VP1 Expression Research Articles

Generation of infectious recombinant Adeno-associated virus in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae has been successfully employed to establish model systems for a number of viruses. Such model systems are powerful tools to study the virus biology and in particular for the identification and characterization of host factors playing a role in the viral infection cycle. Adeno-associated viruses (AAV) are heavily studied due to their use as gene delivery vectors. AAV relies on other helper viruses for successful replication and on host factors for several aspects of the viral life cycle. However the role of host and helper viral factors is only partially known. Production of recombinant AAV (rAAV) vectors for gene delivery applications depends on knowledge of AAV biology and the limited understanding of host and helper viral factors may be precluding efficient production, particularly in heterologous systems. Model systems in simpler eukaryotes like the yeast S. cerevisiae would be useful tools to identify and study the role of host factors in AAV biology. Here we show that expression of AAV2 viral proteins VP1, VP2, VP3, AAP, Rep78, Rep52 and an ITR-flanked DNA in yeast leads to capsid formation, DNA replication and encapsidation, resulting in formation of infectious particles. Many of the AAV characteristics observed in yeast resemble those in other systems, making it a suitable model system. Future findings in the yeast system could be translatable to other AAV host systems and aid in more efficient production of rAAV vectors.

OneBac 2.0: Sf9 Cell Lines for Production of AAV1, AAV2, and AAV8 Vectors with Minimal Encapsidation of Foreign DNA.

Recombinant adeno-associated viral (rAAV) vectors for human gene therapy require efficient and economical production methods to keep pace with the rapidly increasing clinical demand. In addition, the manufacturing process must ensure high vector quality and biological safety. The OneBac system offers easily scalable rAAV vector production in insect Sf9-derived AAV rep/cap-expressing producer cell lines infected with a single baculovirus that carries the rAAV backbone. For most AAV serotypes high burst sizes per cell were achieved, combined with high infectivity rates. OneBac 2.0 represents a 2-fold advancement: First, enhanced VP1 proportions in AAV5 capsids lead to vastly increased per-particle infectivity rates. Second, collateral packaging of foreign DNA is suppressed by removal of the Rep-binding element (RBE). In this study we show that this advancement of AAV5 packaging can be translated to OneBac 2.0-derived packaging systems for alternative AAV serotypes. By removal of the RBE, collateral packaging of nonvector DNA was drastically reduced in all newly tested serotypes (AAV1, AAV2, and AAV8). However, the splicing-based strategy to enhance VP1 expression in order to increase AAV5 infectivity hardly improved infectivity rates of AAV-1, -2, or -8 compared with the original OneBac cell lines. Our results emphasize that OneBac 2.0 represents an advancement for scalable, high-titer production of various AAV serotypes, leading to AAV particles with minimal packaging of foreign DNA.

Structural Protein VP2 of African Horse Sickness Virus Is Not Essential for Virus Replication In Vitro.

Members of the Reoviridae family cause major health problems worldwide, ranging from lethal diarrhea caused by rotavirus in humans to economic losses in livestock production caused by different orbiviruses. The Orbivirus genus contains many virus species, of which bluetongue virus, epizootic hemorrhagic disease virus, and African horse sickness virus (AHSV) cause notifiable diseases according to the World Organization of Animal Health. Recently, it has been shown that nonstructural proteins NS3/NS3a and NS4 are not essential for virus replication in vitro, whereas it is generally assumed that structural proteins VP1 to -7 of these nonenveloped, architecturally complex virus particles are essential. Here we demonstrate for the first time that structural protein VP2 of AHSV is not essential for virus replication in vitro Our findings are very important for virologists working in the field of nonenveloped viruses, in particular reoviruses.

