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Natalia Freitas
CIRI – Centre International de Recherche en Infectiologie, Univ Lyon, Université Claude Bernard Lyon 1, Inserm, U1111, CNRS, UMR5308, ENS Lyon, Lyon, France

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Journal article
Published: 30 June 2021 in eLife
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Cell entry of enveloped viruses relies on the fusion between the viral and plasma or endosomal membranes, through a mechanism that is triggered by a cellular signal. Here we used a combination of computational and experimental approaches to unravel the main determinants of hepatitis B virus (HBV) membrane fusion process. We discovered that ERp57 is a host factor critically involved in triggering HBV fusion and infection. Then, through modelling approaches, we uncovered a putative allosteric cross-strand disulfide (CSD) bond in the HBV S glycoprotein and we demonstrate that its stabilization could prevent membrane fusion. Finally, we identified and characterized a potential fusion peptide in the preS1 domain of the HBV L glycoprotein. These results underscore a membrane fusion mechanism that could be triggered by ERp57, allowing a thiol/disulfide exchange reaction to occur and regulate isomerization of a critical CSD, which ultimately leads to the exposition of the fusion peptide.

ACS Style

Jimena Pérez-Vargas; Elin Teppa; Fouzia Amirache; Bertrand Boson; Rémi Pereira de Oliveira; Christophe Combet; Anja Böckmann; Floriane Fusil; Natalia Freitas; Alessandra Carbone; François-Loïc Cosset. A fusion peptide in preS1 and the human protein-disulfide isomerase ERp57 are involved in HBV membrane fusion process. eLife 2021, 10, 1 .

AMA Style

Jimena Pérez-Vargas, Elin Teppa, Fouzia Amirache, Bertrand Boson, Rémi Pereira de Oliveira, Christophe Combet, Anja Böckmann, Floriane Fusil, Natalia Freitas, Alessandra Carbone, François-Loïc Cosset. A fusion peptide in preS1 and the human protein-disulfide isomerase ERp57 are involved in HBV membrane fusion process. eLife. 2021; 10 ():1.

Chicago/Turabian Style

Jimena Pérez-Vargas; Elin Teppa; Fouzia Amirache; Bertrand Boson; Rémi Pereira de Oliveira; Christophe Combet; Anja Böckmann; Floriane Fusil; Natalia Freitas; Alessandra Carbone; François-Loïc Cosset. 2021. "A fusion peptide in preS1 and the human protein-disulfide isomerase ERp57 are involved in HBV membrane fusion process." eLife 10, no. : 1.

Review
Published: 23 June 2021 in Viruses
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Hepatitis delta virus (HDV) is a defective human virus that lacks the ability to produce its own envelope proteins and is thus dependent on the presence of a helper virus, which provides its surface proteins to produce infectious particles. Hepatitis B virus (HBV) was so far thought to be the only helper virus described to be associated with HDV. However, recent studies showed that divergent HDV-like viruses could be detected in fishes, birds, amphibians, and invertebrates, without evidence of any HBV-like agent supporting infection. Another recent study demonstrated that HDV can be transmitted and propagated in experimental infections ex vivo and in vivo by different enveloped viruses unrelated to HBV, including hepatitis C virus (HCV) and flaviviruses such as Dengue and West Nile virus. All this new evidence, in addition to the identification of novel virus species within a large range of hosts in absence of HBV, suggests that deltaviruses may take advantage of a large spectrum of helper viruses and raises questions about HDV origins and evolution.

ACS Style

Jimena Pérez-Vargas; Rémi Pereira de Oliveira; Stéphanie Jacquet; Dominique Pontier; François-Loïc Cosset; Natalia Freitas. HDV-Like Viruses. Viruses 2021, 13, 1207 .

AMA Style

Jimena Pérez-Vargas, Rémi Pereira de Oliveira, Stéphanie Jacquet, Dominique Pontier, François-Loïc Cosset, Natalia Freitas. HDV-Like Viruses. Viruses. 2021; 13 (7):1207.

Chicago/Turabian Style

Jimena Pérez-Vargas; Rémi Pereira de Oliveira; Stéphanie Jacquet; Dominique Pontier; François-Loïc Cosset; Natalia Freitas. 2021. "HDV-Like Viruses." Viruses 13, no. 7: 1207.

Review
Published: 28 April 2021 in Viruses
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The Bunyavirales order comprises more than 500 viruses (generally defined as bunyaviruses) classified into 12 families. Some of these are highly pathogenic viruses infecting different hosts, including humans, mammals, reptiles, arthropods, birds, and/or plants. Host cell sensing of infection activates the innate immune system that aims at inhibiting viral replication and propagation. Upon recognition of pathogen-associated molecular patterns (PAMPs) by cellular pattern recognition receptors (PRRs), numerous signaling cascades are activated, leading to the production of interferons (IFNs). IFNs act in an autocrine and paracrine manner to establish an antiviral state by inducing the expression of hundreds of IFN-stimulated genes (ISGs). Some of these ISGs are known to restrict bunyavirus infection. Along with other constitutively expressed host cellular factors with antiviral activity, these proteins (hereafter referred to as “restriction factors”) target different steps of the viral cycle, including viral entry, genome transcription and replication, and virion egress. In reaction to this, bunyaviruses have developed strategies to circumvent this antiviral response, by avoiding cellular recognition of PAMPs, inhibiting IFN production or interfering with the IFN-mediated response. Herein, we review the current knowledge on host cellular factors that were shown to restrict infections by bunyaviruses. Moreover, we focus on the strategies developed by bunyaviruses in order to escape the antiviral state developed by the infected cells.

