Alterations in N-glycosylation of HCV E2 Protein in Children Patients with IFN-RBV Therapy Failure.

The glycosylation of viral envelope proteins plays an important role in virus biology and the immune response of the host to infection. Hepatitis C virus (HCV) envelope proteins E1 and E2, key players in virus entry and spread, are highly N-glycosylated and possess 4 (5 in certain genotypes) to 11 conserved glycosylation sites, respectively. Many published results based on recombinant proteins indicate that the glycan shield can mask the epitopes targeted by neutralizing antibodies. Glycan shifting within the conserved linear E2 region (412-423) could be one of the escape strategies used by HCV. In the present report, we isolated E2 genes from samples (collected before the IFN-RBV therapy) originating from pediatric patients infected with HCV gt 1a. We analyzed the biochemical properties of cloned E2 glycoprotein variants and investigated their glycosylation status. The sequencing of E2 genes isolated from patients who did not respond to therapy revealed mutations at N-glycosylation sites, thus leading to a lower molecular weight and a low affinity to both linear and conformational neutralizing antibodies. The loss of the glycosylation site within the conserved epitope (amino acid 417) impaired the binding with AP33, an antibody that potently neutralizes all genotypes of HCV. Our findings, based on clinical samples, confirm the influence of N-glycosylation aberrations on the antigenic and conformational properties of HCV E1/E2, which may possibly correlate with the outcome of therapy in patients.

The herpesvirus UL49.5 protein hijacks a cellular C-degron pathway to drive TAP transporter degradation.

The transporter associated with antigen processing (TAP) is a key player in the major histocompatibility class I-restricted antigen presentation and an attractive target for immune evasion by viruses. Bovine herpesvirus 1 impairs TAP-dependent antigenic peptide transport through a two-pronged mechanism in which binding of the UL49.5 gene product to TAP both inhibits peptide transport and triggers its proteasomal degradation. How UL49.5 promotes TAP degradation has, so far, remained unknown. Here, we use high-content siRNA and genome-wide CRISPR-Cas9 screening to identify CLR2KLHDC3 as the E3 ligase responsible for UL49.5-triggered TAP disposal. We propose that the C terminus of UL49.5 mimics a C-end rule degron that recruits the E3 to TAP and engages the cullin-RING E3 ligase in endoplasmic reticulum-associated degradation.

The herpesvirus UL49.5 protein hijacks a cellular C-degron pathway to drive TAP transporter degradation.

The transporter associated with antigen processing (TAP) is a key player in the MHC class I-restricted antigen presentation and an attractive target for immune evasion by viruses. Bovine herpesvirus 1 (BoHV-1) impairs TAP-dependent antigenic peptide transport through a two-pronged mechanism in which binding of the UL49.5 gene product to TAP both inhibits peptide transport and promotes its proteasomal degradation. How UL49.5 promotes TAP degradation is unknown. Here, we use high-content siRNA and genome-wide CRISPR-Cas9 screening to identify CLR2KLHDC3 as the E3 ligase responsible for UL49.5-triggered TAP disposal in human cells. We propose that the C-terminus of UL49.5 mimics a C-end rule degron that recruits the E3 to TAP and engages the CRL2 E3 in ER-associated degradation.

The N-terminal Proline Hinge Motif Controls the Structure of Bovine Herpesvirus 1-encoded Inhibitor of the Transporter Associated with Antigen Processing Required for its Immunomodulatory Function.

Due to unique features, proline residues may control protein structure and function. Here, we investigated the role of 52PPQ54 residues, indicated by the recently established experimental 3D structure of bovine herpesvirus 1-encoded UL49.5 protein as forming a characteristic proline hinge motif in its N-terminal domain. UL49.5 acts as a potent inhibitor of the transporter associated with antigen processing (TAP), which alters the antiviral immune response. Mechanisms employed by UL49.5 to affect TAP remain undetermined on a molecular level. We found that mutations in the 52PPQ54 region had a vast impact on its immunomodulatory function, increasing cell surface MHC class I expression, TAP levels, and peptide transport efficiency. This inhibitory effect was specific for UL49.5 activity towards TAP but not towards the viral glycoprotein M. To get an insight into the impact of proline hinge modifications on structure and dynamics, we performed all-atom and coarse-grained molecular dynamics studies on the native protein and PPQ mutants. The results demonstrated that the proline hinge sequence with its highly rigid conformation served as an anchor into the membrane. This anchor was responsible for the structural and dynamical behavior of the whole protein, constraining the mobility of the C-terminus, increasing the mobility of the transmembrane region, and controlling the accessibility of the C-terminal residues to the cytoplasmic environment. Those features appear crucial for TAP binding and inhibition. Our findings significantly advance the structural understanding of the UL49.5 protein and its functional regions and support the importance of proline motifs for the protein structure.