Incorporation of a bi-functional protein FimH enhances the immunoprotection of chitosan-pVP1 vaccine against coxsackievirus B3-induced myocarditis

Viral myocarditis is a common clinical cardiovascular disease mainly induced by coxsackievirus B3 (CVB3) with no effective therapeutic measures. Induction of efficient mucosal immune responses is very critical against CVB3-induced myocarditis. FimH is an Escherichia coli (E. coli)-derived protein, which possesses an M cell-targeting property and functions as a TLR4 agonist. In this study, we introduced the recombinant FimH protein, into our previously developed CVB3 mucosal vaccine chitosan (CS)-pVP1, aiming to provoke more efficient mucosal immune responses and immunoprotection against CVB3-induced myocarditis. Compared with the CS-pVP1 vaccine, immunization with FimH-CS-pVP1 remarkably increased the levels and neutralizing titers of CVB3-specific protective secretory IgA (sIgA), enhanced the frequency of CVB3-specific IgA-producing B cells and amplified mucosal T-cell immune responses in mesenteric lymph nodes (MLNs), although failing to significantly amplify CVB3-specific systemic immune responses. Consistently, FimH-CS-pVP1 group showed the enhanced immunoprotection against CVB3-induced myocarditis, evidenced by the indices of limited myocardial injury, reduced viral loads and enhanced survival rate. Further study showed that this enhanced immunoprotection was not only ascribed to its M cell-targeting property, which led to the enhanced mucosal antigen VP1 expression, but also associated with the mucosal adjuvant effect of FimH, which facilitated the formation of germinal centers (GCs), production of IgA-inducing factors and maturation of antigen-presenting cells (APCs). Taken together, here we developed a bi-functional mucosal vaccine FimH-CS-pVP1, which simultaneously possessed the M cell-targeting property and mucosal adjuvant ability, and we showed that FimH-CS-pVP1 could efficiently induce the higher levels of CVB3-specific protective mucosal immune responses and provide better prophylactic effects against CVB3-induced myocarditis than CS-pVP1.

In vitro antiviral efficacy of moroxydine hydrochloride and ribavirin against grass carp reovirus and giant salamander iridovirus.

Moroxydine hydrochloride (Mor) and ribavirin (Rib) have been reported to exhibit multi-antiviral activities against DNA and RNA viruses, but their antiviral activities and pharmacologies have seldom been studied in aquaculture. This paper has selected 3 aquatic viruses including a double-stranded RNA virus (grass carp reovirus, GCRV), a single-stranded RNA virus (spring viraemia of carp virus, SVCV) and a DNA virus (giant salamander iridovirus, GSIV) for antiviral testing. The results showed that Mor and Rib can effectively control the infection of GCRV and GSIV in respective host cells. Further study was undertaken to explore the antivirus efficiencies and pharmacological mechanisms of Mor and Rib on GCRV and GSIV in vitro. Briefly, compounds showed over 50% protective effects at 15.9 µg ml-1 except for the group of GSIV-infected epithelioma papulosum cyprinid (EPC) cells treated with Mor. Moreover, Mor and Rib blocked the virus-induced cytopathic effects and apoptosis in host cells to keep the normal cellular structure. The expression of VP1 (GCRV) and major capsid protein (MCP; GSIV) gene was also significantly inhibited in the virus-infected cells when treated with Mor and Rib. Cytotoxicity assay verified the 2 compounds had no toxic effects on grass carp ovary (GCO) cells and EPC cells at ≤96 µg ml-1. In conclusion, these results indicated that exposing GCRV-infected GCO cells and GSIV-infected EPC cells to Mor and Rib could elicit significant antiviral responses, and the 2 compounds have been shown to be promising agents for viral control in the aquaculture industry.

