Virus Secretion Research Articles

Induction of interleukin-11 mediated by RhoA GTPase during human cytomegalovirus lytic infection

Human cytomegalovirus (HCMV) is a ubiquitous pathogen which periodically reactivates, causing severe clinical consequences in immunosuppressed patients, organ and stem cell transplant recipients or newborn babies with congenital infections. HCMV infection stimulates the expression of several proinflammatory cytokines, which may contribute to the pathogenesis of the infection. Rho GTPases mediate cytokine expression while increasing evidence implicates them in important aspects of HCMV life cycle. Here, we studied the role of RhoA on the interleukin 11 (IL-11) release in HCMV-infected fibroblasts. Human fibroblasts, either endogenously expressing or silenced for RhoA, were infected by HCMV or UV-inactivated virus and IL-11 transcription and secretion were evaluated. We found that HCMV lytic infection increased the IL-11 levels, both in terms of transcription and translation. Both infectious and non-infectious HCMV particles were able to induce the IL-11 production. The depletion of RhoA resulted in an even higher release of IL-11, revealing the implication of this specific Rho isoform in this biological event. Finally, infection of cells in the presence of the HCMV DNA replication inhibitor, ganciclovir, significantly reduced the secretion of IL-11, strongly associating its induction with active viral DNA replication. Collectively, these data demonstrate, for the first time, a novel role of RhoA GTPase during HCMV lytic infection, regulating the activation of an immune response through IL-11.

COVID-19 and ophthalmology: An environmental work hazard.

COVID-19 and ophthalmology: An environmental work hazard.

The Novel Nuclear Targeting and BFRF1-Interacting Domains of BFLF2 Are Essential for Efficient Epstein-Barr Virus Virion Release.

Epstein-Barr virus (EBV) genomic DNA is replicated and packaged into procapsids in the nucleus to form nucleocapsids, which are then transported into the cytoplasm for tegumentation and final maturation. The process is facilitated by the coordination of the viral nuclear egress complex (NEC), which consists of BFLF2 and BFRF1. By expression alone, BFLF2 is distributed mainly in the nucleus. However, it colocalizes with BFRF1 at the nuclear rim and in cytoplasmic nuclear envelope-derived vesicles in coexpressing cells, suggesting temporal control of the interaction between BFLF2 and BFRF1 is critical for their proper function. The N-terminal sequence of BFLF2 is less conserved than that of alpha- and betaherpesvirus homologs. Here, we found that BFLF2 amino acids (aa) 2 to 102 are required for both nuclear targeting and its interaction with BFRF1. Coimmunoprecipitation and confocal analysis indicated that aa 82 to 106 of BFLF2 are important for its interaction with BFRF1. Three crucial amino acids (R47, K50, and R52) and several noncontinuous arginine and histidine residues within aa 59 to 80 function together as a noncanonical nuclear localization signal (NLS), which can be transferred onto yellow fluorescent protein (YFP)-LacZ for nuclear targeting in an importin β-dependent manner. Virion secretion is defective in 293 cells harboring a BFLF2 knockout EBV bacmid upon lytic induction and is restored by trans-complementation of wild-type BFLF2, but not NLS or BFRF1-interacting defective mutants. In addition, multiple domains of BFRF1 were found to bind BFLF2, suggesting multiple contact regions within BFRF1 and BFLF2 are required for proper nuclear egress of EBV nucleocapsids.IMPORTANCE Although Epstein-Barr virus (EBV) BFRF1 and BFLF2 are homologs of conserved viral nuclear egress complex (NEC) in all human herpesviruses, unique amino acid sequences and functions were identified in both proteins. In this study, the nuclear targeting and BFRF1-interacting domains were found within the N terminus of BFLF2. We showed that amino acids (aa) 82 to 106 are the major region required for BFLF2 to interact with BFRF1. However, the coimmunoprecipitation (Co-IP) data and glutathione transferase (GST) pulldown experiments revealed that multiple regions of both proteins contribute to reciprocal interactions. Different from the canonical nuclear localization signal (NLS) in other herpes viral homologs, BFLF2 contains a novel importin-dependent nuclear localization signal, including R47, K50, and R52 and several neighboring discontinuous arginine and histidine residues. Using a bacmid complementation system, we show that both the nuclear targeting and the novel nuclear localization signal within aa 82 to 106 of BFLF2 are required for virion secretion.