ACS Style

Solène Lerolle; Natalia Freitas; François-Loïc Cosset; Vincent Legros. Host Cell Restriction Factors of Bunyaviruses and Viral Countermeasures. Viruses 2021, 13, 784 .

AMA Style

Solène Lerolle, Natalia Freitas, François-Loïc Cosset, Vincent Legros. Host Cell Restriction Factors of Bunyaviruses and Viral Countermeasures. Viruses. 2021; 13 (5):784.

Chicago/Turabian Style

Solène Lerolle; Natalia Freitas; François-Loïc Cosset; Vincent Legros. 2021. "Host Cell Restriction Factors of Bunyaviruses and Viral Countermeasures." Viruses 13, no. 5: 784.

Microbiology
Published: 21 September 2020 in PLOS Pathogens
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Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne orthonairovirus that has become a serious threat to the public health. CCHFV has a single-stranded, tripartite RNA genome composed of L, M, and S segments. Cleavage of the M polyprotein precursor generates the two envelope glycoproteins (GPs) as well as three secreted nonstructural proteins GP38 and GP85 or GP160, representing GP38 only or GP38 linked to a mucin-like protein (MLD), and a double-membrane-spanning protein called NSm. Here, we examined the relevance of each M-segment non-structural proteins in virus assembly, egress and infectivity using a well-established CCHFV virus-like-particle system (tc-VLP). Deletion of MLD protein had no impact on infectivity although it reduced by 60% incorporation of GPs into particles. Additional deletion of GP38 abolished production of infectious tc-VLPs. The loss of infectivity was associated with impaired Gc maturation and exclusion from the Golgi, showing that Gn is not sufficient to target CCHFV GPs to the site of assembly. Consistent with this, efficient complementation was achieved in cells expressing MLD-GP38 in trans with increased levels of preGc to Gc conversion, co-targeting to the Golgi, resulting in particle incorporation and restored infectivity. Contrastingly, a MLD-GP38 variant retained in the ER allowed preGc cleavage but failed to rescue miss-localization or infectivity. NSm deletion, conversely, did not affect trafficking of Gc but interfered with Gc processing, particle formation and secretion. NSm expression affected N-glycosylation of different viral proteins most likely due to increased speed of trafficking through the secretory pathway. This highlights a potential role of NSm in overcoming Golgi retention and facilitating CCHFV egress. Thus, deletions of GP38 or NSm demonstrate their important role on CCHFV particle production and infectivity. GP85 is an essential viral factor for preGc cleavage, trafficking and Gc incorporation into particles, whereas NSm protein is involved in CCHFV assembly and virion secretion. Orthonairoviruses, like the lethal Crimean-Congo hemorrhagic fever virus (CCHFV), encode secreted glycoproteins, such as GP38, in addition to virion envelope glycoproteins (Gn and Gc) that are processed by internal cleavage of the viral M segment encoded polyprotein. CCHFV MLD-GP38 proteins (GP160/GP85) also include an N-terminal domain encompassing a mucin-like protein that is released from GP38 by Furin. The protective effect of non-neutralizing monoclonal antibodies targeting GP38 against lethal CCHFV challenge previously highlighted the importance of GP38 in CCHFV replication. CCHFV also encodes a double-membrane-spanning protein (NSm) of unknown function, located between the Gn and Gc on the polyprotein. To investigate the roles of these so-called accessory proteins encoded by the CCHFV M-segment in virus formation and infectivity, we generated several M-segment deletion mutants and tested them in a CCHFV transcription-entry-competent virus-like particle (tc-VLP) system. Here, we demonstrate that GP38 is crucial for Gc biogenesis, interaction with Gn and trafficking to the Golgi, and that its deletion abrogates formation of infectious particles. We also show that NSm increases the rate of protein trafficking through the secretory pathway with altered N-glycosylation profiles that are advantageous for efficient virus release. These data advanced our understanding of GP38 and NSm roles and CCHFV-host interactions.

ACS Style

Natalia Freitas; Margot Enguehard; Solène Denolly; Camille Levy; Gregory Neveu; Solène Lerolle; Stephanie Devignot; Friedemann Weber; Eric Bergeron; Vincent Legros; François-Loïc Cosset. The interplays between Crimean-Congo hemorrhagic fever virus (CCHFV) M segment-encoded accessory proteins and structural proteins promote virus assembly and infectivity. PLOS Pathogens 2020, 16, e1008850 .

AMA Style

Natalia Freitas, Margot Enguehard, Solène Denolly, Camille Levy, Gregory Neveu, Solène Lerolle, Stephanie Devignot, Friedemann Weber, Eric Bergeron, Vincent Legros, François-Loïc Cosset. The interplays between Crimean-Congo hemorrhagic fever virus (CCHFV) M segment-encoded accessory proteins and structural proteins promote virus assembly and infectivity. PLOS Pathogens. 2020; 16 (9):e1008850.