Effect of Glycan Shift on Antibodies against Hepatitis C Virus E2 412-425 Epitope Elicited by Chimeric sHBsAg-Based Virus-Like Particles.

Two of the most important mechanisms of hepatitis C virus (HCV) immune evasion are the high variability of the amino acid sequence and epitope shielding via heavy glycosylation of the envelope (E) proteins. Previously, we showed that chimeric sHBsAg (hepatitis B virus [HBV] small surface antigen)-based virus-like particles (VLPs) carrying highly conserved epitope I from the HCV E2 glycoprotein (sHBsAg_412-425) elicit broadly neutralizing antibodies (bnAbs). However, many reports have identified escape mutations for such bnAbs that shift the N-glycosylation site from N417 to N415. This shift effectively masks the recognition of epitope I by antibodies raised against the wild-type glycoprotein. To investigate if glycan-shift-mediated immune evasion could be overcome by targeted vaccination strategies, we designed sHBsAg-based VLPs carrying epitope I with an N417S change (sHBsAg_N417S). Studies in BALB/c mice revealed that both sHBsAg_412-425 and sHBsAg_N417S VLPs were immunogenic, eliciting antibodies that recognized peptides encompassing epitope I regardless of the N417S change. However, we observed substantial differences in E1E2 glycoprotein binding and cell culture-derived HCV (HCVcc) neutralization between the sera elicited by sHBsAg_412-425 and those elicited by sHBsAg_N417S VLPs. Our results suggest a complex interplay among antibodies targeting epitope I, the E1E2 glycosylation status, and the epitope or global E1E2 conformation. Additionally, we observed striking similarities in the E1E2 glycoprotein binding patterns and HCVcc neutralization between sHBsAg_412-425 sera and AP33, suggesting that the immunization of mice with sHBsAg_412-425 VLPs can elicit AP33-like antibodies. This study emphasizes the role of antibodies against epitope I and represents an initial effort toward designing an antigen that elicits an immune response against epitope I with a glycan shift change. IMPORTANCE Epitope I, located within amino acids 412 to 423 of the HCV E2 glycoprotein, is an important target for an epitope-based HCV vaccine. One interesting feature of epitope I is the N417 glycosylation site, where a single change to S417 or T417 can shift the glycosylation site to position N415. This shift can effectively prevent the binding of broadly neutralizing antibodies targeting epitope I. Aiming to overcome glycan-shift-mediated immune evasion, we constructed sHBsAg_N417S VLPs carrying E2 epitope I, with N417S, and compared them with VLPs carrying wild-type epitope I. We show that antibodies elicited by the sHBsAg-based VLPs presenting two variants of the 412-425 epitope targeted two distinct glycan variants of the HCV E1E2 heterodimer. Our study suggests that due to the conformational flexibility of the E2 glycoprotein and epitope I, future vaccine antigens should elicit antibodies targeting more than one conformation and glycosylation variant of the 412-423 epitope.

Functional Analysis of a Frontal miRNA Cluster Located in the Large Latency Transcript of Pseudorabies Virus.

MicroRNAs (miRNAs) have been identified as a class of crucial regulators of virus-host crosstalk, modulating such processes as viral replication, antiviral immune response, viral latency, and pathogenesis. Pseudorabies virus (PRV), a model for the study of alphaherpesvirus biology, codes for 11 distinct miRNAs mapped to the ~4.6 kb intron of Large Latency Transcript (LLT). Recent studies have revealed the role of clusters consisting of nine and eleven miRNA genes in the replication and virulence of PRV. The function of separate miRNA species in regulating PRV biology has not been thoroughly investigated. To analyze the regulatory potential of three PRV miRNAs located in the frontal cluster of the LLT intron, we generated a research model based on the constitutive expression of viral miRNAs in swine testis cells (ST_LLT [1-3] cell line). Using a cell culture system providing a stable production of individual miRNAs at high levels, we demonstrated that the LLT [1-3] miRNA cluster significantly downregulated IE180, EP0, and gE at the early stages of PRV infection. It was further determined that LLT [1-3] miRNAs could regulate the infection process, leading to a slight distortion in transmission and proliferation ability. Collectively, our findings indicate the potential of LLT [1-3] miRNAs to retard the host responses by reducing viral antigenic load and suppressing the expansion of progeny viruses at the early stages of infection.