Hepatitis A virus structural protein pX interacts with ALIX and promotes the secretion of virions and foreign proteins through exosome-like vesicles

ABSTRACT Hepatitis A virus (HAV), a classic nonenveloped virus, has recently been found to be released mainly in the form of quasi-enveloped HAV (eHAV) by hijacking host endosomal sorting complexes required for transport (ESCRT) complexes. Unlike the nonenveloped virion, eHAV contains the viral protein pX on the surface of the HAV capsid as an extension of VP1. How HAV capsids acquire the host envelope and whether the pX protein is involved in this process were previously unknown. Here, we analyse the role of pX in foreign protein secretion in exosome-like extracellular vesicles (EVs) and the formation of eHAV. Fusion of pX to eGFP guided eGFP into exosome-like EVs through directing eGFP into multivesicular bodies (MVBs), and apoptosis-linked gene 2-interacting protein X (ALIX) release was significantly enhanced. Coimmunoprecipitation (co-IP) demonstrated the interaction between pX and the ALIX V domain. Removal of the C-terminal half of pX abolished eHAV release and reduced the interaction between the HAV virion and ALIX. Finally, the C-terminal half of pX alone was sufficient for loading eGFP into EVs by interacting with ALIX. In conclusion, the C-terminal part of pX is important for eHAV production and may have potential for large protein complex loading into exosome-like EVs for therapeutic purposes.

Epstein-Barr Virus BBRF2 Is Required for Maximum Infectivity.

Epstein-Barr virus (EBV) is a member of the gammaherpesvirinae, which causes infectious mononucleosis and several types of cancer. BBRF2 is an uncharacterized gene of EBV and is expressed during the lytic phase. To evaluate its function, BBRF2-knockout EBV was prepared using bacterial artificial chromosome (BAC) technology and the CRISPR/Cas9 system. Although viral gene expression, DNA synthesis, and progeny secretion were not affected, the infectivity of progeny viruses was significantly reduced by the disruption of BBRF2. When expressed alone, BBRF2 protein localized to the nucleus and cytoplasm, while the coexpression of an interacting partner, BSRF1, resulted in its relocalization to the cytoplasm. Interestingly, the coexpression of BBRF2 protected BSRF1 from proteasome/ubiquitin-dependent degradation. Therefore, BBRF2, together with BSRF1, augments viral infectivity.

Dengue proteins with their role in pathogenesis, and strategies for developing an effective anti-dengue treatment: A review.

Dengue virus is an arbovirus belonging to class Flaviviridae Its clinical manifestation ranges from asymptomatic to extreme conditions (dengue hemorrhagic fever/dengue shock syndrome). A lot of research has been done on this ailment, yet there is no effective treatment available for the disease. This review provides the systematic understanding of all dengue proteins, role of its structural proteins (C-protein, E-protein, prM) in virus entry, assembly, and secretion in host cell, and nonstructural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5) in viral assembly, replication, and immune evasion during dengue progression and pathogenesis. Furthermore, the review has highlighted the controversies related to the only commercially available dengue vaccine, that is, Dengvaxia, and the risk associated with it. Lastly, it provides an insight regarding various approaches for developing an effective anti-dengue treatment.

Differential Roles of Lipin1 and Lipin2 in the Hepatitis C Virus Replication Cycle.

Although their origin, nature and structure are not identical, a common feature of positive-strand RNA viruses is their ability to subvert host lipids and intracellular membranes to generate replication and assembly complexes. Recently, lipin1, a cellular enzyme that converts phosphatidic acid into diacylglycerol, has been implicated in the formation of the membranous web that hosts hepatitis C virus (HCV) replicase. In the liver, lipin1 cooperates with lipin2 to maintain glycerolipid homeostasis. We extended our previous study of the lipin family on HCV infection, by determining the impact of the lipin2 silencing on viral replication. Our data reveal that lipin2 silencing interferes with HCV virion secretion at late stages of the infection, without significantly affecting viral replication or assembly. Moreover, uninfected lipin2-, but not lipin1-deficient cells display alterations in mitochondrial and Golgi apparatus morphology, suggesting that lipin2 contributes to the maintenance of the overall organelle architecture. Finally, our data suggest a broader function of lipin2 for replication of HCV and other RNA viruses, in contrast with the specific impact of lipin1 silencing on HCV replication. Overall, this study reveals distinctive functions of lipin1 and lipin2 in cells of hepatic origin, a context in which they are often considered functionally redundant.