Chicago/Turabian Style

Natalia Freitas; Margot Enguehard; Solène Denolly; Camille Levy; Gregory Neveu; Solène Lerolle; Stephanie Devignot; Friedemann Weber; Eric Bergeron; Vincent Legros; François-Loïc Cosset. 2020. "The interplays between Crimean-Congo hemorrhagic fever virus (CCHFV) M segment-encoded accessory proteins and structural proteins promote virus assembly and infectivity." PLOS Pathogens 16, no. 9: e1008850.

Journal article
Published: 08 May 2019 in Nature Communications
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Hepatitis D virus (HDV) doesn’t encode envelope proteins for packaging of its ribonucleoprotein (RNP) and typically relies on the surface glycoproteins (GPs) from hepatitis B virus (HBV) for virion assembly, envelopment and cellular transmission. HDV RNA genome can efficiently replicate in different tissues and species, raising the possibility that it evolved, and/or is still able to transmit, independently of HBV. Here we show that alternative, HBV-unrelated viruses can act as helper viruses for HDV. In vitro, envelope GPs from several virus genera, including vesiculovirus, flavivirus and hepacivirus, can package HDV RNPs, allowing efficient egress of HDV particles in the extracellular milieu of co-infected cells and subsequent entry into cells expressing the relevant receptors. Furthermore, HCV can propagate HDV infection in the liver of co-infected humanized mice for several months. Further work is necessary to evaluate whether HDV is currently transmitted by HBV-unrelated viruses in humans. Hepatitis D virus (HDV) relies on a helper virus to package and transmit its ribonucleoprotein (RNP). Here, Perez-Vargas et al. show that HDV can use envelope proteins from HBV-unrelated viruses, including HCV and flaviviruses, to propagate in vitro and in humanized mice.

ACS Style

Jimena Perez-Vargas; Fouzia Amirache; Bertrand Boson; Chloé Mialon; Natalia Maria Bezerra de Freitas; Camille Sureau; Floriane Fusil; François-Loïc Cosset. Enveloped viruses distinct from HBV induce dissemination of hepatitis D virus in vivo. Nature Communications 2019, 10, 1 -15.

AMA Style

Jimena Perez-Vargas, Fouzia Amirache, Bertrand Boson, Chloé Mialon, Natalia Maria Bezerra de Freitas, Camille Sureau, Floriane Fusil, François-Loïc Cosset. Enveloped viruses distinct from HBV induce dissemination of hepatitis D virus in vivo. Nature Communications. 2019; 10 (1):1-15.

Chicago/Turabian Style

Jimena Perez-Vargas; Fouzia Amirache; Bertrand Boson; Chloé Mialon; Natalia Maria Bezerra de Freitas; Camille Sureau; Floriane Fusil; François-Loïc Cosset. 2019. "Enveloped viruses distinct from HBV induce dissemination of hepatitis D virus in vivo." Nature Communications 10, no. 1: 1-15.

Protocol
Published: 29 December 2018 in Advanced Structural Safety Studies
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The membrane fusion properties of HCV envelope glycoproteins can be evaluated using several assays. Fusion assays generally require contacts between glycoproteins expressed on a donor membrane, such as those from a cell or a viral particle, and an acceptor membrane that may or may not express cognate viral receptor, such as those from an indicator cell or a liposome. In this chapter, we describe three well-established methods in the field that use either cell surface expression of glycoproteins, HCV pseudoparticles (HCVpp), or cell culture-grown HCV (HCVcc) particles for donor membrane and cells or liposomes as acceptor membrane in which specific components can be included to monitor and quantify fusion. We provide details of cell-cell fusion assay, virus-liposome fusion assay, and finally virus-plasma membrane fusion assay. We also describe inhibitors that can block HCV envelope membrane fusion.

ACS Style

Solène Denolly; François-Loïc Cosset; Natalia Maria Bezerra de Freitas. Membrane Fusion Assays for Studying Entry Hepatitis C Virus into Cells. Advanced Structural Safety Studies 2018, 219 -234.

AMA Style

Solène Denolly, François-Loïc Cosset, Natalia Maria Bezerra de Freitas. Membrane Fusion Assays for Studying Entry Hepatitis C Virus into Cells. Advanced Structural Safety Studies. 2018; ():219-234.

Chicago/Turabian Style

Solène Denolly; François-Loïc Cosset; Natalia Maria Bezerra de Freitas. 2018. "Membrane Fusion Assays for Studying Entry Hepatitis C Virus into Cells." Advanced Structural Safety Studies , no. : 219-234.

