Establishment and Characterization of Free-Floating 3D Macrophage Programming Model in the Presence of Cancer Cell Spheroids.

Reprogramming of tumor-associated macrophages (TAMs) is a promising strategy for cancer immunotherapy. Several studies have shown that cancer cells induce/support the formation of immunosuppressive TAMs phenotypes. However, the specific factors that orchestrate this immunosuppressive process are unknown or poorly studied. In vivo studies are expensive, complex, and ethically constrained. Therefore, 3D cell interaction models could become a unique framework for the identification of important TAMs programming factors. In this study, we have established and characterized a new in vitro 3D model for macrophage programming in the presence of cancer cell spheroids. First, it was demonstrated that the profile of cytokines, chemokines, and surface markers of 3D-cultured macrophages did not differ conceptually from monolayer-cultured M1 and M2-programmed macrophages. Second, the possibility of reprogramming macrophages in 3D conditions was investigated. In total, the dynamic changes in 6 surface markers, 11 cytokines, and 22 chemokines were analyzed upon macrophage programming (M1 and M2) and reprogramming (M1→M2 and M2→M1). According to the findings, the reprogramming resulted in a mixed macrophage phenotype that expressed both immunosuppressive and anti-cancer immunostimulatory features. Third, cancer cell spheroids were shown to stimulate the production of immunosuppressive M2 markers as well as pro-tumor cytokines and chemokines. In summary, the newly developed 3D model of cancer cell spheroid/macrophage co-culture under free-floating conditions can be used for studies on macrophage plasticity and for the development of targeted cancer immunotherapy.

N-Terminal Modification of Gly-His-Tagged Proteins with Azidogluconolactone.

Site-specific protein modifications are vital for biopharmaceutical drug development. Gluconoylation is a non-enzymatic, post-translational modification of N-terminal HisTags. We report high-yield, site-selective in vitro α-aminoacylation of peptides, glycoproteins, antibodies, and virus-like particles (VLPs) with azidogluconolactone at pH 7.5 in 1 h. Conjugates slowly hydrolyse, but diol-masking with borate esters inhibits reversibility. In an example, we multimerise azidogluconoylated SARS-CoV-2 receptor-binding domain (RBD) onto VLPs via click-chemistry, to give a COVID-19 vaccine. Compared to yeast antigen, HEK-derived RBD was immunologically superior, likely due to observed differences in glycosylation. We show the benefits of ordered over randomly oriented multimeric antigen display, by demonstrating single-shot seroconversion and best virus-neutralizing antibodies. Azidogluconoylation is simple, fast and robust chemistry, and should accelerate research and development.

SI-CLP inhibits the growth of mouse mammary adenocarcinoma by preventing recruitment of tumor-associated macrophages.

Chitinase-like proteins (CLP) are chitin-binding proteins that lack chitin hydrolyzing activity, but possess cytokine-like and growth factor-like properties, and play crucial role in intercellular crosstalk. Both human and mice express two members of CLP family: YKL-40 and stabilin-1 interacting chitinase-like protein (SI-CLP). Despite numerous reports indicating the role of YKL-40 in the support of angiogenesis, tumor cell proliferation, invasion and metastasis, the role of its structurally related protein SI-CLP in cancer was not reported. Using gain-of-function approach, we demonstrate in the current study that the expression of recombinant SI-CLP in mouse TS/A mammary adenocarcinoma cells results in significant and persistent inhibition of in vivo tumor growth. Using quantitative immunohistochemistry, we show that on the cellular level this phenomenon is associated with reduced infiltration of tumor-associated macrophages (TAMs), CD4+ and FoxP3+ cells in SI-CLP expressing tumors. Gene expression analysis in TAM isolated from SI-CLP-expressing and control tumors demonstrated that SI-CLP does not affect macrophage phenotype. However, SI-CLP significantly inhibited migration of murine bone-marrow derived macrophages and human primary monocytes toward monocyte-recruiting chemokine CCL2 produced in the tumor microenvironment (TME). Mechanistically, SI-CLP did not affect CCL2/CCR2 interaction, but suppressed cytoskeletal rearrangements in response to CCL2. Altogether, our data indicate that SI-CLP functions as a tumor growth inhibitor in mouse breast cancer by altering cellular composition of TME and blocking cytokine-induced TAM recruitment. Taking into consideration weak to absent expression of SI-CLP in human breast cancer, it can be considered as a therapeutic protein to block TAM-mediated support of breast tumor growth.

