPyV infected cells both rapamycin and INK128 reduced early expression, however, INK128 resulted in higher late-mRNA levels when compared to rapamycin treatment. ConclusionsOur results demonstrate that mTOR1-inhibitors are able to reduce early-expression of wildtype and rearranged NCCRs, which might contribute to previously described inhibition of BKPyV-replication. Dual mTOR1/2-inhibitors, however, additionally might shift viral early into late-expression promoting synthesis of viral structural proteins and particle production.

Modeling the physicochemical properties of DNA:PEI polyplexes on transient gene expression: A DoE-based approach

Cell-based production of viral particles has gain interest in the last years due to promising therapeutic applications. In the field of vaccination, novel vaccines are required to face the new pathogen outbreaks, and rapid and more efficient processes are required to respond as fast as possible to the vaccination demand worldwide. On the other hand, viral vector-driven gene therapies have demonstrated to be efficient and safe in numerous clinical trials, and three of them are already approved for commercialization. However, viral vector production is still a bottleneck in the road to the clinic. Although batch and fed-batch culture modes are preferred in industry, continuous culture strategies have demonstrated to improve viral titers and to reduce the bioprocessing costs. Therefore, there is increasing interest in exploring and optimizing continuous strategies in order to intensify viral vector production bioprocesses. This review is a summary of how continuous cultures have been applied to viral particle production (viral vaccines and viral vectors), the improvements achieved so far and the future perspectives in this field.

Suppression of Hepatitis C Virus Genome Replication and Particle Production by a Novel Diacylglycerol Acyltransferases Inhibitor.

Diacylglycerol acyltransferases (DGATs) play a critical role in the biosynthesis of endogenous triglycerides (TGs) and formation of lipid droplets (LDs) in the liver. In particular, one member of DGATs, DGAT-1 was reported to be an essential host factor for the efficient production of hepatitis C virus (HCV) particles. By utilizing our previously characterized three different groups of twelve DGAT inhibitors, we found that one of the DGAT inhibitors, a 2-((4-adamantylphenoxy) methyl)-N-(furan-2-ylmethyl)-1H-benzo[d]imidazole-5-carboxam (10j) is a potent suppressor of both HCV genome replication and particle production. 10j was able to induce inhibition of these two critical viral functions in a mutually separate manner. Abrogation of the viral genome replication by 10j led to a significant reduction in the viral protein expression as well. Interestingly, we found that its antiviral effect did not depend on the reduction of TG biosynthesis by 10j. This suggests that the inhibitory activity of 10j against DGATs may not be directly related with its antiviral action.

The effect of delay in viral production in within-host models during early infection

ABSTRACTDelay in viral production may have a significant impact on the early stages of infection. During the eclipse phase, the time from viral entry until active production of viral particles, no viruses are produced. This delay affects the probability that a viral infection becomes established and timing of the peak viral load. Deterministic and stochastic models are formulated with either multiple latent stages or a fixed delay for the eclipse phase. The deterministic model with multiple latent stages approaches in the limit the model with a fixed delay as the number of stages approaches infinity. The deterministic model framework is used to formulate continuous-time Markov chain and stochastic differential equation models. The probability of a minor infection with rapid viral clearance as opposed to a major full-blown infection with a high viral load is estimated from a branching process approximation of the Markov chain model and the results are confirmed through numerical simulations. In addition, parameter values for influenza A are used to numerically estimate the time to peak viral infection and peak viral load for the deterministic and stochastic models. Although the average length of the eclipse phase is the same in each of the models, as the number of latent stages increases, the numerical results show that the time to viral peak and the peak viral load increase.

Regulation of Apolipoprotein E Trafficking by Hepatitis C Virus-Induced Autophagy.