The Role of IFITM Proteins in Tick-Borne Encephalitis Virus Infection.

Tick-borne encephalitis virus (TBEV), of the genus Flavivirus, is a causative agent of severe encephalitis in regions of endemicity of northern Asia and central and northern Europe. Interferon-induced transmembrane proteins (IFITMs) are restriction factors that inhibit the replication cycles of numerous viruses, including flaviviruses such as West Nile virus, dengue virus, and Zika virus. Here, we demonstrate the role of IFITM1, IFITM2, and IFITM3 in the inhibition of TBEV infection and in protection against virus-induced cell death. We show that the most significant role is that of IFITM3, including the dissection of its functional motifs by mutagenesis. Furthermore, through the use of CRISPR-Cas9-generated IFITM1/3-knockout monoclonal cell lines, we confirm the role and additive action of endogenous IFITMs in TBEV suppression. However, the results of coculture assays suggest that TBEV might partially escape interferon- and IFITM-mediated suppression during high-density coculture infection when the virus enters naive cells directly from infected donor cells. Thus, cell-to-cell spread may constitute a strategy for virus escape from innate host defenses. IMPORTANCE TBEV infection may result in encephalitis, chronic illness, or death. TBEV is endemic in northern Asia and Europe; however, due to climate change, new centers of endemicity have arisen. Although effective TBEV vaccines have been approved, vaccination coverage is low, and due to the lack of specific therapeutics, infected individuals depend on their immune responses to control the infection. IFITM proteins are components of the innate antiviral defenses that suppress cell entry of many viral pathogens. However, no studies on the role of IFITM proteins in TBEV infection have been published thus far. Understanding antiviral innate immune responses is crucial for the future development of antiviral strategies. Here, we show the important role of IFITM proteins in the inhibition of TBEV infection and virus-mediated cell death. However, our data suggest that TBEV cell-to-cell spread may be less prone to both interferon- and IFITM-mediated suppression, potentially facilitating escape from IFITM-mediated immunity.

Zoonotic spill-over of SARS-CoV-2: mink-adapted virus in humans.

OBJECTIVES: This work aimed to analyse possible zoonotic spill-over of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We report the spill-over of mink-adapted SARS-CoV-2 from farmed mink to humans after adaptation that lasted at least 3 months. METHODS: Next-generation sequencing and a bioinformatic approach were applied to analyse the data. RESULTS: In an isolate obtained from an asymptomatic patient testing positive for SARS-CoV-2, we found four distinguishing mutations in the S gene that gave rise to the mink-adapted variant (G75V, M177T, Y453F, and C1247F) and others. CONCLUSIONS: Zoonotic spill-over of SARS-CoV-2 can occur from mink to human.

Immune response against SARS-CoV-2 variants: the role of neutralization assays.

Since the emergence of the novel coronavirus SARS-CoV-2 in late 2019, the COVID-19 pandemic has hindered social life and global economic activity. As of July 2021, SARS-CoV-2 has caused over four million deaths. The rapid spread and high mortality of the disease demanded the international scientific community to develop effective vaccines in a matter of months. However, unease about vaccine efficacy has arisen with the spread of the SARS-CoV-2 variants of concern (VOCs). Time- and cost-efficient in vitro neutralization assays are widely used to measure neutralizing antibody responses against VOCs. However, the extent to which in vitro neutralization reflects protection from infection remains unclear. Here, we describe common neutralization assays based on infectious and pseudotyped viruses and evaluate their role in testing neutralizing responses against new SARS-CoV-2 variants. Additionally, we briefly review the recent findings on the immune response elicited by available vaccines against major SARS-CoV-2 variants, including Alpha, Beta, Gamma, and Delta.

Expansion of a SARS-CoV-2 Delta variant with an 872 nt deletion encompassing ORF7a, ORF7b and ORF8, Poland, July to August 2021.