High efficiency of alphaviral gene transfer in combination with 5-fluorouracil in a mouse mammary tumor model.

BACKGROUND: The combination of virotherapy and chemotherapy may enable efficient tumor regression that would be unachievable using either therapy alone. In this study, we investigated the efficiency of transgene delivery and the cytotoxic effects of alphaviral vector in combination with 5-fluorouracil (5-FU) in a mouse mammary tumor model (4 T1). METHODS: Replication-deficient Semliki Forest virus (SFV) vectors carrying genes encoding fluorescent proteins were used to infect 4 T1 cell cultures treated with different doses of 5-FU. The efficiency of infection was monitored via fluorescence microscopy and quantified by fluorometry. The cytotoxicity of the combined treatment with 5-FU and alphaviral vector was measured using an MTT-based cell viability assay. In vivo experiments were performed in a subcutaneous 4 T1 mouse mammary tumor model with different 5-FU doses and an SFV vector encoding firefly luciferase. RESULTS: Infection of 4 T1 cells with SFV prior to 5-FU treatment did not produce a synergistic anti-proliferative effect. An alternative treatment strategy, in which 5-FU was used prior to virus infection, strongly inhibited SFV expression. Nevertheless, in vivo experiments showed a significant enhancement in SFV-driven transgene (luciferase) expression upon intratumoral and intraperitoneal vector administration in 4 T1 tumor-bearing mice pretreated with 5-FU: here, we observed a positive correlation between 5-FU dose and the level of luciferase expression. CONCLUSIONS: Although 5-FU inhibited SFV-mediated transgene expression in 4 T1 cells in vitro, application of the drug in a mouse model revealed a significant enhancement of intratumoral transgene synthesis compared with 5-FU untreated mice. These results may have implications for efficient transgene delivery and the development of potent cancer treatment strategies using alphaviral vectors and 5-FU.

Toward a SARS-CoV-2 VLP Vaccine: HBc/G as a Carrier for SARS-CoV-2 Spike RBM and Nucleocapsid Protein-Derived Peptides.

Virus-like particles (VLPs) offer an attractive possibility for the development of vaccines. Recombinant core antigen (HBc) of Hepatitis B virus (HBV) was expressed in different systems, and the E. coli expression system was shown to be effective for the production of HBc VLPs. Here, we used HBc of the HBV genotype G (HBc/G) as a technologically promising VLP carrier for the presentation of spike RBM and nucleocapsid protein-derived peptides of the SARS-CoV-2 Delta variant for subsequent immunological evaluations of obtained fusion proteins. The major immunodominant region (MIR) of the HBc/G protein was modified through the insertion of a receptor binding motif (RBM) from the S protein or B-cell epitope-containing peptide from the N protein. The C-terminus of the two truncated HBc/G proteins was used for the insertion of a group of five cytotoxic T lymphocyte (CTL) epitopes from the N protein. After expression in E. coli, the MIR-derived proteins were found to be insoluble and were recovered through step-wise solubilization with urea, followed by refolding. Despite the lack of correct VLPs, the chimeric proteins induced high levels of antibodies in BALB/c mice. These antibodies specifically recognized either eukaryotically expressed hRBD or bacterially expressed N protein (2-220) of SARS-CoV-2. CTL-epitope-containing proteins were purified as VLPs. The production of cytokines was analyzed through flow cytometry after stimulation of T-cells with target CTL peptides. Only a protein with a deleted polyarginine (PA) domain was able to induce the specific activation of T-cells. At the same time, the T-cell response against the carrier HBc/G protein was detected for both proteins. The neutralization of SARS-CoV-2 pseudotyped murine retrovirus with anti-HBc/G-RBM sera was found to be low.