Journal article
Published: 15 May 2018 in Journal of Virology
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Five matching sets of nonmalignant liver tissues and hepatocellular carcinoma (HCC) samples from individuals chronically infected with hepatitis B virus (HBV) were examined. The HBV genomic sequences were determined by using overlapping PCR amplicons covering the entire viral genome. Four pairs of tissues were infected with HBV genotype C, while one pair was infected with HBV genotype B. HBV replication markers were found in all tissues. In the majority of HCC samples, the levels of pregenomic/precore RNA (pgRNA) and covalently closed circular DNA (cccDNA) were lower than those in liver tissue counterparts. Regardless of the presence of HBV replication markers, (i) integrant-derived HBV RNAs (id-RNAs) were found in all tissues by reverse transcription-PCR (RT-PCR) analysis and were considerably abundant or predominant in 6/10 tissue samples (2 liver and 4 HCC samples), (ii) RNAs that were polyadenylated using the cryptic HBV polyadenylation signal and therefore could be produced by HBV replication or derived from integrated HBV DNA were found in 5/10 samples (3 liver and 2 HCC samples) and were considerably abundant species in 3/10 tissues (2 livers and 1 HCC), and (iii) cccDNA-transcribed RNAs polyadenylated near position 1931 were not abundant in 7/10 tissues (2 liver and 5 HCC samples) and were predominant in only two liver samples. Subsequent RNA sequencing analysis of selected liver/HCC samples also showed relative abundance of id-RNAs in most of the examined tissues. Our findings suggesting that id-RNAs could represent a significant source of HBV envelope proteins, which is independent of viral replication, are discussed in the context of the possible contribution of id-RNAs to the HBV life cycle. IMPORTANCE The relative abundance of integrant-derived HBV RNAs (id-RNAs) in chronically infected tissues suggest that id-RNAs coding for the envelope proteins may facilitate the production of a considerable fraction of surface antigens (HBsAg) in infected cells bearing HBV integrants. If the same cells support HBV replication, then a significant fraction of assembled HBV virions could bear id-RNA-derived HBsAg as a major component of their envelopes. Therefore, the infectivity of these HBV virions and their ability to facilitate virus cell-to-cell spread could be determined mainly by the properties of id-RNA-derived envelope proteins and not by the properties of replication-derived HBsAg. These interpretations suggest that id-RNAs may play a role in the maintenance of chronic HBV infection and therefore contribute to the HBV life cycle. Furthermore, the production of HBsAg from id-RNAs independently of viral replication may explain at least in part why treatment with interferon or nucleos(t)ides in most cases fails to achieve a loss of serum HBsAg.

ACS Style

Natalia Freitas; Tetyana Lukash; Sumedha Gunewardena; Benjamin Chappell; Betty L. Slagle; Severin O. Gudima. Relative Abundance of Integrant-Derived Viral RNAs in Infected Tissues Harvested from Chronic Hepatitis B Virus Carriers. Journal of Virology 2018, 92, 1 .

AMA Style

Natalia Freitas, Tetyana Lukash, Sumedha Gunewardena, Benjamin Chappell, Betty L. Slagle, Severin O. Gudima. Relative Abundance of Integrant-Derived Viral RNAs in Infected Tissues Harvested from Chronic Hepatitis B Virus Carriers. Journal of Virology. 2018; 92 (10):1.

Chicago/Turabian Style

Natalia Freitas; Tetyana Lukash; Sumedha Gunewardena; Benjamin Chappell; Betty L. Slagle; Severin O. Gudima. 2018. "Relative Abundance of Integrant-Derived Viral RNAs in Infected Tissues Harvested from Chronic Hepatitis B Virus Carriers." Journal of Virology 92, no. 10: 1.

Journal article
Published: 10 June 2015 in Journal of Virology
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The infectivity of hepadnavirus virions produced during either acute or chronic stages of infection was compared by testing the ability of the virions of woodchuck hepatitis virus (WHV) to induce productive acute infection in naive adult woodchucks. Serum WHV collected during acute infection was compared to virions harvested from WHV-infected woodchucks during either (i) early chronic infection, when WHV-induced hepatocellular carcinoma (HCC) was not yet developed, or (ii) late chronic infection, when established HCC was terminal. All tested types of WHV inoculum were related, because they were collected from woodchucks that originally were infected with standardized WHV7 inoculum. Despite the individual differences between animals, the kinetics of accumulation of serum relaxed circular DNA of WHV demonstrated that the virions produced during early or late chronic infection are fully capable of inducing productive acute infection with long-lasting high viremia. These findings were further supported by the analysis of such intrahepatic markers of WHV infection as replicative intermediate DNA, covalently closed circular DNA, pregenomic RNA, and the percentage of WHV core antigen-positive hepatocytes measured at several time points over the course of 17.5 weeks after the inoculation. In addition, the observed relationship between the production of antibodies against WHV surface antigens and parameters of WHV infection appears to be complex. Taken together, the generated data suggest thatin vivohepadnavirus virions produced during different phases of chronic infection did not demonstrate any considerable deficiencies in infectivity compared to that of virions generated during the acute phase of infection.IMPORTANCEThe generated data suggest that infectivity of virions produced during the early or late stages of chronic hepadnavirus infection is not compromised. Our novel results provided several lines of further evidence supporting the idea that during the state of chronic infectionin vivo, the limitations of hepadnavirus cell-to-cell spread/superinfection (observed recently in the woodchuck model) are not due to the diminished infectivity of the virions circulating in the blood and likely are (i) related to the properties of hepatocytes (i.e., their capacity to support hepadnavirus infection/replication) and (ii) influenced by the immune system. The obtained results further extend the understanding of the mechanisms regulating the persistence of hepadnavirus infection. Follow-up studies that will further investigate hepadnavirus cell-to-cell spread as a potential regulator of the chronic state of the infection are warranted.

ACS Style

Natalia Maria Bezerra de Freitas; Tetyana Lukash; Louise Rodrigues; Sam Litwin; Bhaskar V. Kallakury; Stephan Menne; Severin O. Gudima. Infection Patterns Induced in Naive Adult Woodchucks by Virions of Woodchuck Hepatitis Virus Collected during either the Acute or Chronic Phase of Infection. Journal of Virology 2015, 89, 8749 -8763.