Direct interaction between the hepatitis B virus core and envelope proteins analyzed in a cellular context

Hepatitis B virus (HBV) production requires intricate interactions between the envelope and core proteins. Analyses of mutants of these proteins have made it possible to map regions involved in the formation and secretion of virions. Tests of binding between core and envelope peptides have also been performed in cell-free conditions, to study the interactions potentially underlying these mechanisms. We investigated the residues essential for core-envelope interaction in a cellular context in more detail, by transiently producing mutant or wild-type L, S, or core proteins separately or in combination, in Huh7 cells. The colocalization and interaction of these proteins were studied by confocal microscopy and co-immunoprecipitation, respectively. The L protein was shown to constitute a molecular platform for the recruitment of S and core proteins in a perinuclear environment. Several core amino acids were found to be essential for direct interaction with L, including residue Y132, known to be crucial for capsid formation, and residues L60, L95, K96 and I126. Our results confirm the key role of L in the tripartite core-S-L interaction and identify the residues involved in direct core-L interaction. This model may be valuable for studies of the potential of drugs to inhibit HBV core-envelope interaction.

Ethiopathogenetic aspects and laboratory diagnostics of viral respiratory-intestinal infections of calves

The results of a virological and serological study of calves, patients and deaths with symptoms of diarrhea and pneumoenteritis are presented. In the pathological material of 8 calves of 2–8 days of age, sick and deaths with signs of diarrhea, antigens of rotavirus B (62.5%) and betacoronavirus 1 (100%) were identified in direct immunofluorescence test, including 62.5% of animals with associated infection. Serological research of 17 calf convalescents revealed a diagnostic increase in antibodies to rotavirus B (52.9%) and betacoronavirus 1 (70.6%), including 23.5% of animals with seroconversion to two viruses. In a research of blood sera and colostrum on the first day, 25 cows were found to have antibodies to rotavirus B (20.0%) and beta-coronavirus 1 (32.0%) in hemagglutination inhibition test. The relationship between antibody titers in the serum and colostrum of maternal cows and blood serum of newborn calves was established. Antigens of rotaviruses A and C, ungulate boсaparvоvirus 1 and specific antibodies to them for diarrheal calf disease have not been established. In the pathological material of 11 calves of 15–20 days of age, sick and deaths with signs of pneumoenteritis, antigens of bovine mastadenovirus B (72.7%) and bovine atadenovirus D (45.5%) were identified in direct immunofluorescence test, including 18.2% of animals are associated with two viruses. In a serological research of 48 calf-convalescents, a diagnostic increase in antibody titers to bovine mastadenovirus B (66.7%) and bovine atadenovirus D (50.0%) was detected in hemagglutination inhibition test. In a study of colostrum on the first day of 22 cows, antibodies to bovine mastadenovirus B (27.3%) and bovine atadenovirus D (18.2%) were detected. Antigens of bovine mastаdenovirus A and C, pestiviruses A and B, mammalian orthoreovirus, ungulate boсaparvоvirus 1 and specific antibodies to them for pneumoenteritis of calves have not been established. Therefore, in the etiopathogenesis of diarrheal disease of newborn calves, the involvement of rotavirus B and betacoronavirus 1 was established. The presence of colostrum antibodies did not protect calves from rotavirus and coronavirus infections, did not block the secretion of viruses from feces, and correlated with their mire contents. Colostrum antibodies inhibited the body's immunological response to infection. The participation of bovine mastodenovirus and bovine atadenovirus D has been found in the etiopathogenesis of calf pneumoenteritis. These viruses latently infect adult animals as a source of infectious agents for calves.