Recombinant Virus Quantification Using Single-Cell Droplet Digital PCR: A Method for Infectious Titer Quantification.

The quantification of viruses is necessary for both research and clinical applications. The methods available for RNA virus quantification possess several drawbacks, including sensitivity to inhibitors and the necessity of a standard curve generation. The main purpose of this study was to develop and validate a method for the quantification of recombinant, replication-deficient Semliki Forest virus (SFV) vectors using droplet digital PCR (ddPCR). This technique demonstrated stability and reproducibility using various sets of primers that targeted inserted transgenes, as well as the nsP1 and nsP4 genes of the SFV genome. Furthermore, the genome titers in the mixture of two types of replication-deficient recombinant virus particles were successfully measured after optimizing the annealing/extension temperature and virus:virus ratios. To measure the infectious units, we developed a single-cell ddPCR, adding the whole infected cells to the droplet PCR mixture. Cell distribution in the droplets was investigated, and ß-actin primers were used to normalize the quantification. As a result, the number of infected cells and the virus infectious units were quantified. Potentially, the proposed single-cell ddPCR approach could be used to quantify infected cells for clinical applications.

Styrylpyridinium Derivatives for Fluorescent Cell Imaging.

A set of styrylpyridinium (SP) compounds was synthesised in order to study their spectroscopic and cell labelling properties. The compounds comprised different electron donating parts (julolidine, p-dimethylaminophenyl, p-methoxyphenyl, 3,4,5-trimethoxyphenyl), conjugated linkers (vinyl, divinyl), and an electron-withdrawing N-alkylpyridinium part. Geminal or bis-compounds incorporating two styrylpyridinium (bis-SP) moieties at the 1,3-trimethylene unit were synthesised. Compounds comprising a divinyl linker and powerful electron-donating julolidine donor parts possessed intensive fluorescence in the near-infrared region (maximum at ~760 nm). The compounds had rather high cytotoxicity towards the cancerous cell lines HT-1080 and MH-22A; at the same time, basal cytotoxicity towards the NIH3T3 fibroblast cell line ranged from toxic to harmful. SP compound 6e had IC50 values of 1.0 ± 0.03 µg/mL to the cell line HT-1080 and 0.4 µg/mL to MH-22A; however, the basal toxicity LD50 was 477 mg/kg (harmful). The compounds showed large Stokes' shifts, including 195 nm for 6a,b, 240 nm for 6e, and 325 and 352 nm for 6d and 6c, respectively. The highest photoluminescence quantum yield (PLQY) values were observed for 6a,b, which were 15.1 and 12.2%, respectively. The PLQY values for the SP derivatives 6d,e (those with a julolidinyl moiety) were 0.5 and 0.7%, respectively. Cell staining with compound 6e revealed a strong fluorescent signal localised in the cell cytoplasm, whereas the cell nuclei were not stained. SP compound 6e possessed self-assembling properties and formed liposomes with an average diameter of 118 nm. The obtained novel data on near-infrared fluorescent probes could be useful for the development of biocompatible dyes for biomedical applications.

Recombinant Viral Vectors for Therapeutic Programming of Tumour Microenvironment: Advantages and Limitations.

Viral vectors have been widely investigated as tools for cancer immunotherapy. Although many preclinical studies demonstrate significant virus-mediated tumour inhibition in synergy with immune checkpoint molecules and other drugs, the clinical success of viral vector applications in cancer therapy currently is limited. A number of challenges have to be solved to translate promising vectors to clinics. One of the key elements of successful virus-based cancer immunotherapy is the understanding of the tumour immune state and the development of vectors to modify the immunosuppressive tumour microenvironment (TME). Tumour-associated immune cells, as the main component of TME, support tumour progression through multiple pathways inducing resistance to treatment and promoting cancer cell escape mechanisms. In this review, we consider DNA and RNA virus vectors delivering immunomodulatory genes (cytokines, chemokines, co-stimulatory molecules, antibodies, etc.) and discuss how these viruses break an immunosuppressive cell development and switch TME to an immune-responsive "hot" state. We highlight the advantages and limitations of virus vectors for targeted therapeutic programming of tumour immune cell populations and tumour stroma, and propose future steps to establish viral vectors as a standard, efficient, safe, and non-toxic cancer immunotherapy approach that can complement other promising treatment strategies, e.g., checkpoint inhibitors, CAR-T, and advanced chemotherapeutics.