Apolipoprotein E (ApoE) plays an important role in the maturation and infectivity of hepatitis C virus (HCV). By analyzing the subcellular localization of ApoE in Huh7 hepatoma cells that harbored an HCV subgenomic RNA replicon, we found that ApoE colocalized with autophagosomes. This colocalization was marginally detected in HCV-infected cells, apparently due to the depletion of ApoE by HCV, as treatment with bafilomycin A1 (BafA1), a vacuolar ATPase inhibitor that inhibits autophagic protein degradation, partially restored the ApoE level and enhanced its colocalization with autophagosomes in HCV-infected cells. The role of HCV-induced autophagy in the degradation of ApoE was further supported by the observations that nutrient starvation, which induces autophagic protein degradation, led to the loss of ApoE in HCV subgenomic RNA replicon cells and that the knockdown of ATG7, a protein essential for the formation of autophagic vacuoles, increased the ApoE level in cells with productive HCV replication. Interestingly, the inhibition of autophagy by ATG7 knockdown reduced the colocalization of ApoE with the HCV E2 envelope protein and the HCV titers released from cells. In contrast, the treatment of cells with BafA1 enhanced the colocalization of ApoE and HCV E2 and increased both intracellular and extracellular HCV titers. These results indicated that autophagy played an important role in the trafficking of ApoE in HCV-infected cells. While it led to autophagic degradation of ApoE, it also promoted the interaction between ApoE and HCV E2 to enhance the production of infectious progeny viral particles.IMPORTANCE Hepatitis C virus (HCV) is one of the most important human pathogens. Its virion is associated with apolipoprotein E (ApoE), which enhances its infectivity. HCV induces autophagy to enhance its replication. In this report, we demonstrate that autophagy plays an important role in the trafficking of ApoE in HCV-infected cells. This leads to the degradation of ApoE by autophagy. However, if the autophagic protein degradation is inhibited, ApoE is stabilized and colocalized with autophagosomes. This leads to its enhanced colocalization with the HCV E2 envelope protein and increased production of infectious progeny viral particles. If autophagy is inhibited by suppressing the expression of ATG7, a gene essential for the formation of autophagosomes, the colocalization of ApoE with E2 is reduced, resulting in the reduction of progeny viral titers. These results indicate an important role of autophagy in the transport of ApoE to promote the production of infectious HCV particles.

Description and characterization of a novel live-attenuated tri-segmented Machupo virus in Guinea pigs

BackgroundMachupo virus (MACV) is a member of the Mammarenavirus genus, Arenaviridae family and is the etiologic agent of Bolivian hemorrhagic fever, which causes small outbreaks or sporadic cases. Several other arenaviruses in South America Junín virus (JUNV) in Argentina, Guanarito in Venezuela, Sabiá in Brazil and Chapare in Bolivia, also are responsible for human hemorrhagic fevers. Among these arenaviruses, JUNV caused thousands of human cases until 1991, when the live attenuated Candid #1 vaccine, was used. Other than Candid #1 vaccine, few other therapeutic or prophylactic treatments exist. Therefore, new strategies for production of safe countermeasures with broad spectrum activity are needed.FindingsWe tested a tri-segmented MACV, a potential vaccine candidate with several mutations, (r3MACV). In cell culture, r3MACV showed a 2-log reduction in infectious virus particle production and the MACV inhibition of INF-1β was removed from the construct and produced by infected cells. Furthermore, in an animal experiment, r3MACV was able to protect 50% of guinea pigs from a simultaneous lethal JUNV challenge. Protected animals didn’t display clinical symptoms nor were virus particles found in peripheral blood (day 14) or in organs (day 28 post-inoculation). The r3MACV provided a higher protection than the Candid #1 vaccine.ConclusionsThe r3MACV provides a potential countermeasure against two South America arenaviruses responsible of human hemorrhagic fever.

PO-031 Sensitising P53 mutant HNSCC to oncolytic reovirus (RT3D) by targeting the unfolded protein response

Introduction Oncolytic viruses are able to enter and selectively replicate in cancer cells. These self-propagating agents kill through multiple mechanisms and have the potential to promote anti-tumour immune responses. Reovirus strain type 3 Dearing (RT3D) is an oncolytic virus actively under clinical investigation. RT3D has been shown to induce ER stress and trigger the downstream unfolded protein response (UPR) in a variety of tumour types. Recent evidence has indicated that proteasome inhibition in multiple myeloma, and BRAF inhibition in BRAF mutant melanoma, sensitise to RT3D through perturbation of the normal cellular response to ER stress. We sought to investigate if more direct modulation of UPR signalling could sensitise head and neck squamous cell carcinoma (HNSCC) to RT3D. Material and methods ER stress agents including novel agents targeting UPR signalling were screened for sensitisation to RT3D in a panel of HNSCC cell lines. Alterations to reovirus protein levels and viable virus particle production was assessed. A 3D tumour spheroid model was used as a more accurate method to study stress in vitro. Recombinant protein expression and shRNA techniques were utilised to study how the unfolded protein response impacts reovirus replication. ER stress measured by GRP78 expression was also assessed in HNSCC patient samples. Results and discussions The PERK inhibitor GSK2606414 and the canonical ER stress agent thapsigargin were shown to sensitise HNSCC to RT3D. PERK inhibition resulted in increased viral capsid protein levels and increased viable RT3D production from target cells. PERK inhibition enhanced the ability of RT3D to eradicate HNSCC 3D tumour spheroids. PERK inhibition increased RT3D spread in 3D spheroids while perturbing normal ER chaperone levels and inhibiting RT3D induced LC3A/B. In HNSCC patient samples, GRP78 was found to be elevated in tumour vs stroma with significant variation between patients. This highlights the importance in understanding the link between ER stress levels/UPR signalling and reovirus efficacy. Conclusion We demonstrate that modulators of the ER stress response represent a novel mechanism of sensitisation to RT3D in head and neck.