AMA Style

Natalia Maria Bezerra de Freitas, Tetyana Lukash, Louise Rodrigues, Sam Litwin, Bhaskar V. Kallakury, Stephan Menne, Severin O. Gudima. Infection Patterns Induced in Naive Adult Woodchucks by Virions of Woodchuck Hepatitis Virus Collected during either the Acute or Chronic Phase of Infection. Journal of Virology. 2015; 89 (17):8749-8763.

Chicago/Turabian Style

Natalia Maria Bezerra de Freitas; Tetyana Lukash; Louise Rodrigues; Sam Litwin; Bhaskar V. Kallakury; Stephan Menne; Severin O. Gudima. 2015. "Infection Patterns Induced in Naive Adult Woodchucks by Virions of Woodchuck Hepatitis Virus Collected during either the Acute or Chronic Phase of Infection." Journal of Virology 89, no. 17: 8749-8763.

Journal article
Published: 12 May 2015 in Virus Research
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Woodchuck hepatitis virus (WHV) is often used as surrogate to study mechanism of HBV infection. Currently, most infections are conducted using strains WHV7 or WHV8 that have very high sequence identity. This study focused on natural strain WHVNY that is more genetically distant from WHV7. Three naive adult woodchucks inoculated with WHVNY developed productive acute infection with long lasting viremia. However, only one of two woodchucks infected with WHV7 at the same multiplicity demonstrated productive liver infection. Quantification of intracellular WHV RNA and DNA replication intermediates; percentages of core antigen-positive hepatocytes; and serum relaxed circular DNA showed that strains WHVNY and WHV7 displayed comparable replication levels and capacities to induce acute infection in naive adult woodchucks. Strain WHVNY was therefore validated as valuable reagent to analyze the mechanism of hepadnavirus infection, especially in co- and super-infection settings, which required discrimination between two related virus genomes replicating in the same liver.

ACS Style

Natalia Freitas; Tetyana Lukash; Megan Dudek; Sam Litwin; Stephan Menne; Severin O. Gudima. Capacity of a natural strain of woodchuck hepatitis virus, WHVNY, to induce acute infection in naive adult woodchucks. Virus Research 2015, 205, 12 -21.

AMA Style

Natalia Freitas, Tetyana Lukash, Megan Dudek, Sam Litwin, Stephan Menne, Severin O. Gudima. Capacity of a natural strain of woodchuck hepatitis virus, WHVNY, to induce acute infection in naive adult woodchucks. Virus Research. 2015; 205 ():12-21.

Chicago/Turabian Style

Natalia Freitas; Tetyana Lukash; Megan Dudek; Sam Litwin; Stephan Menne; Severin O. Gudima. 2015. "Capacity of a natural strain of woodchuck hepatitis virus, WHVNY, to induce acute infection in naive adult woodchucks." Virus Research 205, no. : 12-21.

Journal article
Published: 15 October 2014 in Journal of Virology
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The determinants of the maintenance of chronic hepadnaviral infection are yet to be fully understood. A long-standing unresolved argument in the hepatitis B virus (HBV) research field suggests that during chronic hepadnaviral infection, cell-to-cell spread of hepadnavirus is at least very inefficient (if it occurs at all), virus superinfection is an unlikely event, and chronic hepadnavirus infection can be maintained exclusively via division of infected hepatocytes in the absence of virus spread. Superinfection exclusion was previously shown for duck HBV, but it was not demonstrated for HBV or HBV-related woodchuck hepatitis virus (WHV). Three woodchucks, which were chronically infected with the strain WHV7 and already developed WHV-induced hepatocellular carcinomas (HCCs), were superinfected with another WHV strain, WHVNY. Six weeks after the superinfection, the woodchucks were sacrificed and tissues of the livers and HCCs were examined. The WHVNY superinfection was demonstrated by using WHV strain-specific PCR assays and (i) finding WHVNY relaxed circular DNA in the serum samples collected from all superinfected animals during weeks one through six after the superinfection, (ii) detecting replication-derived WHVNY RNA in the tissue samples of the livers and HCCs collected from three superinfected woodchucks, and (iii) finding WHVNY DNA replication intermediates in tissues harvested after the superinfection. The results are consistent with the occurrence of continuous but inefficient hepadnavirus cell-to-cell spread and superinfection during chronic infection and suggest that the replication space occupied by the superinfecting hepadnavirus in chronically infected livers is limited. The findings are discussed in the context of the mechanism of chronic hepadnavirus infection.IMPORTANCEThis study aimed to better understand the determinants of the maintenance of chronic hepadnavirus infection. The generated data suggest that in the livers chronically infected with woodchuck hepatitis virus, (i) hepadnavirus superinfection and cell-to-cell spread likely continue to occur and (ii) the virus spread is apparently inefficient, which is consistent with the interpretation that a limited number of cells in the livers facilitates the spread of hepadnavirus. The limitations of the cell-to-cell virus spread most likely are mediated at the level of the cells and do not reflect the properties of the virus. Our results further advance the understanding of the mechanism of chronic hepadnavirus infection. The significance of the continuous but limited hepadnavirus spread and superinfection for the maintenance of the chronic state of infection should be further evaluated in follow-up studies in order to determine whether blocking the virus spread would facilitate the suppression of chronic hepadnavirus infection.