UV inactivation of Semliki Forest virus and E. coli bacteria by alternative light sources.

The quick spreading of the SARS-CoV-2 virus, initiating the global pandemic with a significant impact on economics and health, highlighted an urgent need for effective and sustainable restriction mechanisms of pathogenic microorganisms. UV-C radiation, causing inactivation of many viruses and bacteria, is one of the tools for disinfection of different surfaces, liquids, and air; however, mainly mercury 254 nm line is commonly used for it. In this paper, we report our results of the experiments with newly elaborated special type polychromatic non-mercury UV light sources, having spectral lines in the spectral region from 190 nm to 280 nm. Inactivation tests were performed with both Escherichia coli (E.coli) bacteria and Semliki Forest virus (SFV) as a representative of human enveloped RNA viruses. In addition, the effect of prepared lamps on virus samples in liquid and dry form (dried virus-containing solution) was tested. Reduction of 4 log10 of E.coli was obtained after 10 min of irradiation with both thallium-antimony and arsenic high-frequency electrodeless lamps. High reduction results for the arsenic light source demonstrated sensitivity of E. coli to wavelengths below 230 nm, including spectral lines around 200 nm. For the Semliki Forest virus, the thallium-antimony light source showed virus inactivation efficiency with a high virus reduction rate in the range of 3.10 to > 4.99 log10 within 5 min of exposure. Thus, the new thallium-antimony light source showed the most promising disinfection effect in bacteria and viruses, and arsenic light sources for bacteria inactivation, opening doors for many applications in disinfection systems, including for pathogenic human RNA viruses.

Design and Synthesis of Hepatitis B Virus (HBV) Capsid Assembly Modulators and Evaluation of Their Activity in Mammalian Cell Model.

Capsid assembly modulators (CAMs) have emerged as a promising class of antiviral agents. We studied the effects of twenty-one newly designed and synthesized CAMs including heteroaryldihydropyrimidine compounds (HAPs), their analogs and standard compounds on hepatitis B virus (HBV) capsid assembly. Cytoplasmic expression of the HBV core (HBc) gene driven by the exogenously delivered recombinant alphavirus RNA replicon was used for high level production of the full-length HBc protein in mammalian cells. HBV capsid assembly was assessed by native agarose gel immunoblot analysis, electron microscopy and inhibition of virion secretion in HepG2.2.15 HBV producing cell line. Induced fit docking simulation was applied for modelling the structural relationships of the synthesized compounds and HBc. The most efficient were the HAP class compounds-dihydropyrimidine 5-carboxylic acid n-alkoxyalkyl esters, which induced the formation of incorrectly assembled capsid products and their accumulation within the cells. HBc product accumulation in the cells was not detected with the reference HAP compound Bay 41-4109, suggesting different modes of action. A significant antiviral effect and substantially reduced toxicity were revealed for two of the synthesized compounds. Two new HAP compounds revealed a significant antiviral effect and a favorable toxicity profile that allows these compounds to be considered promising leads and drug candidates for the treatment of HBV infection. The established alphavirus based HBc expression approach allows for the specific selection of capsid assembly modulators directly in the natural cell environment.

Detection of SARS-CoV-2 RNA in wastewater and importance of population size assessment in smaller cities: An exploratory case study from two municipalities in Latvia.