Experimental Analysis of Mimivirus Translation Initiation Factor 4a Reveals Its Importance in Viral Protein Translation during Infection of Acanthamoeba polyphaga.

The Acanthamoeba polyphaga mimivirus is the first giant virus ever described, with a 1.2-Mb genome which encodes 979 proteins, including central components of the translation apparatus. One of these proteins, R458, was predicted to initiate translation, although its specific role remains unknown. We silenced the R458 gene using small interfering RNA (siRNA) and compared levels of viral fitness and protein expression in silenced versus wild-type mimivirus. Silencing decreased the growth rate, but viral particle production at the end of the viral cycle was unaffected. A comparative proteomic approach using two-dimensional difference-in-gel electrophoresis (2D-DIGE) revealed deregulation of the expression of 32 proteins in silenced mimivirus, which were defined as up- or downregulated. Besides revealing proteins with unknown functions, silencing R458 also revealed deregulation in proteins associated with viral particle structures, transcriptional machinery, oxidative pathways, modification of proteins/lipids, and DNA topology/repair. Most of these proteins belong to genes transcribed at the end of the viral cycle. Overall, our data suggest that the R458 protein regulates the expression of mimivirus proteins and, thus, that mimivirus translational proteins may not be strictly redundant in relation to those from the amoeba host. As is the case for eukaryotic initiation factor 4a (eIF4a), the R458 protein is the prototypical member of the ATP-dependent DEAD box RNA helicase mechanism. We suggest that the R458 protein is required to unwind the secondary structures at the 5' ends of mRNAs and to bind the mRNA to the ribosome, making it possible to scan for the start codon. These data are the first experimental evidence of mimivirus translation-related genes, predicted to initiate protein biosynthesis.IMPORTANCE The presence in the genome of a mimivirus of genes coding for many translational processes, with the exception of ribosome constituents, has been the subject of debate since its discovery in 2003. In this work, we focused on the R458 mimivirus gene, predicted to initiate protein biosynthesis. After silencing was performed, we observed that it has no major effect on mimivirus multiplication but that it affects protein expression and fitness. This suggests that it is effectively used by mimivirus during its developmental cycle. Until large-scale genetic manipulation of giant viruses becomes possible, the silencing strategy used here on mimivirus translation-related factors will open the way to understanding the functions of these translational genes.

Identification and characterization of host proteins bound to dengue virus 3' UTR reveal an antiviral role for quaking proteins.

The four dengue viruses (DENV1-4) are rapidly reemerging infectious RNA viruses. These positive-strand viral genomes contain structured 3′ untranslated regions (UTRs) that interact with various host RNA binding proteins (RBPs). These RBPs are functionally important in viral replication, pathogenesis, and defense against host immune mechanisms. Here, we combined RNA chromatography and quantitative mass spectrometry to identify proteins interacting with DENV1-4 3′ UTRs. As expected, RBPs displayed distinct binding specificity. Among them, we focused on quaking (QKI) because of its preference for the DENV4 3′ UTR (DENV-4/SG/06K2270DK1/2005). RNA immunoprecipitation experiments demonstrated that QKI interacted with DENV4 genomes in infected cells. Moreover, QKI depletion enhanced infectious particle production of DENV4. On the contrary, QKI did not interact with DENV2 3′ UTR, and DENV2 replication was not affected consistently by QKI depletion. Next, we mapped the QKI interaction site and identified a QKI response element (QRE) in DENV4 3′ UTR. Interestingly, removal of QRE from DENV4 3′ UTR abolished this interaction and increased DENV4 viral particle production. Introduction of the QRE to DENV2 3′ UTR led to QKI binding and reduced DENV2 infectious particle production. Finally, reporter assays suggest that QKI reduced translation efficiency of viral RNA. Our work describes a novel function of QKI in restricting viral replication.