Persistence of Hepatitis B Virus DNA and the Tempos between Virion Secretion and Genome Maturation in a Mouse Model.

Hepatitis B virus (HBV) core protein (HBc) accumulates frequent mutations in natural infection. Wild-type HBV is known to secrete predominantly virions containing mature DNA genome. However, a frequent naturally occurring HBc variant, I97L, changing from an isoleucine to a leucine at amino acid 97, exhibited an immature secretion phenotype in culture, which preferentially secretes virions containing immature genomes. In contrast, mutant P130T, changing from a proline to a threonine at amino acid 130, exhibited a hypermaturation phenotype by accumulating an excessive amount of intracellular fully mature DNA genome. Using a hydrodynamic delivery mouse model, we studied the in vivo behaviors of these two mutants, I97L and P130T. We detected no naked core particles in all hydrodynamically injected mice. Mutant I97L in mice exhibited pleiotropic phenotypes: (i) excessive numbers of serum HBV virions containing immature genomes, (ii) significantly reduced numbers of intracellular relaxed-circle and single-stranded DNAs, and (iii) less persistent intrahepatic and secreted HBV DNAs than wild-type HBV. These pleiotropic phenotypes were observed in both immunocompetent and immunodeficient mice. Although mutant P130T also displayed a hypermaturation phenotype in vivo, it cannot efficiently rescue the immature virion secretion of mutant I97L. Unexpectedly, the single mutant P130T exhibited in vivo a novel phenotype in prolonging the persistence of HBV genome in hepatocytes. Taken together, our studies provide a plausible rationale for HBV to regulate envelopment morphogenesis and virion secretion via genome maturity, which is likely to play an important role in the persistence of viral DNA in this mouse model.IMPORTANCE Chronic infection with human hepatitis B virus (HBV) could lead to cirrhosis and hepatoma. At present, there is no effective treatment to eradicate the virus from patients. HBV in chronic carriers does not exist as a single homogeneous population. The most frequent naturally occurring mutation in HBV core protein occurs at amino acid 97, changing an isoleucine to leucine (I97L). One dogma in the field is that only virions containing a mature genome are preferentially secreted into the medium. Here, we demonstrated that mutant I97L can secrete immature genome in mice. Although viral DNA of mutant I97L with immature genome is less persistent than wild-type HBV in time course experiments, viral DNA of mutant P130T with genome hypermaturation, surprisingly, is more persistent. Therefore, virion secretion regulated by genome maturity could influence viral persistence. It remains an open issue whether virion secretion could be a drug target for HBV therapy.

ER-shaping atlastin proteins act as central hubs to promote flavivirus replication and virion assembly.

Flaviviruses, including dengue virus (DV) and Zika virus, extensively remodel the cellular endomembrane network to generate replication organelles that promote viral genome replication and virus production. However, it remains unclear how these membranes and associated cellular proteins act during the virus cycle. Here, we show that atlastins (ATLs), a subset of ER resident proteins involved in neurodegenerative diseases, have dichotomous effects on flaviviruses with ATL2 depletion leading to replication organelle defects and ATL3 depletion to changes in virus production pathways. We characterized non-conserved functional domains in ATL paralogues and show that the ATL interactome is profoundly reprogrammed upon DV infection. Screen analysis confirmed non-redundant ATL functions and identified a specific role for ATL3, and its interactor ARF4, in vesicle trafficking and virion maturation. Our data identify ATLs as central hubs targeted by flaviviruses to establish their replication organelle and to achieve efficient virion maturation and secretion.

Dynamics and Differences in Systemic and Local Immune Responses After Vaccination With Inactivated and Live Commercial Vaccines and Subsequent Subclinical Infection With PRRS Virus.