Wastewater-based epidemiology (WBE) has regained global importance during the COVID-19 pandemic. The mobility of people and other factors, such as precipitation and irregular inflow of industrial wastewater, are complicating the estimation of the disease prevalence through WBE, which is crucial for proper crisis management. These estimations are particularly challenging in urban areas with moderate or low numbers of inhabitants in situations where movement restrictions are not adopted (as in the case of Latvia) because residents of smaller municipalities tend to be more mobile and less strict in following the rules and measures of disease containment. Thus, population movement can influence the outcome of WBE measurements significantly and may not reflect the actual epidemiological situation in the respective area. Here, we demonstrate that by combining the data of detected SARS-CoV-2 RNA copy number, 5-hydroxyindoleacetic acid (5-HIAA) analyses in wastewater and mobile call detail records it was possible to provide an accurate assessment of the COVID-19 epidemiological situation in towns that are small (COVID-19 28-day cumulative incidence r = 0.609 and 35-day cumulative incidence r = 0.89, p < 0.05) and medium-sized towns (COVID-19 21-day cumulative incidence r = 0.997, 28-day cumulative incidence r = 0.98 and 35-day cumulative incidence r = 0.997, p < 0.05). This is the first study demonstrating WBE for monitoring COVID-19 outbreaks in Latvia. We demonstrate that the application of population size estimation measurements such as total 5-HIAA and call detail record data improve the accuracy of the WBE approach.

Characterization of Novel Biopolymer Blend Mycocel from Plant Cellulose and Fungal Fibers.

In this study unique blended biopolymer mycocel from naturally derived biomass was developed. Softwood Kraft (KF) or hemp (HF) cellulose fibers were mixed with fungal fibers (FF) in different ratios and the obtained materials were characterized regarding microstructure, air permeability, mechanical properties, and virus filtration efficiency. The fibers from screened Basidiomycota fungi Ganoderma applanatum (Ga), Fomes fomentarius (Ff), Agaricus bisporus (Ab), and Trametes versicolor (Tv) were applicable for blending with cellulose fibers. Fungi with trimitic hyphal system (Ga, Ff) in combinations with KF formed a microporous membrane with increased air permeability (>8820 mL/min) and limited mechanical strength (tensile index 9-14 Nm/g). HF combination with trimitic fungal hyphae formed a dense fibrillary net with low air permeability (77-115 mL/min) and higher strength 31-36 Nm/g. The hyphal bundles of monomitic fibers of Tv mycelium and Ab stipes made a tight structure with KF with increased strength (26-43 Nm/g) and limited air permeability (14-1630 mL/min). The blends KF FF (Ga) and KF FF (Tv) revealed relatively high virus filtration capacity: the log10 virus titer reduction values (LRV) corresponded to 4.54 LRV and 2.12 LRV, respectively. Mycocel biopolymers are biodegradable and have potential to be used in water microfiltration, food packaging, and virus filtration membranes.

Alphavirus-Driven Interferon Gamma (IFNg) Expression Inhibits Tumor Growth in Orthotopic 4T1 Breast Cancer Model.

Interferon gamma (IFNg) is a pleiotropic cytokine that can potentially reprogram the tumor microenvironment; however, the antitumor immunomodulatory properties of IFNg still need to be validated due to variable therapeutic outcomes in preclinical and clinical studies. We developed a replication-deficient Semliki Forest virus vector expressing IFNg (SFV/IFNg) and evaluated its immunomodulatory antitumor potential in vitro in a model of 3D spheroids and in vivo in an immunocompetent 4T1 mouse breast cancer model. We demonstrated that SFV-derived, IFN-g-stimulated bone marrow macrophages can be used to acquire the tumoricidal M1 phenotype in 3D nonattached conditions. Coculturing SFV/IFNg-infected 4T1 spheroids with BMDMs inhibited spheroid growth. In the orthotopic 4T1 mouse model, intratumoral administration of SFV/IFNg virus particles alone or in combination with the Pam3CSK4 TLR2/1 ligand led to significant inhibition of tumor growth compared to the administration of the control SFV/Luc virus particles. Analysis of the composition of intratumoral lymphoid cells isolated from tumors after SFV/IFNg treatment revealed increased CD4+ and CD8+ and decreased T-reg (CD4+/CD25+/FoxP3+) cell populations. Furthermore, a significant decrease in the populations of cells bearing myeloid cell markers CD11b, CD38, and CD206 was observed. In conclusion, the SFV/IFNg vector induces a therapeutic antitumor T-cell response and inhibits myeloid cell infiltration in treated tumors.