Cell-to-Cell Measles Virus Spread between Human Neurons Is Dependent on Hemagglutinin and Hyperfusogenic Fusion Protein.

Measles virus (MV) usually causes acute infection but in rare cases persists in the brain, resulting in subacute sclerosing panencephalitis (SSPE). Since human neurons, an important target affected in the disease, do not express the known MV receptors (signaling lymphocyte activation molecule [SLAM] and nectin 4), how MV infects neurons and spreads between them is unknown. Recent studies have shown that many virus strains isolated from SSPE patients possess substitutions in the extracellular domain of the fusion (F) protein which confer enhanced fusion activity. Hyperfusogenic viruses with such mutations, unlike the wild-type MV, can induce cell-cell fusion even in SLAM- and nectin 4-negative cells and spread efficiently in human primary neurons and the brains of animal models. We show here that a hyperfusogenic mutant MV, IC323-F(T461I)-EGFP (IC323 with a fusion-enhancing T461I substitution in the F protein and expressing enhanced green fluorescent protein), but not the wild-type MV, spreads in differentiated NT2 cells, a widely used human neuron model. Confocal time-lapse imaging revealed the cell-to-cell spread of IC323-F(T461I)-EGFP between NT2 neurons without syncytium formation. The production of virus particles was strongly suppressed in NT2 neurons, also supporting cell-to-cell viral transmission. The spread of IC323-F(T461I)-EGFP was inhibited by a fusion inhibitor peptide as well as by some but not all of the anti-hemagglutinin antibodies which neutralize SLAM- or nectin-4-dependent MV infection, suggesting the presence of a distinct neuronal receptor. Our results indicate that MV spreads in a cell-to-cell manner between human neurons without causing syncytium formation and that the spread is dependent on the hyperfusogenic F protein, the hemagglutinin, and the putative neuronal receptor for MV.IMPORTANCE Measles virus (MV), in rare cases, persists in the human central nervous system (CNS) and causes subacute sclerosing panencephalitis (SSPE) several years after acute infection. This neurological complication is almost always fatal, and there is currently no effective treatment for it. Mechanisms by which MV invades the CNS and causes the disease remain to be elucidated. We have previously shown that fusion-enhancing substitutions in the fusion protein of MVs isolated from SSPE patients contribute to MV spread in neurons. In this study, we demonstrate that MV bearing the hyperfusogenic mutant fusion protein spreads between human neurons in a cell-to-cell manner. Spread of the virus was inhibited by a fusion inhibitor peptide and antibodies against the MV hemagglutinin, indicating that both the hemagglutinin and hyperfusogenic fusion protein play important roles in MV spread between human neurons. The findings help us better understand the disease process of SSPE.

Multiplex CRISPR/Cas9 system impairs HCMV replication by excising an essential viral gene.

Anti-HCMV treatments used in immunosuppressed patients reduce viral replication, but resistant viral strains can emerge. Moreover, these drugs do not target latently infected cells. We designed two anti-viral CRISPR/Cas9 strategies to target the UL122/123 gene, a key regulator of lytic replication and reactivation from latency. The singleplex strategy contains one gRNA to target the start codon. The multiplex strategy contains three gRNAs to excise the complete UL122/123 gene. Primary fibroblasts and U-251 MG cells were transduced with lentiviral vectors encoding Cas9 and one or three gRNAs. Both strategies induced mutations in the target gene and a concomitant reduction of immediate early (IE) protein expression in primary fibroblasts. Further detailed analysis in U-251 MG cells showed that the singleplex strategy induced 50% of indels in the viral genome, leading to a reduction in IE protein expression. The multiplex strategy excised the IE gene in 90% of all viral genomes and thus led to the inhibition of IE protein expression. Consequently, viral genome replication and late protein expression were reduced by 90%. Finally, the production of new viral particles was nearly abrogated. In conclusion, the multiplex anti-UL122/123 CRISPR/Cas9 system can target the viral genome efficiently enough to significantly prevent viral replication.