ACS Style

Louise Rodrigues; Natalia Maria Bezerra de Freitas; Bhaskar V. Kallakury; Stephan Menne; Severin O. Gudima. Superinfection with Woodchuck Hepatitis Virus Strain WHVNY of Livers Chronically Infected with Strain WHV7. Journal of Virology 2014, 89, 384 -405.

AMA Style

Louise Rodrigues, Natalia Maria Bezerra de Freitas, Bhaskar V. Kallakury, Stephan Menne, Severin O. Gudima. Superinfection with Woodchuck Hepatitis Virus Strain WHVNY of Livers Chronically Infected with Strain WHV7. Journal of Virology. 2014; 89 (1):384-405.

Chicago/Turabian Style

Louise Rodrigues; Natalia Maria Bezerra de Freitas; Bhaskar V. Kallakury; Stephan Menne; Severin O. Gudima. 2014. "Superinfection with Woodchuck Hepatitis Virus Strain WHVNY of Livers Chronically Infected with Strain WHV7." Journal of Virology 89, no. 1: 384-405.

Journal article
Published: 19 March 2014 in Journal of Virology
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This study examined how the envelope proteins of 25 variants of hepatitis B virus (HBV) genotypes A to I support hepatitis delta virus (HDV) infectivity. The assembled virions bore the same HDV ribonucleoprotein and differed only by the HBV variant-specific envelope proteins coating the particles. The total HDV yields varied within a 122-fold range. A residue Y (position 374) in the HDV binding site was identified as critical for HDV assembly. Virions that bound antibodies, which recognize the region that includes the HBV matrix domain and predominantly but not exclusively immunoprecipitate the PreS1-containing virions, were termed PreS1*-HDVs. Using in vitro infection of primary human hepatocytes (PHH), we measured the specific infectivity (SI), which is the number of HDV genomes/cell produced by infection and normalized by the PreS1*-MOI, which is the multiplicity of infection that reflects the number of PreS1*-HDVs per cell in the inoculum used. The SI values varied within a 160-fold range and indicated a probable HBV genotype-specific trend of D > B > E > A in supporting HDV infectivity. Three variants, of genotypes B, C, and D, supported the highest SI values. We also determined the normalized index (NI) of infected PHH, which is the percentage of HDV-infected hepatocytes normalized by the PreS1*-MOI. Comparison of the SI and NI values revealed that, while a particular HBV variant may facilitate the infection of a relatively significant fraction of PHH, it may not always result in a considerable number of genomes that initiated replication after entry. The potential implications of these findings are discussed in the context of the mechanism of attachment/entry of HBV and HDV.

ACS Style

Natalia Maria Bezerra de Freitas; Kenji Abe; Celso Cunha; Stephan Menne; Severin O. Gudima. Support of the Infectivity of Hepatitis Delta Virus Particles by the Envelope Proteins of Different Genotypes of Hepatitis B Virus. Journal of Virology 2014, 88, 6255 -6267.

AMA Style

Natalia Maria Bezerra de Freitas, Kenji Abe, Celso Cunha, Stephan Menne, Severin O. Gudima. Support of the Infectivity of Hepatitis Delta Virus Particles by the Envelope Proteins of Different Genotypes of Hepatitis B Virus. Journal of Virology. 2014; 88 (11):6255-6267.

Chicago/Turabian Style

Natalia Maria Bezerra de Freitas; Kenji Abe; Celso Cunha; Stephan Menne; Severin O. Gudima. 2014. "Support of the Infectivity of Hepatitis Delta Virus Particles by the Envelope Proteins of Different Genotypes of Hepatitis B Virus." Journal of Virology 88, no. 11: 6255-6267.

Journal article
Published: 12 March 2014 in Journal of Virology
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A natural subviral agent of human hepatitis B virus (HBV), hepatitis delta virus (HDV), requires only the envelope proteins from HBV in order to maintain persistent infection. HBV surface antigens (HBsAgs) can be produced either by HBV replication or from integrated HBV DNA regardless of replication. The functional properties of the integrant-generated HBsAgs were examined using two human hepatocellular carcinoma-derived cell lines, Hep3B and PLC/PRF/5, that contain HBV integrants but do not produce HBV virions and have no signs of HBV replication. Both cell lines were able to support HDV replication and assembly/egress of HDV virions. Neither of the cell lines was able to produce substantial amounts of the pre-S1-containing HDV particles. HDV virions assembled in PLC/PRF/5 cells were able to infect primary human hepatocytes, while Hep3B-derived HDV appeared to be noninfectious. These results correlate with the findings that the entire open reading frame (ORF) for the large (L) envelope protein that is essential for infectivity is present on HBV RNAs from PLC/PRF/5 cells, while an L protein ORF that was truncated and fused to inverted precore sequences was found using RNAs from Hep3B cells. This study demonstrates for the first time that at least some of the HBV DNA sequence naturally integrated during infection can produce functional small and large envelope proteins capable of the formation of infectious HDV virions. Our data indicate that in vivo chronic HDV infection can persist in the absence of HBV replication (or when HBV replication is profoundly suppressed) if functional envelope proteins are supplied from HBV integrants. The study addresses the unique mechanism of HDV persistence in the absence of ongoing HBV replication, advances our understanding of HDV-HBV interactions, and supports the implementation of treatments directly targeting HDV for HDV/HBV-infected individuals.