Application of Alphaviral Vectors for Immunomodulation in Cancer Therapy.

BACKGROUND: The lack of specific and efficient cancer therapies has influenced the development of novel approaches, such as immunotherapy, which from its original application of immunogenic protein delivery has developed into the use of more sophisticated recombinant gene delivery methods to achieve better safety and efficacy profiles. This approach involves viral and non-viral delivery systems. METHODS: Expression vectors have been engineered for alphaviruses, including Semliki Forest virus, Sindbis virus and Venezuelan equine encephalitis virus. For immunotherapeutic applications, recombinant particles, RNA replicons and layered DNA vectors that express tumor-associated antigens (TAAs) and cytokines have been studied in animal models and in a few clinical trials. RESULTS: Immunization studies with TAAs and cytokines have elicited strong antibody responses and vaccination has provided protection against challenges with tumor cells in mouse models. Furthermore, the combination of TAAs and cytokines, antibodies and growth factors and the co-administration of chemotherapeutics and bacteriabased adjuvants have enhanced immunogenicity. Intratumoral and systemic delivery of recombinant alphavirus particles has demonstrated significant tumor regression and prolonged survival rates in rodent tumor models. CONCLUSION: Alphavirus-based immunotherapy represents a rapid and efficient method for prophylactic and therapeutic applications in animal models.

Magnetic nanoparticles for efficient cell transduction with Semliki Forest virus.

Semliki Forest virus (SFV) is a potential cancer gene therapy vector capable of providing high and transient expression of heterologous proteins in mammalian cells. However, SFV has shown suboptimal transduction levels in several cancer cell types as well as wide biodistribution of SFV has been observed after in vivo applications. Magnetic nanoparticles (MNPs) have been shown to increase cell transduction with several viral vectors in vitro under an external magnetic field and enhance magnetically guided viral vector delivery. Here, we examined a panel of MNPs for enhanced cancer cell transduction with SFV vector. Magneto-transduction using positively charged MNPs increased Semliki Forest virus transduction in TS/A mouse mammary carcinoma cells in vitro in the presence of fetal bovine serum. Positively charged MNPs efficiently captured SFV particles independently of capturing medium, and MNPs-SFV complexes were successfully separated from suspension by magnetic precipitation. These results reveal the potential application of MNPs for enhanced gene delivery by SFV vector as well as proposes magnetic precipitation for efficient concentration of SFV particles from different media.

Generation and Functional In Vitro Analysis of Semliki Forest Virus Vectors Encoding TNF-α and IFN-γ.

Cytokine gene delivery by viral vectors is a promising novel strategy for cancer immunotherapy. Semliki Forest virus (SFV) has many advantages as a delivery vector, including the ability to (i) induce p53-independent killing of tumor cells via apoptosis, (ii) elicit a type-I interferon (IFN) response, and (iii) express high levels of the transgene. SFV vectors encoding cytokines such as interleukin (IL)-12 have shown promising therapeutic responses in experimental tumor models. Here, we developed two new recombinant SFV vectors encoding either murine tumor necrosis factor-α (TNF-α) or murine interferon-γ (IFN-γ), two cytokines with documented immunostimulatory and antitumor activity. The SFV vector showed high infection rate and cytotoxicity in mouse and human lung carcinoma cells in vitro. By contrast, mouse and human macrophages were resistant to infection with SFV. The recombinant SFV vectors directly inhibited mouse lung carcinoma cell growth in vitro, while exploiting the cancer cells for production of SFV vector-encoded cytokines. The functionality of SFV vector-derived TNF-α was confirmed through successful induction of cell death in TNF-α-sensitive fibroblasts in a concentration-dependent manner. SFV vector-derived IFN-γ activated macrophages toward a tumoricidal phenotype leading to suppressed Lewis lung carcinoma cell growth in vitro in a concentration-dependent manner. The ability of SFV to provide functional cytokines and infect tumor cells but not macrophages suggests that SFV may be very useful for cancer immunotherapy employing tumor-infiltrating macrophages.