The goals of our study were to compare the immune response to different killed and modified live vaccines against PRRS virus and to monitor the antibody production and the cell mediated immunity both at the systemic and local level. In the experiment, we immunized four groups of piglets with two commercial inactivated (A1—Progressis, A2—Suivac) and two modified live vaccines (B3—Amervac, B4—Porcilis). Twenty-one days after the final vaccination, all piglets, including the control non-immunized group (C5), were i.n., infected with the Lelystad strain of PRRS virus. The serum antibody response (IgM and IgG) was the strongest in group A1 followed by two MLV (B3 and B4) groups. Locally, we demonstrated the highest level of IgG antibodies in bronchoalveolar lavages (BALF), and saliva in group A1, whereas low IgA antibody responses in BALF and feces were detected in all groups. We have found virus neutralization antibody at DPV 21 (days post vaccination) and higher levels in all groups including the control at DPI 21 (days post infection). Positive antigen specific cell-mediated response in lymphocyte transformation test (LTT) was observed in groups B3 and B4 at DPV 7 and in group B4 at DPV 21 and in all intervals after infection. The IFN-γ producing lymphocytes after antigen stimulation were found in CD4−CD8+ and CD4+CD8+ subsets of all immunized groups 7 days after infection. After infection, there were obvious differences in virus excretion. The virus was detected in all groups of piglets in serum, saliva, and occasionally in feces at DPI 3. Significantly lower virus load was found in groups A1 and B3 at DPI 21. Negative samples appeared at DPI 21 in B3 group in saliva. It can be concluded that antibodies after immunization and infection, and the virus after infection can be detected in all the compartments monitored. Immunization with inactivated vaccine A1—Progressis induces high levels of antibodies produced both systemically and locally. Immunization with MLV-vaccines (Amervac and Porcilis) produces sufficient antibody levels and also cell-mediated immunity. After infection virus secretion gradually decreases in group B3, indicating tendency to induce sterile immunity.

Iminosugars counteract the downregulation of the interferon γ receptor by dengue virus

The antiviral mechanism of action of iminosugars against many enveloped viruses is hypothesized to be a consequence of misfolding of viral N-linked glycoproteins through inhibition of host endoplasmic reticulum α-glucosidase enzymes. Iminosugar treatment of dengue virus (DENV) infection results in reduced secretion of virions and hence lower viral titres in vitro and in vivo. We investigated whether iminosugars might also affect host receptors important in DENV attachment and uptake and immune responses to DENV.Using a primary human macrophage model of DENV infection, we investigated the effects of maturation with IL-4, DENV-infection and treatment with N-butyl-1-deoxynojirimycin (NB-DNJ) or N-(9-methoxynonyl)-1-DNJ (MON-DNJ) on expression of 11 macrophage receptors.Whereas iminosugars did not affect surface expression of any of the receptors examined, DENV infection significantly reduced surface IFNγ receptor amongst other changes to total receptor expression. This effect required infectious DENV and was reversed by iminosugar treatment. Treatment also affected signalling of the IFNγ receptor and TNFα receptor. In addition, iminosugars reduced ligand binding to the carbohydrate receptor-binding domain of the mannose receptor. This work demonstrates that iminosugar treatment of primary macrophages affects expression and functionality of some key glycosylated host immune receptors important in the dengue life cycle.

Experimental infection of dromedary camels with virulent virus of Peste des Petits Ruminants

Peste des Petits Ruminants Virus (PPRV) causes a severe contagious disease of sheep and goats and has spread extensively in last years through Asia and Africa. PPRV, known to infect exclusively small ruminants, has been recently reported in camels in Iran and Sudan. Reported clinical symptoms are similar to those observed in small ruminants, fatality rate still unknown. However most of the authors reported seropositive camels without clinical signs. Camel sensitivity to PPRV is still controversial and more investigation need to be performed. In this study, we tested camel susceptibility by an experimental infection using a virulent PPRV strain belonging to lineage IV. Young dromedary camels were infected intravenously and observed one month for clinical symptoms. Viraemia and virus secretion charge in swabs were evaluated by PCR. Seroconversion was assessed by ELISA and virus neutralisation test. Infected animals did not manifest any clinical symptoms of the disease and no virus was detected in secretions. Seroconversion was observed from day 14 post infection.

Formation of semi-enveloped particles as a unique feature of a hepatitis B virus PreS1 deletion mutant.