Routine genomic surveillance on samples from COVID-19 patients collected in Poland during summer 2021 revealed the emergence of a SARS-CoV-2 Delta variant with a large 872 nt deletion. This change, confirmed by Sanger and deep sequencing, causes complete loss of ORF7a, ORF7b, and ORF8 genes. The index case carrying the deletion is unknown. The standard pipeline for sequencing may mask this deletion with a long stretch of N's. Effects of this deletion on phenotype or immune evasion needs further study.

Palmitoylated mNeonGreen Protein as a Tool for Visualization and Uptake Studies of Extracellular Vesicles.

Extracellular vesicles (EVs) are membranous nanoparticles released by cells as vital mediators of intercellular communication. As such, EVs have become an attractive target for pathogens and cancer cells, which can take control over their cargo composition, as well as their trafficking, shaping the pathogenesis. Despite almost four decades of research on EVs, the number of specific and efficient EV labeling methods is limited, and there is still no universal method for the visualization of their transport in living cells. Lipophilic dyes that non-specifically intercalate into the EVs membranes may diffuse to other membranes, leading to the misinterpretation of the results. Here, we propose a palmitoylated fluorescent mNeonGreen (palmNG) protein as an alternative to chemical dyes for EVs visualization. The Branchiostoma lanceolatum-derived mNeonGreen is a brighter, more stable, and less sensitive to laser-induced bleaching alternative to green fluorescent protein (GFP), which makes it a more potent tag in a variety of fluorescence-based techniques. A palmNG-expressing stable human melanoma cell line was generated using retrovirus gene transfer and cell sorting. This protein partially localizes to cellular membranes, and can be detected inside size-exclusion (SEC)-purified EVs. With the use of flow cytometry and fluorescent confocal microscopy, we performed qualitative and quantitative analyses of palmNG-EVs uptake in recipient human hepatoma cells, in comparison to PKH67-labeled vesicles. Our findings confirm that membrane-embedded mNeonGreen can be successfully applied as a tool in EVs transfer and uptake studies.

Specific Antibodies Induced by Immunization with Hepatitis B Virus-Like Particles Carrying Hepatitis C Virus Envelope Glycoprotein 2 Epitopes Show Differential Neutralization Efficiency.

Hepatitis C virus (HCV) infection with associated chronic liver diseases is a major health problem worldwide. Here, we designed hepatitis B virus (HBV) small surface antigen (sHBsAg) virus-like particles (VLPs) presenting different epitopes derived from the HCV E2 glycoprotein (residues 412-425, 434-446, 502-520, and 523-535 of isolate H77C). Epitopes were selected based on their amino acid sequence conservation and were previously reported as targets of HCV neutralizing antibodies. Chimeric VLPs obtained in the Leishmania tarentolae expression system, in combination with the adjuvant Addavax, were used to immunize mice. Although all VLPs induced strong humoral responses, only antibodies directed against HCV 412-425 and 523-535 epitopes were able to react with the native E1E2 glycoprotein complexes of different HCV genotypes in ELISA. Neutralization assays against genotype 1-6 cell culture infectious HCV (HCVcc), revealed that only VLPs carrying the 412-425 epitope induced efficient HCV cross-neutralizing antibodies, but with isolate specific variations in efficacy that could not necessarily be explained by differences in epitope sequences. In contrast, antibodies targeting 434-446, 502-520, and 523-535 epitopes were not neutralizing HCVcc, highlighting the importance of conformational antibodies for efficient virus neutralization. Thus, 412-425 remains the most promising linear E2 epitope for further bivalent, rationally designed vaccine research.

Alphaherpesvirus gB Homologs Are Targeted to Extracellular Vesicles, but They Differentially Affect MHC Class II Molecules.