Effects of different promoters on the protective efficacy of recombinant Marek’s disease virus type 1 expressing the VP2 gene of infectious bursal disease virus

The vaccine efficacy of recombinant viruses can be influenced by many factors. Accordingly, the activity of promoters has been one of the major factors affecting the antigen expression and protection rate. In the present study, two recombinant Marek’s disease virus type 1 (MDV1) vaccines containing the VP2 gene of infectious bursal disease virus (IBDV) under control of different promoters were generated from overlapping fosmid DNAs. The rMDV-Pec-VP2 virus containing the VP2 gene under control of the Pec promoter (CMV enhancer and chicken β-actin chimera promoter) demonstrated higher VP2 expression and stronger antibody response against IBDV in chickens than the rMDV-CMV-VP2 virus using the CMV promoter. After IBDV lethal challenge in specific-pathogen-free chickens, rMDV-Pec-VP2 provided complete protection against developing mortality, clinical signs, and the formation of bursal lesions, which was better than that provided by rMDV-CMV-VP2. Our findings indicate that the protective efficacy of the recombinant MDV1 vaccine against IBDV highly correlates with VP2 expression. This recombinant MDV1 vaccine expressing VP2 could have significant potential as a bivalent vaccine against both virulent IBDV and MDV infections in chickens.

Fructus Amomi Cardamomi Extract Inhibit Coxsackievirus-B3 Induced Myocarditis in Murine Myocarditis Model.

Coxsackievirus B3 (CVB3) is the main cause of acute myocarditis and dilated cardiomyopathy. Plant extracts are considered as useful materials to develop new antiviral drugs. We had previously selected candidate plant extracts, which showed anti-inflammatory effects. We examined the antiviral effects by using a HeLa cell survival assay. Among these extracts, we chose the Amomi Cardamomi (Amomi) extract, which showed strong antiviral effect and preserved cell survival in CVB3 infection. We investigated the mechanisms underlying the ability of Amomi extract to inhibit CVB3 infection and replication. HeLa cells were infected by CVB3 with or without Amomi extract. Erk and Akt activities, and their correlation with virus replication were observed. Live virus titers in cell supernatants and viral positive- and negative-strand RNA amplification were measured. Amomi extract significantly increased HeLa cell survival in different concentrations (100-10 µg/ml). CVB3 capsid protein VP1 expression (76%) and viral protease 2A-induced eIF4G1 cleavage (70%) were significantly decreased in Amomi extract (100 µg/ml) treated cells. The levels of positive- (20%) and negative-strand (80%) RNA were dramatically decreased compared with the control, as revealed by reverse transcription-PCR. In addition, Amomi extract improved mice survival (51% vs 26%) and dramatically reduced heart inflammation in a CVB3-induced myocarditis mouse model. These results suggested that Amomi extract significantly inhibited Enterovirus replication and myocarditis damage. Amomi may be developed as a therapeutic drug for Enterovirus.

Integrated molecular and bioprocess engineering for bacterially produced immunogenic modular virus-like particle vaccine displaying 18 kDa rotavirus antigen.

A high global burden of rotavirus disease and the unresolved challenges with the marketed rotavirus vaccines, particularly in the developing world, have ignited efforts to develop virus-like particle (VLP) vaccines for rotavirus. While rotavirus-like particles comprising multiple viral proteins can be difficult to process, modular VLPs presenting rotavirus antigenic modules are promising alternatives in reducing process complexity and cost. In this study, integrated molecular and bioprocess engineering approaches were used to simplify the production of modular murine polyomavirus capsomeres and VLPs presenting a rotavirus 18 kDa VP8* antigen. A single construct was generated for dual expression of non-tagged murine polyomavirus capsid protein VP1 and modular VP1 inserted with VP8*, for co-expression in Escherichia coli. Co-expressed proteins assembled into pentameric capsomeres in E. coli. A selective salting-out precipitation and a polishing size exclusion chromatography step allowed the recovery of stable modular capsomeres from cell lysates at high purity, and modular capsomeres were successfully translated into modular VLPs when assembled in vitro. Immunogenicity study in mice showed that modular capsomeres and VLPs induced high levels of VP8*-specific antibodies. Our results demonstrate that a multipronged synthetic biology approach combining molecular and bioprocess engineering enabled simple and low-cost production of highly immunogenic modular capsomeres and VLPs presenting conformational VP8* antigenic modules. This strategy potentially provides a cost-effective production route for modular capsomere and VLP vaccines against rotavirus, highly suitable to manufacturing economics for the developing world. Biotechnol. Bioeng. 2017;114: 397-406. © 2016 Wiley Periodicals, Inc.