Naturally occurring variants with deletions or mutations in the C-terminal PreS1 domain from hepatitis B virus (HBV) chronically infected patients have been shown to promote HBsAg retention, inhibit HBsAg secretion and change the extracellular appearance of PreS1-containing HBV particles (filaments and virions). To study the impact of N-terminal deletion in preS1 domain on viral secretion and morphogenesis. An HBV mutant with 15 amino acids (aa 25-39) deletion in N-terminal preS1 was isolated. Intracellular and extracellular HBsAg were quantified by Western blot. Subcellular HBsAg distribution was analysed by confocal laser scanning microscopy. The viral morphology was characterised by sucrose density gradient ultracentrifugation, Western blot, electron microscopy, HBV mixed ELISA and HBV particle gel essay. Expression of this mutant genome released higher amounts of HBsAg in the form of shorter filaments. A significant fraction of semi-enveloped virions was observed in the supernatant that has been unprecedented so far. Stepwise insertion of aa 25-31, aa 32-39 and aa 25-39 increased the length of filaments. The rescue of aa 25-31 and aa 25-39 drastically reduced the amounts of extracellular HBsAg and semi-enveloped virions, while such effects could not be observed after insertion of aa 32-39, arguing against a simple spacer function of this region. The deletion and rescued mutants do not differ in subcellular HBsAg distribution and colocalisation with ER, Golgi and multivesicular bodies markers arguing against differences in release pathways. N-terminal PreS1-domain (aa 25-31) determines HBsAg secretion and triggers proper assembly of PreS1-containing particles.

Complementation of Wild-Type and Drug-Resistant Hepatitis B Virus Genomes to Maintain Viral Replication and Rescue Virion Production under Nucleos(t)ide Analogs.

As the open reading frames of hepatitis B virus (HBV) genomes are overlapping, resistance mutations (MTs) in HBV polymerase may result in stop codon MTs in hepatitis B surface proteins, which are usually detected as a mixed population with wild-type (WT) HBV. The question was raised how the coexistence of nucleos(t)ide analogs (NAs) resistance MTs and WT sequences affects HBV replication. In the present study, HBV genomes with frequently detected reverse transcriptase (RT)/surface truncation MTs, rtA181T/sW172*, rtV191I/sW182* and rtM204I/sW196*, were phenotypically characterized alone or together with their WT counterparts in different ratios by transient transfection in the absence or presence of NAs. In the absence of NAs, RT/surface truncation MTs impaired the expression and secretion of HBV surface proteins, and had a dose-dependent negative effect on WT HBV virion secretion. However, in the presence of NAs, coexistence of MTs with WT maintained viral replication, and the presence of WT was able to rescue the production of MT HBV virions. Our findings reveal that complementation of WT and MT HBV genomes is highly effective under drug treatment.

Hepatitis C virus sequence divergence preserves p7 viroporin structural and dynamic features

The hepatitis C virus (HCV) viroporin p7 oligomerizes to form ion channels, which are required for the assembly and secretion of infectious viruses. The 63-amino acid p7 monomer has two putative transmembrane domains connected by a cytosolic loop, and has both N- and C- termini exposed to the endoplasmic reticulum (ER) lumen. NMR studies have indicated differences between p7 structures of distantly related HCV genotypes. A critical question is whether these differences arise from the high sequence variation between the different isolates and if so, how the divergent structures can support similar biological functions. Here, we present a side-by-side characterization of p7 derived from genotype 1b (isolate J4) in the detergent 6-cyclohexyl-1-hexylphosphocholine (Cyclofos-6) and p7 derived from genotype 5a (isolate EUH1480) in n-dodecylphosphocholine (DPC). The 5a isolate p7 in conditions previously associated with a disputed oligomeric form exhibits secondary structure, dynamics, and solvent accessibility broadly like those of the monomeric 1b isolate p7. The largest differences occur at the start of the second transmembrane domain, which is destabilized in the 5a isolate. The results show a broad consensus among the p7 variants that have been studied under a range of different conditions and indicate that distantly related HCVs preserve key features of structure and dynamics.