Herpesvirus envelope glycoprotein B (gB) is one of the best-documented extracellular vesicle (EVs)-incorporated viral proteins. Regarding the sequence and structure conservation between gB homologs, we asked whether bovine herpesvirus-1 (BoHV-1) and pseudorabies virus (PRV)-encoded gB share the property of herpes simplex-1 (HSV-1) gB to be trafficked to EVs and affect major histocompatibility complex (MHC) class II. Our data highlight some conserved and differential features of the three gBs. We demonstrate that mature, fully processed BoHV-1 and PRV gBs localize to EVs isolated from constructed stable cell lines and EVs-enriched fractions from virus-infected cells. gB also shares the ability to co-localize with CD63 and MHC II in late endosomes. However, we report here a differential effect of the HSV-1, BoHV-1, and PRV glycoprotein on the surface MHC II levels, and MHC II loading to EVs in stable cell lines, which may result from their adverse ability to bind HLA-DR, with PRV gB being the most divergent. BoHV-1 and HSV-1 gB could retard HLA-DR exports to the plasma membrane. Our results confirm that the differential effect of gB on MHC II may require various mechanisms, either dependent on its complex formation or on inducing general alterations to the vesicular transport. EVs from virus-infected cells also contained other viral glycoproteins, like gD or gE, and they were enriched in MHC II. As shown for BoHV-1 gB- or BoHV-1-infected cell-derived vesicles, those EVs could bind anti-virus antibodies in ELISA, which supports the immunoregulatory potential of alphaherpesvirus gB.

Fluorescent TAP as a Platform for Virus-Induced Degradation of the Antigenic Peptide Transporter.

Transporter associated with antigen processing (TAP), a key player in the major histocompatibility complex class I-restricted antigen presentation, makes an attractive target for viruses that aim to escape the immune system. Mechanisms of TAP inhibition vary among virus species. Bovine herpesvirus 1 (BoHV-1) is unique in its ability to target TAP for proteasomal degradation following conformational arrest by the UL49.5 gene product. The exact mechanism of TAP removal still requires elucidation. For this purpose, a TAP-GFP (green fluorescent protein) fusion protein is instrumental, yet GFP-tagging may affect UL49.5-induced degradation. Therefore, we constructed a series of TAP-GFP variants using various linkers to obtain an optimal cellular fluorescent TAP platform. Mel JuSo (MJS) cells with CRISPR/Cas9 TAP1 or TAP2 knockouts were reconstituted with TAP-GFP constructs. Our results point towards a critical role of GFP localization on fluorescent properties of the fusion proteins and, in concert with the type of a linker, on the susceptibility to virally-induced inhibition and degradation. The fluorescent TAP platform was also used to re-evaluate TAP stability in the presence of other known viral TAP inhibitors, among which only UL49.5 was able to reduce TAP levels. Finally, we provide evidence that BoHV-1 UL49.5-induced TAP removal is p97-dependent, which indicates its degradation via endoplasmic reticulum-associated degradation (ERAD).

Transmembrane regions of bovine herpesvirus 1-encoded UL49.5 and glycoprotein M regulate complex maturation and ER-Golgi trafficking.

Bovine herpesvirus 1 (BoHV-1)-encoded UL49.5 (a homologue of herpesvirus glycoprotein N) can combine different functions, regulated by complex formation with viral glycoprotein M (gM). We aimed to identify the mechanisms governing the immunomodulatory activity of BoHV-1 UL49.5. In this study, we addressed the impact of gM/UL49.5-specific regions on heterodimer formation, folding and trafficking from the endoplasmic reticulum (ER) to the trans-Golgi network (TGN) - events previously found to be responsible for abrogation of the UL49.5-mediated inhibition of the transporter associated with antigen processing (TAP). We first established, using viral mutants, that no other viral protein could efficiently compensate for the chaperone function of UL49.5 within the complex. The cytoplasmic tail of gM, containing putative trafficking signals, was dispensable either for ER retention of gM or for the release of the complex. We constructed cell lines with stable co-expression of BoHV-1 gM with chimeric UL49.5 variants, composed of the BoHV-1 N-terminal domain fused to the transmembrane region (TM) from UL49.5 of varicella-zoster virus or TM and the cytoplasmic tail of influenza virus haemagglutinin. Those membrane-anchored N-terminal domains of UL49.5 were sufficient to form a complex, yet gM/UL49.5 folding and ER-TGN trafficking could be affected by the UL49.5 TM sequence. Finally, we found that leucine substitutions in putative glycine zipper motifs within TM helices of gM resulted in strong reduction of complex formation and decreased ability of gM to interfere with UL49.5-mediated major histocompatibility class I downregulation. These findings highlight the importance of gM/UL49.5 transmembrane domains for the biology of this conserved herpesvirus protein complex.

Recombinant Flag-tagged E1E2 glycoproteins from three hepatitis C virus genotypes are biologically functional and elicit cross-reactive neutralizing antibodies in mice.