Solubility as a limiting factor for expression of hepatitis A virus proteins in insect cell-baculovirus system

The use of recombinant proteins may represent an alternative model to inactivated vaccines against hepatitis A virus (HAV). The present study aimed to express the VP1 protein of HAV in baculovirus expression vector system (BEVS). The VP1 was expressed intracellularly with molecular mass of 35 kDa. The VP1 was detected both in the soluble fraction and in the insoluble fraction of the lysate. The extracellular expression of VP1 was also attempted, but the protein remained inside the cell. To verify if hydrophobic characteristics would also be present in the HAV structural polyprotein, the expression of P1-2A protein was evaluated. The P1-2A polyprotein remained insoluble in the cellular extract, even in the early infection stages. These results suggest that HAV structural proteins are prone to form insoluble aggregates. The low solubility represents a drawback for production of large amounts of HAV proteins in BEVS.

Discovery of 3-benzyl-1,3-benzoxazine-2,4-dione analogues as allosteric mitogen-activated kinase kinase (MEK) inhibitors and anti-enterovirus 71 (EV71) agents

Enterovirus 71 (EV71) is a kind of RNA virus and one of the two causes of Hand, foot and mouth disease (HFMD). Inhibitors that target key components of Ras/Raf/MEK/ERK pathway in host cells could impair replication of EV71. A series of 3-benzyl-1,3-benzoxazine-2,4-diones were designed from a specific MEK inhibitor G8935, by replacing the double bond between C3 and C4 within the coumarin scaffold with amide bond. One compound (9f) showed submicromolar inhibitory activity among the 12 derivatives. Further optimization on 9f led to two active compounds (9k and 9m) with nanomolar bioactivities (55nM and 60nM). The results of enzymatic assays also demonstrated that this series of compounds were allosteric inhibitors of unphosphorylated MEK1. The binding mode of compound 9k was predicted by molecular dynamic simulation and the key interactions were same as published MEK1/2 allosteric inhibitors. In the cell-based assays, compounds 9k and 9m could effectively suppress the ERK1/2 pathway, expression of EV71 VP1, and EV71 induced cytopathic effect (CPE) in rhabdomyosarcoma (RD) cells.

Expression of cDNA of the gene for the capsid protein VP2 of German cockroach densovirus in the transgenic strain of Drosophila melanogaster

Transgenic strains of Drosophila melanogaster capable of expressing a cDNA fragment corresponding to open reading frame (ORF) of the gene for the German cockroach densonucleosis virus capsid protein VP2 (ORF VP2) in specific tissues and at a certain stage of development depending on the type of chosen driver strains (GAL-UAS system) were obtained. The ORF VP2 transcription was examined at the imago stage after crossing the obtained transgenic Drosophila with the driver line expressing the inducer protein (GAL4) under control of actin promoter (the ORF VP2 expression is induced in all tissues of the first-generation Drosophila). It was demonstrated that the greater part of transcribed foreign RNA was represented by three spliced variants in which RNA fragments either between nucleotides 137 and 353 or between nucleotides 609 and 1925 were excised; the third spliced variant was represented by RNA lacking both introns. Using the next-generation sequencing (NGS) technique, the proportion of unspliced form relative to spliced variants of the analyzed RNA was assessed. It was shown that the ratio of unspliced form to the identified spliced variants of the analyzed RNA was approximately 1:6. It is suggested that splicing of viral RNA foreign to Drosophila can be a sort of defense mechanism preventing the large-scale production of the capsid protein, potentially hazardous to the host organism.