Recapitulation of HDV infection in a fully permissive hepatoma cell line allows efficient drug evaluation

Hepatitis delta virus (HDV) depends on the helper function of hepatitis B virus (HBV), which provides the envelope proteins for progeny virus secretion. Current infection-competent cell culture models do not support assembly and secretion of HDV. By stably transducing HepG2 cells with genes encoding the NTCP-receptor and the HBV envelope proteins we produce a cell line (HepNB2.7) that allows continuous secretion of infectious progeny HDV following primary infection. Evaluation of antiviral drugs shows that the entry inhibitor Myrcludex B (IC50: 1.4 nM) and interferon-α (IC50: 28 IU/ml, but max. 60–80% inhibition) interfere with primary infection. Lonafarnib inhibits virus secretion (IC50: 36 nM) but leads to a substantial intracellular accumulation of large hepatitis delta antigen and replicative intermediates, accompanied by the induction of innate immune responses. This work provides a cell line that supports the complete HDV replication cycle and presents a convenient tool for antiviral drug evaluation.

Parvulin 14 and Parvulin 17 Bind to HBx and cccDNA and Upregulate Hepatitis B Virus Replication from cccDNA to Virion in an HBx-Dependent Manner.

The parvulin 14 (Par14) and parvulin 17 (Par17) proteins, which are both encoded by the PIN4 gene, play roles in protein folding, chromatin remodeling, DNA binding, ribosome biogenesis, and cell cycle progression. However, the effects of Par14 and Par17 on viral replication have never been explored. In this study, we found that, in the presence of HBx, either Par14 or Par17 could upregulate hepatitis B virus (HBV) replication, whereas in the absence of HBx, neither Par14 nor Par17 had any effect on replication. Overexpression of Par14/Par17 markedly increased the formation of covalently closed circular DNA (cccDNA), synthesis of HBV RNA and DNA, and virion secretion. Conversely, PIN4 knockdown significantly decreased HBV replication in HBV-transfected and -infected cells. Coimmunoprecipitation revealed that Par14/Par17 engaged in direct physical interactions with HBx in the cytoplasm, nucleus, and mitochondria, possibly mediated through substrate-binding residues on Par14/Par17 (E46/D74 and E71/D99, respectively) and conserved 19R20P-28R29P motifs on HBx. Furthermore, these interactions enhanced HBx stability, promoted HBx translocation to the nuclear and mitochondrial fractions, and increased HBV replication. Chromatin immunoprecipitation assays revealed that, in the presence of HBx, Par14/Par17 were efficiently recruited to cccDNA and promoted transcriptional activation via specific DNA-binding residues (S19/44). In contrast, in the absence of HBx, Par14/Par17 bound cccDNA only at the basal level and did not promote transcriptional activation. Taken together, our results demonstrate that Par14 and Par17 upregulate HBV RNA transcription and DNA synthesis, thereby increasing the HBV cccDNA level, through formation of the cccDNA-Par14/17-HBx complex.IMPORTANCE The HBx protein plays an essential regulatory role in HBV replication. We found that substrate-binding residues on the human parvulin peptidylprolyl cis/trans isomerase proteins Par14 and Par17 bound to conserved arginine-proline (RP) motifs on HBx in the cytoplasm, nucleus, and mitochondria. The HBx-Par14/Par17 interaction stabilized HBx; promoted its translocation to the nucleus and mitochondria; and stimulated multiple steps of HBV replication, including cccDNA formation, HBV RNA and DNA synthesis, and virion secretion. In addition, in the presence of HBx, the Par14 and Par17 proteins bound to cccDNA and promoted its transcriptional activation. Our results suggest that inhibition or knockdown of Par14 and Par17 may represent a novel therapeutic option against HBV infection.

Secretion of Empty or Complete Hepatitis B Virions: Envelopment of Empty Capsids Versus Mature Nucleocapsids

HBV replicates its DNA genome, a partially double-stranded, relaxed circular DNA, via reverse transcription of an RNA intermediate called pre-genomic RNA by its reverse transcriptase. A major characteristic of HBV replication is the selective envelopment and secretion of relaxed circular DNA-containing mature capsids and empty capsids with no DNA or RNA, but not those containing pre-genomic RNA or the single-stranded DNA replication intermediate. In this review, the potential mechanisms of HBV virion morphogenesis will be discussed, with a focus on key determinants of both the capsid and envelope proteins for the selective secretion of complete and empty virions.