Hepatitis C virus (HCV) is a globally disseminated human pathogen for which no vaccine is currently available. HCV is highly diverse genetically and can be classified into 7 genotypes and multiple sub-types. Due to this antigenic variation, the induction of cross-reactive and at the same time neutralizing antibodies is a challenge in vaccine production. Here we report the analysis of immunogenicity of recombinant HCV envelope glycoproteins from genotypes 1a, 1b and 2a, with a Flag tag inserted in the hypervariable region 1 of E2. This modification did not affect protein expression or conformation or its capacity to bind the crucial virus entry factor, CD81. Importantly, in immunogenicity studies on mice, the purified E2-Flag mutants elicited high-titer, cross-reactive antibodies that were able to neutralize HCV infectious particles from two genotypes tested (1a and 2a). These findings indicate that E1E2-Flag envelope glycoproteins could be important immunogen candidates for vaccine aiming to induce broad HCV-neutralizing responses.

Tunneling Nanotubes as a Novel Route of Cell-to-Cell Spread of Herpesviruses.

Various types of intercellular connections that are essential for communication between cells are often utilized by pathogens. Recently, a new type of cellular connection, consisting of long, thin, actin-rich membrane extensions named tunneling nanotubes (TNTs), has been shown to play an important role in cell-to-cell spread of HIV and influenza virus. In the present report, we show that TNTs are frequently formed by cells infected by an alphaherpesvirus, bovine herpesvirus 1 (BoHV-1). Viral proteins, such as envelope glycoprotein E (gE), capsid protein VP26, and tegument protein Us3, as well as cellular organelles (mitochondria) were detected by immunofluorescence and live-cell imaging of nanotubes formed by bovine primary fibroblasts and oropharynx cells (KOP cells). Time-lapse confocal studies of live cells infected with fluorescently labeled viruses showed that viral particles were transmitted via TNTs. This transfer also occurred in the presence of neutralizing antibodies, which prevented free entry of BoHV-1. We conclude that TNT formation contributes to successful cell-to-cell spread of BoHV-1 and demonstrate for the first time the participation of membrane nanotubes in intercellular transfer of a herpesvirus in live cells.IMPORTANCE Efficient transmission of viral particles between cells is an important factor in successful infection by herpesviruses. Herpesviruses can spread by the free-entry mode or direct cell-to-cell transfer via cell junctions and long extensions of neuronal cells. In this report, we show for the first time that an alphaherpesvirus can also spread between various types of cells using tunneling nanotubes, intercellular connections that are utilized by HIV and other viruses. Live-cell monitoring revealed that viral transmission occurs between the cells of the same type as well as between epithelial cells and fibroblasts. This newly discovered route of herpesviruses spread may contribute to efficient transmission despite the presence of host immune responses, especially after reactivation from latency that developed after primary infection. Long-range communication provided by TNTs may facilitate the spread of herpesviruses between many tissues and organs of an infected organism.

HtrA3 is a cellular partner of cytoskeleton proteins and TCP1α chaperonin.