Interaction between Simian Virus 40 Major Capsid Protein VP1 and Cell Surface Ganglioside GM1 Triggers Vacuole Formation.

ABSTRACTSimian virus 40 (SV40), a polyomavirus that has served as an important model to understand many aspects of biology, induces dramatic cytoplasmic vacuolization late during productive infection of monkey host cells. Although this activity led to the discovery of the virus in 1960, the mechanism of vacuolization is still not known. Pentamers of the major SV40 capsid protein VP1 bind to the ganglioside GM1, which serves as the cellular receptor for the virus. In this report, we show that binding of VP1 to cell surface GM1 plays a key role in SV40 infection-induced vacuolization. We previously showed that SV40 VP1 mutants defective for GM1 binding fail to induce vacuolization, even though they replicate efficiently. Here, we show that interfering with GM1-VP1 binding by knockdown of GM1 after infection is established abrogates vacuolization by wild-type SV40. Vacuole formation during permissive infection requires efficient virus release, and conditioned medium harvested late during SV40 infection rapidly induces vacuoles in a VP1- and GM1-dependent fashion. Furthermore, vacuolization can also be induced by a nonreplicating SV40 pseudovirus in a GM1-dependent manner, and a mutation in BK pseudovirus VP1 that generates GM1 binding confers vacuole-inducing activity. Vacuolization can also be triggered by purified pentamers of wild-type SV40 VP1, but not by GM1 binding-defective pentamers or by intracellular expression of VP1. These results demonstrate that SV40 infection-induced vacuolization is caused by the binding of released progeny viruses to GM1, thereby identifying the molecular trigger for the activity that led to the discovery of SV40.

Facilitation of rice stripe virus accumulation in the insect vector by Himetobi P virus VP1.

The small brown planthopper (SBPH) is the main vector for rice stripe virus (RSV), which causes serious rice stripe disease in East Asia. To characterize the virus-vector interactions, the SBPH cDNA library was screened with RSV ribonucleoprotein (RNP) as bait using a GAL4-based yeast two-hybrid system. The interaction between RSV-RNP and the Himetobi P virus (HiPV, an insect picorna-like virus) VP1 protein was identified. The relationships between HiPV and RSV in SBPH were further investigated, and the results showed that the titer of RSV was commonly higher in single insect that exhibited more VP1 expression. After the VP1 gene was repressed by RNA silencing, the accumulation of RSV decreased significantly in the insect, whereas the virus acquisition ability of SBPH was unaffected, which suggests that HiPV VP1 potentially facilitates the accumulation of RSV in SBPH.

Development of Lactobacillus paracasei harboring nucleic acid-hydrolyzing 3D8 scFv as a preventive probiotic against murine norovirus infection.

The protein 3D8 single-chain variable fragment (3D8 scFv) has potential anti-viral activity due to its ability to penetrate into cells and hydrolyze nucleic acids. Probiotic Lactobacillus paracasei engineered to secrete 3D8 scFv for oral administration was used to test the anti-viral effects of 3D8 scFv against gastrointestinal virus infections. We found that injection of 3D8 scFv into the intestinal lumen resulted in the penetration of 3D8 scFv into the intestinal villi and lamina propria. 3D8 scFv secreted from engineered L. paracasei retained its cell-penetrating and nucleic acid-hydrolyzing activities, which were previously shown with 3D8 scFv expressed in Escherichia coli. Pretreatment of RAW264.7 cells with 3D8 scFv purified from L. paracasei prevented apoptosis induction by murine norovirus infection and decreased messenger RNA (mRNA) expression of the viral capsid protein VP1. In a mouse model, oral administration of the engineered L. paracasei prior to murine norovirus infection reduced the expression level of mRNA encoding viral polymerase. Taken together, these results suggest that L. paracasei secreting 3D8 scFv provides a basis for the development of ingestible anti-viral probiotics active against gastrointestinal viral infection.

