Hepatitis B virus (HBV) is hyperendemic to southern Africa, the Asia-Pacific region, and the Amazon Basin. HBV causes both acute, and chronic infection of the liver that result in a wide spectrum of liver diseases ranging from acute mild subclinical infection, and an asymptomatic carrier state (ASC) to severe clinical manifestations, including, severe acute and, chronic hepatitis, which can progress to cirrhosis, and the development of hepatocellular carcinoma (HCC). Several viral factors have been implicated in the activation, and inhibition of apoptosis. The development, and progression of this wide spectrum of liver diseases are associated with an unregulated increase or decrease in hepatocyte apoptosis as well as a loss of balance between cell proliferation, and apoptosis. In southern Africa, genotype A of HBV is the predominant genotype, with subgenotype A1 prevailing. Individuals infected with subgenotype A1 have several unique characteristics, including relatively lower levels of HBV DNA, and early seroconversion of hepatitis B e antigen (HBeAg) to antibodies against HBeAg during the course of HBV infection. Infection with this subgenotype is associated with rapid disease progression, and high frequency of HCC development. Moreover, patients infected with subgenotype A1 had increased levels of apoptosis when compared to the other subgenotypes. The G1862T mutation occurs most frequently in subgenotype A1. In the clinical setting, South African HCC patients infected with G1862T subgenotype A1 strains had higher levels of apoptosis while ASCs patients infected with G1862T subgenotype A1 strains had lower levels of apoptosis, when compared to those infected with wild-type. To date, G1862T has been functionally characterized in subgenotype A2, genotype B and in a genotype D HBV genome containing a subgenotype A1 precore (PreC) region. The objectives of our study were to construct 1.28 mer replication competent clones containing an endogenous HBV promoter for wild-type subgenotypes A1, A2, and D3 as well as mutant G1862T subgenotype A1, and to functionally characterize these strains in tissue culture. Transfection of Huh7 cells was used to follow the viral replication, expression of HBeAg, activation of the unfolded protein response (UPR), and subsequent apoptosis. The strategy used for the generation of these replication competent clones had several advantages. Very few PCR errors were introduced, and carry-over of enzymes, nonspecific products, and reaction reagents downstream was prevented. The clones contained endogenous HBV promoters, and enhancers, and were generated from a single complete genome of HBV. These replication competent clones may be used in future studies for the establishment of stable cell lines that constitutively express HBV proteins without the need for further manipulation. This strategy can be used for the generation of replication competent clones belonging to genotypes A to D, and G, and with a few minor modifications, for genotypes E, F, and H. Using the newly generated clones, their replication competence was demonstrated using transfection of Huh7 cells. Even in the absence of an exogenous promoter, these clones were able to support the expression of intracellular, and extracellular HBV DNA at levels of 108 to 109 genome copies/ml. HBcAg, HBeAg, and HBsAg were expressed for a period of five days, and the order of expression was similar to that seen during acute HBV infection. Comparison of transfection with a replication competent clone containing an exogenous HBV promoter demonstrated higher expression of HBV DNA, and proteins, as well as an earlier expression, and accumulation of HBeAg in the endoplasmic reticulum (ER) relative to the clone containing an endogenous HBV promoter. This initial increased accumulation of HBeAg in the ER did not affect the level of activation of the UPR, but led to an increased level of total cell death as a consequence of necrosis. When comparing the different subgenotypes following transfection into Huh7 cells, subgenotype D3 replicated at a lower level, as measured by HBsAg, and HBV DNA levels, with HBeAg passing through the secretory pathway earlier, when compared to cells transfected with genotype A. There was no difference in the intracellular, and extracellular HBsAg between cells transfected with either subgenotype A1 or A2. However, cells transfected with subgenotype A1 had higher levels of intracellular replicative intermediates, HBeAg, and HBcAg, and lower extracellular expression of HBeAg from days 