Challenging a DNA Packaging Motor with a Modified Substrate

The DNA packaging motor of the bacteriophage phi29 is a powerful molecular machine that couples the free energy of ATP hydrolysis with DNA translocation in order to complete the production of new viral particles. The active part of this motor is a pentameric ring ATPase which mechanochemical cycle has been described with exquisite detail using hi-resolution optical tweezers. It was described that in each turn of the cycle, the motor packages DNA taking discrete steps of 10 base pairs (bp) each, in what is called a burst of translocation. At the same time it was shown that this 10 bp burst is composed of four 2.5 bp sub-steps, presumably reflecting the power stroke of the individual ATPases. Several models can explain what is the origin of the burst size: the helical pitch of B-form DNA is 10.5 bp/turn of the double helix, suggesting that the structure of the substrate is what determines the burst size; however, the non-integer nature of the sub-steps within the burst allows to hypothesize that is the ATPase’s conformational change what sets the burst size. Yet another possibility is that the DNA packaging motor switches the local conformation of the DNA substrate from B-form to A-form during packaging (DNA scrunching). To test the above hypotheses we challenged the phi29 DNA packaging motor with a double-stranded RNA substrate (that adopts the A-form of nucleic acids) and measured the packaging activity using optical tweezers.

A role for domain I of the hepatitis C virus NS5A protein in virus assembly.

The NS5A protein of hepatitis C virus (HCV) plays roles in both virus genome replication and assembly. NS5A comprises three domains, of these domain I is believed to be involved exclusively in genome replication. In contrast, domains II and III are required for the production of infectious virus particles and are largely dispensable for genome replication. Domain I is highly conserved between HCV and related hepaciviruses, and is highly structured, exhibiting different dimeric conformations. To investigate the functions of domain I in more detail, we conducted a mutagenic study of 12 absolutely conserved and surface-exposed residues within the context of a JFH-1-derived sub-genomic replicon and infectious virus. Whilst most of these abrogated genome replication, three mutants (P35A, V67A and P145A) retained the ability to replicate but showed defects in virus assembly. P35A exhibited a modest reduction in infectivity, however V67A and P145A produced no infectious virus. Using a combination of density gradient fractionation, biochemical analysis and high resolution confocal microscopy we demonstrate that V67A and P145A disrupted the localisation of NS5A to lipid droplets. In addition, the localisation and size of lipid droplets in cells infected with these two mutants were perturbed compared to wildtype HCV. Biophysical analysis revealed that V67A and P145A abrogated the ability of purified domain I to dimerize and resulted in an increased affinity of binding to HCV 3’UTR RNA. Taken together, we propose that domain I of NS5A plays multiple roles in assembly, binding nascent genomic RNA and transporting it to lipid droplets where it is transferred to Core. Domain I also contributes to a change in lipid droplet morphology, increasing their size. This study reveals novel functions of NS5A domain I in assembly of infectious HCV and provides new perspectives on the virus lifecycle.

Detection of Mouse Mammary Tumor Virus (MMTV) Particles in an Immortalized T Cell Line Based on Electrical Parameters

Label-free detection and characterization of cells producing virus particles is a highly desirable property that can pave the way for direct detection of virally infected cells in body fluids, tissues, and eventually in infected individuals. Identification of such properties can also provide a better understanding of the growth and/or evolution of virally infected cells in real-time experimental setups. This paper takes a closer look at the electrical properties of an immortalized T cell line capable of producing virus particles along with its non-virus producing control cell line using capacitance–voltage ( ${C}$ – ${V}$ ) measurements. In addition, two other important control cell lines were also included in which the ability of the cells to produce virus particles was abrogated by using genetic mutations. The conducted ${C}$ – ${V}$ measurements revealed that it is possible to electrically differentiate between these different cell lines. The cells producing wild-type (WT) virus particles could be distinguished from the control cells in which no DNA was introduced and, hence, were incapable of producing any virus particles. Interestingly, the cells in which two mutant DNAs were introduced that abrogated their ability to produce virus particles showed a similar electrical profile to each other, yet distinct from that of the cells producing the WT virus. These results clearly demonstrate that the electrical technique was able to distinguish between cells expressing virus particles versus cells that do not express virus particles. Data analysis revealed a twofold difference in the interaction capacitance between WT and mutant cells. Characterizing cells using electrical parameters are not laborious and lengthy, as is the case with conventional biochemical methods that typically take several days. Together, these data suggest that it is possible to electrically characterize and differentiate virally infected cells from uninfected cells. It is our hope that eventually these observations could be translated into label-free, electrical detection of virally infected cells in biological samples and individuals for diagnostic purposes.