Inhibition of large T antigen ATPase activity as a potential strategy to develop anti-polyomavirus JC drugs

IntroductionThis study evaluates polyomavirus JC (JCV) large T antigen (LTA) as a potential target for drug development. LTA is a hexameric protein with a helicase activity that is powered by ATP binding and hydrolysis. The helicase and ATPase function is critical for viral replication. MethodsRecombinant JCV LTA was produced in an Escherichia coli based expression plasmid. ATPase activity was measured using the malachite green assay. A high throughput screen was completed using a brain-biased library of 75,000 drug-like compounds selected for physicochemical properties consistent with blood–brain barrier permeability. ResultsFive compounds showed non-competitive inhibition of ATPase activity with an EC50⩽15μM. Modest antiviral activity was demonstrated in an immunofluorescence assay for JCV VP-1 expression in COS7 cells (EC50 15, 18, 20, 27, and 52μM respectively). The compounds also inhibited viral replication in a real time PCR assay at comparable concentrations. LD50 in the MTS96 and Cell TiterGlo assays was >100μM for all compounds in COS7 as well as HEK293 cells. However, two compounds inhibited cell proliferation in culture with IC50 values of 43 and 34μM respectively. Despite substantial amino acid similarity between polyomavirus JC, BK and SV40 proteins, these compounds differ from those previously reported to inhibit SV40 LTA ATPase in chemical structure as well as a non-competitive mechanism of inhibition. ConclusionLTA ATPase is a valid target for discovery. Additional screening and chemical optimization is needed to develop clinically useful compounds with less toxicity, which should be measured by metabolic as well as cell proliferation assays.

In vivo time-related evaluation of a therapeutic neutralization monoclonal antibody against lethal enterovirus 71 infection in a mouse model.

Enterovirus 71 (EV71) is a neurotropic virus capable of inducing severe neurological symptoms and death. No direct targeting antivirals are useful in the treatment of severe EV71 infection. Because of low toxicity and good specificity, monoclonal antibodies (MAb) are a potential candidate for the treatment of viral infections. Therefore, we developed an EV71-specific conformational MAb with high in vitro cross-neutralization activity to heterologous EV71 subgenotypes. The in vivo treatment experiment at different days post-infection indicated that a single treatment of MAb CT11F9 within day 3 post-infection fully protected mice from morbidity and mortality (0% PBS vs. 100% at 10 µg/g per body weight ***P<0.0001). Immunohistochemical and histological analysis confirmed that CT11F9 significantly prohibited EV71 VP1 expression in various tissues and prevented EV71-induced myonecrosis. Moreover, thrice-treatment at day 4, 5, 6 post-infection was associated with an increased survival rate (18.2% single vs. 50% thrice at 20 µg/g per body weight), and the mice recovered from limb paralysis. Competitive ELISA also confirmed that CT11F9-recognized epitopes were immunodominant in humans. In conclusion, MAb CT11F9 is an ideal candidate to be humanized and used in severe EV71 infection.

Cloning and Characterisation of Two H+ Translocating Organic Pyrophos-phatase Genes in Salix and Their Expression Differences in Two Willow Varieties with Different Salt Tolerances.

Willows are one of the most important tree species for landscaping, biofuel and raw timber. Screening salt-tolerant willow varieties is an effective approach to balance wood supply and demand. However, more salt-tolerant willow varieties are required and little is known regarding the mechanism of salt tolerance at the gene expression level. In this paper, two willow varieties were studies in terms of their differences in salt-tolerances and mechanism of salt tolerance at the level of VP1 gene expression. The results showed that Salix L0911 (L0911) had higher biomass than Salix matsudana (SM), and salt injuries were less severe in L0911 than in SM. The activities of peroxidase and superoxide dismutase, as well as the contents of soluble protein and proline, were higher in L0911 than in SM, whereas the contents of Na+ and K+, as well as the Na+/K+ ratio, were lower in L0911 than in SM. Two VP1 genes (VP1.1 and VP1.2) cloned in L0911 and SM had similar sequences and structures. VP1.1 and VP1.2 belonged to different subgroups. Total expression levels of the VP1.1 gene in both roots and leaves of L0911 were higher than that in SM under normal conditions. Under salt stress, expression of VP1 in SM roots initially increased and then decreased, whereas the expression of VP1 in leaves of L0911 and SM, as well as in roots of L0911, decreased with increasing salt concentrations. This study increased our understanding of the salt-tolerance mechanism of willow and may facilitate the selection of salt-tolerant willow resources.