1 to 3, when compared to cells transfected with subgenotype A2. The intracellular retention of the PreC/ core (C) precursor protein in cells transfected with subgenotype A1 was clearly demonstrated by its lower expression in the secretory pathway, and its higher co-localization in the nucleus, using indirect immunofluorescence. This intracellular retention led to greater ER stress, and an earlier, and prolonged activation of the UPR. This correlated well with the higher PERK, ATF6, and IRE1/XBP1 activity seen on days 3 than on day 5. These findings suggest that the prolonged activation of the UPR in cells transfected with subgenotype A1 led to increased apoptosis, and subsequent induction of liver damage, and may therefore, be a contributing factor to the higher hepatocarcinogenic potential of subgenotype A1. Our study demonstrated that G1862T reduced replication, and led to the initial temporal retardation of intracellular core-particle-associated HBV DNA. Although, G1862T did not affect HBsAg expression, it led to a decreased expression of HBcAg, and HBeAg. The decreased expression of extracellular HBeAg was probably as a result of decreased cleavage efficiency by the signal peptide, which consequently led to the retardation of the PreC/C precursor protein in the ER, and ER-Golgi intermediate compartment (ERGIC), and its decreased expression in the nucleus. This retardation, and accumulation led to the earlier activation of all three UPR pathways, but not to increased apoptosis. Therefore, it is evident that G1862T does not completely abolish HBeAg expression, but affects the rate of HBeAg maturation, and its expression through the secretory pathway. These findings suggest that in response to the accumulation of HBeAg in the ER, the UPR was activated resulting in the alteration of the capacity to overcome this stress, consequently leading to a new homeostasis of the ER being reached. The capacity of the ER is increased, with no further activation of the UPR and apoptosis, which facilitates maturation of HBeAg. In conclusion, our study for the first time demonstrated that there are a number of factors that influence the expression of proteins in HBV transfection studies including the type of transcriptional promoter, the different genotypes/subgenotypes of HBV, the use of protein expressing as opposed to replication competent clones, and the presence, and absence of mutations, such as the G1862T. Therefore, when comparing the outcomes of various experiments these factors should be taken into consideration, and the results interpreted with caution, because experiments may not be strictly speaking comparable. Importantly, replication competent clones were generated from strains circulating in southern Africa. The generation of these clones is an important step in further functional characterization of African strains of HBV, and their comparison to strains circulating other geographical regions of the world. These strains, in particular, subgenotype A1 can develop unique mutations, such as the G1862T, which we demonstrated can influence the expression of HBeAg, in a way that it can possibly account for the higher hepatocarcinogenic potential of subgenotype A1.

Essen, Universiẗat, Diss., 2001.

Mouse Hepatitis virus (MHV) is a member of the coronavirus family in the order Nidovirales. The 32 kb genome contains cis-acting sequences necessary for replication of the viral genome. Those cis-acting sequences have been shown to bind host proteins, and binding of those proteins is necessary for virus replication. One of the cis-acting elements is the 3' terminal 42 nucleotide host protein binding element. Previous work has demonstrated that mitochondrial aconitase, mitochondrial heat shock protein 70, heat shock protein 60 and heat shock protein 40 bind to the 3' terminal 42 nucleotide host protein binding element. We demonstrated that RNA secondary structure of the 3' terminal 42 nucleotide host protein binding element is necessary for host protein binding in vitro. We also demonstrate that primary structure of the 3' terminal 42 nucleotide host protein binding element is necessary for viral replication by targeted recombination. DI replication assays infer that the 3' terminal 42 nucleotide host protein binding element plays a role in positive strand synthesis from the negative strand template. Current studies involve the infectious cDNA clone, which will provide definitive answers on the role of the 3' terminal 42 nucleotide host protein binding element in MHV replication.