ACS Style

Natalia Maria Bezerra de Freitas; Celso Cunha; Stephan Menne; Severin O. Gudima. Envelope Proteins Derived from Naturally Integrated Hepatitis B Virus DNA Support Assembly and Release of Infectious Hepatitis Delta Virus Particles. Journal of Virology 2014, 88, 5742 -5754.

AMA Style

Natalia Maria Bezerra de Freitas, Celso Cunha, Stephan Menne, Severin O. Gudima. Envelope Proteins Derived from Naturally Integrated Hepatitis B Virus DNA Support Assembly and Release of Infectious Hepatitis Delta Virus Particles. Journal of Virology. 2014; 88 (10):5742-5754.

Chicago/Turabian Style

Natalia Maria Bezerra de Freitas; Celso Cunha; Stephan Menne; Severin O. Gudima. 2014. "Envelope Proteins Derived from Naturally Integrated Hepatitis B Virus DNA Support Assembly and Release of Infectious Hepatitis Delta Virus Particles." Journal of Virology 88, no. 10: 5742-5754.

Journal article
Published: 01 January 2013 in World Journal of Virology
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To search for the presence of cis elements in hepatitis D virus (HDV) genomic and antigenomic RNA capable of promoting nuclear export. We made use of a well characterized chloramphenicol acetyl-transferase reporter system based on plasmid pDM138. Twenty cDNA fragments corresponding to different HDV genomic and antigenomic RNA sequences were inserted in plasmid pDM138, and used in transfection experiments in Huh7 cells. The relative amounts of HDV RNA in nuclear and cytoplasmic fractions were then determined by real-time polymerase chain reaction and Northern blotting. The secondary structure of the RNA sequences that displayed nuclear export ability was further predicted using a web interface. Finally, the sensitivity to leptomycin B was assessed in order to investigate possible cellular pathways involved in HDV RNA nuclear export. Analysis of genomic RNA sequences did not allow identifying an unequivocal nuclear export element. However, two regions were found to promote the export of reporter mRNAs with efficiency higher than the negative controls albeit lower than the positive control. These regions correspond to nucleotides 266-489 and 584-920, respectively. In addition, when analyzing antigenomic RNA sequences a nuclear export element was found in positions 214-417. Export mediated by the nuclear export element of HDV antigenomic RNA is sensitive to leptomycin B suggesting a possible role of CRM1 in this transport pathway. A cis-acting nuclear export element is present in nucleotides 214-417 of HDV antigenomic RNA.

ACS Style

Natalia Maria Bezerra de Freitas; Celso Cunha. Searching for nuclear export elements in hepatitis D virus RNA. World Journal of Virology 2013, 2, 123 -35.

AMA Style

Natalia Maria Bezerra de Freitas, Celso Cunha. Searching for nuclear export elements in hepatitis D virus RNA. World Journal of Virology. 2013; 2 (3):123-35.

Chicago/Turabian Style

Natalia Maria Bezerra de Freitas; Celso Cunha. 2013. "Searching for nuclear export elements in hepatitis D virus RNA." World Journal of Virology 2, no. 3: 123-35.

Viral hepatitis
Published: 15 February 2012 in Hepatology
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Hepatitis delta virus (HDV) is a natural subviral agent of human hepatitis B virus (HBV). HDV enhances liver damage during concomitant infection with HBV. The molecular pathogenesis of HDV infection remains poorly understood. To advance our understanding of the relationship between HDV infection and liver cancer, it was determined whether HDV could infect in vivo the cells of hepadnavirus‐induced hepatocellular carcinoma (HCC). Woodchucks (Marmota monax) that were chronically infected with HBV‐related woodchuck hepatitis virus (WHV) and already developed HCCs were used as an experimental model. The locations of HCCs within the livers were determined using ultrasound imaging followed by open surgery. One week after surgery the WHV carrier woodchucks were superinfected with WHV‐enveloped HDV (wHDV). Six weeks later the animals were sacrificed and HDV replication in normal liver tissues and in center masses of HCCs was evidenced by Northern analysis, real‐time polymerase chain reaction assay, and immunohistochemistry. Based on accumulation levels of HDV RNAs and numbers of infected cells, the efficiency of wHDV infection appears to be comparable in most HCCs and normal liver tissues. Conclusion: Cells of WHV‐induced HCCs are susceptible to HDV infection in vivo, and therefore express functional putative WHV receptors and support the steps of the attachment/entry governed by the hepadnavirus envelope proteins. Because others previously hypothesized that hepadnavirus‐induced HCCs are resistant to reinfection with a hepadnavirus in vivo, our data suggest that if such a resistance exists it likely occurs via a block at the post‐entry step. The demonstrated ability of HDV to infect already formed HCCs may facilitate development of novel strategies further dissecting the mechanism of liver pathogenesis associated with HDV infection. (HEPATOLOGY 2012;56:76–85)

ACS Style

Natalia Freitas; Jessica Salisse; Celso Cunha; Ilia Toshkov; Stephan Menne; Severin O. Gudima. Hepatitis delta virus infects the cells of hepadnavirus-induced hepatocellular carcinoma in woodchucks. Hepatology 2012, 56, 76 -85.