Comparative protein profiling of B16 mouse melanoma cells susceptible and non-susceptible to alphavirus infection: Effect of the tumor microenvironment.

Alphavirus vectors are promising tools for cancer treatment. However, relevant entry mechanisms and interactions with host cells are still not clearly understood. The first step toward a more effective therapy is the identification of novel intracellular alterations that could be associated with cancer aggressiveness and could affect the therapeutic potential of these vectors. In this study, we observed that alphaviruses efficiently infected B16 mouse melanoma tumors/tumor cells in vivo, whereas their transduction efficiency in B16 cells under in vitro conditions was blocked. Therefore, we further aimed to understand the mechanisms pertaining to the differential transduction efficacy of alphaviruses in B16 tumor cells under varying growth conditions. We hypothesized that the tumor microenvironment might alter gene expression in B16 cells, leading to an up-regulation of the expression of virus-binding receptors or factors associated with virus entry and replication. To test our hypothesis, we performed a proteomics analysis of B16 cells cultured in vitro and of B16 cells isolated from tumors, and we identified 277 differentially regulated proteins. A further in-depth analysis to identify the biological and molecular functions of the detected proteins revealed a set of candidate genes that could affect virus infectivity. Importantly, we observed a decrease in the expression of interferon α (IFN-α) in tumor-isolated cells that resulted in the suppression of several IFN-regulated genes, thereby abrogating host cell antiviral defense. Additionally, differences in the expression of genes that regulate cytoskeletal organization caused significant alterations in cell membrane elasticity. Taken together, our findings demonstrated favorable intracellular conditions for alphavirus transduction/replication that occurred during tumor transformation. These results pave the way for optimizing the development of strategies for the application of alphaviral vectors as a potent cancer therapy.

Proteasomal degradation of core protein variants from chronic hepatitis B patients.

The accumulation of complex hepatitis B virus (HBV) variants with internal in-frame deletions in the C gene in immunosuppressed renal transplant recipients is associated with a severe course of the infection leading to end-stage liver disease (ESLD). A set of six HBV C genes with internal in-frame deletions corresponding to the pattern of HBV population in immunosuppressed patients has been expressed in two different eukaryotic cell lines. Synthesis and proteasomal degradation of HBV core (HBc) protein variants were compared with those of the wild-type HBc. In all cases, the steady-state level of internally deleted HBc proteins, predominantly with longer deletions, were considerably lower and turnover was significantly higher in comparison with those of the wild-type HBc, since all deletion variants were degraded rapidly via the proteasome pathway. Involvement and consequences of the proteasomal degradation machinery in the HBc protein turnover during HBV infection with complex HBV variants in the immunosuppressed patients are discussed.

Translation of hepatitis B virus (HBV) surface proteins from the HBV pregenome and precore RNAs in Semliki Forest virus-driven expression.

Hepatitis B virus (HBV) pregenome RNA (pgRNA) serves as a translation template for the HBV core (HBc) protein and viral polymerase (Pol). HBV precore RNA (pcRNA) directs the synthesis of the precore (preC) protein, a precursor of the hepatitis B e antigen (HBeAg). pgRNA and pcRNA were expressed in the Semliki Forest virus (SFV) expression system. Besides the HBc and preC proteins, there was revealed the synthesis of all three forms of HBV surface (HBs) proteins: long (LHBs), middle (MHBs) and short (SHBs), the start codons of which are located more than 1000 nt downstream of the HBc and preC start codons. Moreover, other HBV templates, such as 3'-truncated pgRNA lacking 3' direct repeat and Pol mRNA, both carrying internally the HBs sequences, provided the synthesis of three HBs protein forms in the SFV-driven expression system. Maximal production of the HBs was provided by Pol mRNA, while HBc- and preC-producing templates showed relatively low internal translation of the HBs. These data allow the proposal of a ribosome leaky scanning model of internal translation initiation for HBs proteins. The putative functional role of such exceptional synthesis of the HBs proteins from the pgRNA and pcRNA templates in the natural HBV infection process needs further evaluation.