The human HtrA3 protease is involved in placentation, mitochondrial homeostasis, stimulation of apoptosis and proposed to be a tumor suppressor. Molecular mechanisms of the HtrA3 functions are poorly understood and knowledge concerning its cellular targets is very limited. There are two HtrA3 isoforms, the long (HtrA3L) and short (HtrA3S). Upon stress, their N-terminal domains are removed, resulting in the more active ΔN-HtrA3. By pull down and mass spectrometry techniques, we identified a panel of putative ΔN-HtrA3L/S substrates. We confirmed that ΔN-HtrA3L/S formed complexes with actin, ß-tubulin, vimentin and TCP1α in vitro and in a cell and partially co-localized with the actin and vimentin filaments, microtubules and TCP1α in a cell. In vitro, both isoforms cleaved the cytoskeleton proteins, promoted tubulin polymerization and displayed chaperone-like activity, with ΔN-HtrA3S being more efficient in proteolysis and ΔN-HtrA3L - in polymerization. TCP1α, essential for the actin and tubulin folding, was directly bound by the ΔN-HtrA3L/S but not cleaved. These results indicate that actin, ß-tubulin, vimentin, and TCP1α are HtrA3 cellular partners and suggest that HtrA3 may influence cytoskeleton dynamics. They also suggest different roles of the HtrA3 isoforms and a possibility that HtrA3 protease may also function as a co-chaperone. SIGNIFICANCE: The HtrA3 protease stimulates apoptosis and is proposed to be a tumor suppressor and a therapeutic target, however little is known about its function at the molecular level and very few HtrA3 physiological substrates have been identified so far. Furthermore, HtrA3 is the only member of the HtrA family of proteins which, apart from the long isoform possessing the PD and PDZ domains (HtrA3L), has a short isoform (HtrA3S) lacking the PDZ domain. In this work we identified a large panel (about 150) of the tentative HtrA3L/S cellular partners which provides a good basis for further research concerning the HtrA3 function. We have shown that the cytoskeleton proteins actin, ß-tubulin and vimentin, and the TCP1α chaperonin are cellular partners of both HtrA3 isoforms. Our findings indicate that HtrA3 may promote destabilization of the actin and vimentin cytoskeleton and suggest that it may influence the dynamics of the microtubule network, with the HtrA3S being more efficient in cytoskeleton protein cleavage and HtrA3L - in tubulin polymerization. Also, we have shown for the first time that HtrA3 has a chaperone-like, holdase activity in vitro - activity typical for co-chaperone proteins. The proposed HtrA3 influence on the cytoskeleton dynamics may be one of the ways in which HtrA3 promotes cell death and affects cancerogenesis. We believe that the results of this study provide a new insight into the role of HtrA3 in a cell and further confirm the notion that HtrA3 should be considered as a target of new anti-cancer therapies.

Inhibition of apoptosis in BHV-1-infected cells depends on Us3 serine/threonine kinase and its enzymatic activity.

Us3 protein is a serine/threonine kinase conserved within the Alphaherpesvirinae subfamily of herpesviruses. The Us3 homologs of herpes simplex virus, pseudorabies virus, and bovine herpesvirus type 5 have been shown to block apoptosis triggered by viral infection or exogenous inducers. To determine whether these characteristics are shared by bovine herpesvirus type 1 Us3, we constructed two viral mutants: BHV-1 Us3 deletion mutant (BHV-1ΔUs3) and a kinase-dead mutant (BHV-1KD). Flow cytometry analysis and TUNEL assay clearly demonstrated, that only BHV-1 wild type virus suppressed infection-induced apoptosis and protected cells from apoptosis triggered by exogenous factors: sorbitol or staurosporine. Us3 of BHV-1 was directly capable of blocking apoptosis without the presence of other viral proteins. The presence of Us3 correlated with phosphorylation of BAD, a pro-apoptotic Bcl-2 family member. Our results clearly indicate that BHV-1 Us3 is necessary for efficient blocking of apoptosis triggered by viral infection and exogenous factors.

How short RNAs impact the human ribonuclease Dicer activity: putative regulatory feedback-loops and other RNA-mediated mechanisms controlling microRNA processing.

Ribonuclease Dicer plays a pivotal role in RNA interference pathways by processing long double-stranded RNAs and single-stranded hairpin RNA precursors into small interfering RNAs (siRNAs) and microRNAs (miRNAs), respectively. While details of Dicer regulation by a variety of proteins are being elucidated, less is known about non-protein factors, e.g. RNA molecules, that may influence this enzyme's activity. Therefore, we decided to investigate the question of whether the RNA molecules can function not only as Dicer substrates but also as its regulators. Our previous in vitro studies indicated that the activity of human Dicer can be influenced by short RNA molecules that either bind to Dicer or interact with its substrates, or both. Those studies were carried out with commercial Dicer preparations. Nevertheless, such preparations are usually not homogeneous enough to carry out more detailed RNA-binding studies. Therefore, we have established our own system for the production of human Dicer in insect cells. In this manuscript, we characterize the RNA-binding and RNA-cleavage properties of the obtained preparation. We demonstrate that Dicer can efficiently bind single-stranded RNAs that are longer than ~20-nucleotides. Consequently, we revisit possible scenarios of Dicer regulation by single-stranded RNA species ranging from ~10- to ~60-nucleotides, in the context of their binding to this enzyme. Finally, we show that siRNA/miRNA-sized RNAs may affect miRNA production either by binding to Dicer or by participating in regulatory feedback-loops. Altogether, our studies suggest a broad regulatory role of short RNAs in Dicer functioning.