AMA Style

Natalia Freitas, Jessica Salisse, Celso Cunha, Ilia Toshkov, Stephan Menne, Severin O. Gudima. Hepatitis delta virus infects the cells of hepadnavirus-induced hepatocellular carcinoma in woodchucks. Hepatology. 2012; 56 (1):76-85.

Chicago/Turabian Style

Natalia Freitas; Jessica Salisse; Celso Cunha; Ilia Toshkov; Stephan Menne; Severin O. Gudima. 2012. "Hepatitis delta virus infects the cells of hepadnavirus-induced hepatocellular carcinoma in woodchucks." Hepatology 56, no. 1: 76-85.

Journal article
Published: 01 December 2009 in Current Genomics
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In eukaryotes, the nuclear membrane provides a physical barrier to the passive diffusion of macromolecules from and into the cytoplasm. Nucleocytoplasmic traffic occurs through highly specialized structures known as nuclear pores, and involves the participation of a special class of transport proteins. Active transport across the nuclear pores is an energy-dependent process that relies on the activity of Ran-GTPases both in the nuclear and cytoplasmic compartments.

ACS Style

Natalia Maria Bezerra de Freitas; Celso Cunha. Mechanisms and Signals for the Nuclear Import of Proteins. Current Genomics 2009, 10, 550 -557.

AMA Style

Natalia Maria Bezerra de Freitas, Celso Cunha. Mechanisms and Signals for the Nuclear Import of Proteins. Current Genomics. 2009; 10 (8):550-557.

Chicago/Turabian Style

Natalia Maria Bezerra de Freitas; Celso Cunha. 2009. "Mechanisms and Signals for the Nuclear Import of Proteins." Current Genomics 10, no. 8: 550-557.

Journal article
Published: 02 May 2009 in Journal of Proteomics
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Hepatitis delta virus (HDV) infects human hepatocytes already infected with the hepatitis B virus increasing about ten fold the risk of cirrhosis and fulminant hepatitis. The lack of an appropriate cell culture system capable of supporting virus replication has so far impaired the detailed investigation of the HDV biology including the identification of host factors involved in pathogenesis. Here, we made use of a HDV cDNA stably transfected cell line, Huh7-D12, in a proteomic approach to identify the changes in the protein expression profiles in human liver cells that arise as a consequence of HDV replication. Total protein extracts from Huh7-D12 cells and of the corresponding non transfected human liver carcinoma cell line, Huh7, were separated by 2-DE. Differentially expressed spots were identified by MALDI-TOF followed by database searching. We identified 23 differentially expressed proteins of which 15 were down regulated and 8 up regulated in Huh7-D12 cells. These proteins were found to be involved in different cellular pathways. The down regulation of the histone H1-binding protein and of triosephosphate isomerase was confirmed by Real time PCR, and the up regulation of the La protein and lamin A/C was validated by western blot.

ACS Style

Sérgio Mota; Marta Mendes; Natália Freitas; Deborah Penque; Ana V. Coelho; Celso Cunha. Proteome analysis of a human liver carcinoma cell line stably expressing hepatitis delta virus ribonucleoproteins. Journal of Proteomics 2009, 72, 616 -627.

AMA Style

Sérgio Mota, Marta Mendes, Natália Freitas, Deborah Penque, Ana V. Coelho, Celso Cunha. Proteome analysis of a human liver carcinoma cell line stably expressing hepatitis delta virus ribonucleoproteins. Journal of Proteomics. 2009; 72 (4):616-627.

Chicago/Turabian Style

Sérgio Mota; Marta Mendes; Natália Freitas; Deborah Penque; Ana V. Coelho; Celso Cunha. 2009. "Proteome analysis of a human liver carcinoma cell line stably expressing hepatitis delta virus ribonucleoproteins." Journal of Proteomics 72, no. 4: 616-627.

Journal article
Published: 01 January 2008 in Virology
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The delta antigen (HDAg) is the only protein encoded by the hepatitis delta virus (HDV) RNA genome. The HDAg contains an RNA binding domain, a dimerization domain, and a nuclear localization signal (NLS). The nuclear import of HDV RNPs is thought to be one of the first tasks of the HDAg during the HDV replication cycle. Using c-myc–PK fusions with several regions of the HDAg in transfection assays in Huh7 cells, we found that the HDAg NLS consists of a single stretch of 10 amino acids, EGAPPAKRAR, located in positions 66–75. Deletion and mutation analysis of this region showed that both the acidic glutamic acid residue at position 66 and the basic arginine residue at position 75 are essential for promoting nuclear import

ACS Style

Carolina Alves; Natalia Maria Bezerra de Freitas; Celso Cunha. Characterization of the nuclear localization signal of the hepatitis delta virus antigen. Virology 2008, 370, 12 -21.

AMA Style

Carolina Alves, Natalia Maria Bezerra de Freitas, Celso Cunha. Characterization of the nuclear localization signal of the hepatitis delta virus antigen. Virology. 2008; 370 (1):12-21.

Chicago/Turabian Style

Carolina Alves; Natalia Maria Bezerra de Freitas; Celso Cunha. 2008. "Characterization of the nuclear localization signal of the hepatitis delta virus antigen." Virology 370, no. 1: 12-21.