Antiviral effects of artesunate on JC polyomavirus replication in COS-7 cells.

The human JC polyomavirus (JCPyV) causes the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). A growing number of patients with induced or acquired immunosuppression are at risk for infection, and no effective antiviral therapy is presently available. The widely used antimalarial drug artesunate has shown broad antiviral activity in vitro but limited clinical success. The aim of this study was to investigate the effect of artesunate on JCPyV replication in vitro. The permissivity for JCPyV MAD-4 was first compared in four cell lines, and the monkey kidney cell line COS-7 was selected. Artesunate caused a concentration-dependent decrease in the extracellular JCPyV DNA load 96 h postinfection, with a 50% effective concentration (EC50) of 2.9 μM. This effect correlated with a decreased expression of capsid protein VP1 and a reduced release of infectious viral progeny. For concentrations of <20 μM, transient reductions in cellular DNA replication and proliferation were seen, while for higher concentrations, some cytotoxicity was detected. A selective index of 16.6 was found when cytotoxicity was calculated based on cellular DNA replication in the mock-infected cells, but interestingly, cellular DNA replication in the JCPyV-infected cells was more strongly affected. In conclusion, artesunate is efficacious in inhibiting JCPyV replication at micromolar concentrations, which are achievable in plasma. The inhibition at EC50 probably reflects an effect on cellular proteins and involves transient cytostatic effects. Our results, together with the favorable distribution of the active metabolite dihydroartemisinin to the central nervous system, suggest a potential use for artesunate in patients with PML.

JC virus nucleotides 376-396 are critical for VP1 capsid protein expression.

JC virus (JCV) infection of the brain can cause progressive multifocal leukoencephalopathy, JCV granule cell neuronopathy, and JCV encephalopathy (JCVE). JCVCPN, isolated from the brain of a patient with JCVE, is a naturally occurring strain of JCV with a 143-base pair deletion in the agnogene. Cell culture studies of JCVCPN have shown that the loss of these nucleotides in the agnogene results in impaired expression of VP1 and infectious virion production. To better understand the role of this DNA sequence in JCV replication, we generated a series of deletions in the agnogene on the backbone of a virus which has a mutated agnoprotein start codon preventing agnoprotein expression. We found that deletion of nucleotides 376-396 results in decreased levels of viral DNA replication and a lack of VP1 expression. These results indicate that these nucleotides play a crucial role in JCV replication.

Feline calicivirus can tolerate gross changes of its minor capsid protein expression levels induced by changing translation reinitiation frequency or use of a separate VP2-coding mRNA.

Caliciviruses use reinitiation of translation governed by a ‘termination upstream ribosomal binding site’ (TURBS) for expression of their minor capsid protein VP2. Mutation analysis allowed to identify sequences surrounding the translational start/stop site of the feline calicivirus (FCV) that fine tune reinitiation frequency. A selection of these changes was introduced into the infectious FCV cDNA clone to check the influence of altered VP2 levels on virus replication. In addition, full length constructs were established that displayed a conformation, in which VP2 expression occurred under control of a duplicated subgenomic promoter. Viable viruses recovered from such constructs revealed a rather broad range of VP2 expression levels but comparable growth kinetics showing that caliciviruses can tolerate gross changes of the VP2 expression level.