Murine hepatitis virus type 3 (MHV-3), a member of the coronavirus family, induces a response that varies with the age and genetic background of the host mouse strain. A/J mice are fully resistant to the virus, while Balbc/J are fully susceptible and C3HebFe/J are semi-susceptible, making it possible to predictably reproduce the major human responses to hepatitis viruses. Although there has been considerable discussion of viral pathology in the literature, there has been much less emphasis on pathogenesis. In the experiments described here, histological, biophysical, and immunological techniques have been used to define the processes and cells involved. Transmission electron microscopic observations have confirmed that Kupffer and endothelial cells of hepatic sinusoids show clear changes by 12 hrs post-infection (p.i.), which are more advanced than hepatocellular changes. No replicating virus was seen in altered hepatocytes up to 3 days p.i. Scanning electron microscopy demonstrated that areas of necrosis are focal in nature and at 2-3 days p.i. consist of small spherical areas without flow. In vivo microcirculatory studies confirm the localized nature of the lesion and have shown that red cell velocity can be recorded in individual sinusoids . Velocities were found to vary from zero within a lesion to a normal velocity of 69±31 um/sec over a distance of not more than 3 sinusoids. In-vivo microcirculatory studies also revealed the ability of macrophages to move upstream (against flow) in the hepatic sinusoids. Using fluorescein labelled antibodies to cell surface markers (Thy-1, Lyt-2, and L3T4) it was shown that no T-cells of any subset were present in the areas of hepatocellular necrosis. Furthermore, treatment with cyclosporine A, which would be expected to decrease necrosis due to cell mediated cytotoxicity, did not significantly alter the course of the disease. The only cells which increased in number in the liver post infection were cells of the monocyte/macrophage lineage (Mac 1+), which had increased twofold at 12 hrs (p<.025) p.i. and to greater than twenty fold (p<.005) by 3 days p.i. Resistance in the A/J strain did not reflect an inability of the immunocompetent cells to present and respond to viral antigen. It was demonstrated that MHV-3 infected macrophages from resistant A/J mice are better able to stimulate proliferation of allogeneic and syngeneic lymphocytes than those from the sensitive Balb/cJ strain. In contrast, MHV-3 infection caused a significant enhancement of chemiluminescence from Balb/cJ macrophages, which did not occur in A/J animals. In vivo studies demonstrated a significant increase in free radical reaction products, including conjugated dienes (of long chain free fatty acids and aldehydes), thiobarbituric acid reactive substances, and lipid soluble fluorescent products between 12-72 hours p.i. with MHV-3 in the livers of susceptible Balb/cJ strain mice. All of these are products of oxidative cleavage of cellular and membrane polyunsaturated fatty acids, and result from the action of oxygen free radicals. Free radical inhibitors, or quenchers of free radical reaction products, were able to significantly reduce the liver necrosis in the susceptible mouse strain following infection. Radioimmune assays for antibody to MHV-3 have confirmed the presence of preformed antibodies to (or cross-reactive with) MHV-3 in the sera of both susceptible and resistant mice, pre and post-infection. Immunofluorescent labelled antibodies have also been used to demonstrate the presence of IgG deposits in the sinusoids of the liver both pre and post infection. This suggests the possibility that these mice have been infected with a non-virulent MHV strain prior to these experiments. From these studies, we conclude that the hepatic injury caused by MHV-3 infction in Balb/cJ mice is mediated predominantly by fixed and migratory cells of the mononuclear phagocytic series. Susceptibility and resistance are related to strain dependant differences in the response of macrophages (and Kupffer cells) to infection, and include the release of procoagulant activity (previously shown) and reactive oxygen radicals (and possibly other macrophage activation products such as PAF) that act together to induce hepatocellular necrosis. Preformed non-neutralizing antibody and an intact complement cascade may enhance viral uptake and activation of macrophages in the Balbc/J mice. Resistance to necrosis may be enhanced by a genetic deficiency of C5 in the A/J mice, preventing the formation of the membrane attack complex and hence complement dependant cell lysis, or macrophage activation. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

The non-structural protein 15 (NSP15) locus in Lineage A β-coronaviruses has two important functions during replication. The encoded endoribonuclease is conserved among coronaviruses. The function of the nsp15 protein is still not fully understood, but recent evidence suggests it may be involved in both replication and inhibiting viral sensing of double stranded RNA. In Lineage A β-coronaviruses, the RNA locus contains an inserted packaging signal (P/S). The P/S is essential for selectively packaging viral genomic RNA. While the P/S is required for selective packaging, it is not required for nsp15 protein function or viral replication. Utilizing this region, I studied the interactions of nsp15 protein during infection. Additionally, I studied the effect of selective packaging on virulence. Coronaviruses encode 16 nonstructural proteins in two open reading frames. These proteins are responsible for forming the replication/ transcription complex (RTC) and creating an environment conducive to viral replication. The RTC is an intricate complex of viral and host proteins with a largely unknown composition. While almost all nsps studied to date localize to sites of replication, the interactions between these proteins are not fully understood. In Chapter II, I describe studies of the interactions and localization of Nsp15 by creating an in situ hemagglutinin epitope tag. I found that mouse hepatitis virus nsp15 could tolerate an in situ tag when placed into the P/S (MHVNsp15-HA). MHVNsp15-HA had wild-type like replication in vitro. Nsp15 was localized to sites of replication throughout infection, with no localization to sites of assembly. Finally, nsp15 interacted with the RNA dependent RNA polymerase and putative primase during infection. These data demonstrate that nsp15 is a member of the RTC. During coronavirus replication two species of viral RNA are present, genomic RNA (gRNA) and sub-genomic RNA (sgRNA). These RNAs are co-terminal on both their 5’ and 3’ ends, containing the leader sequence and 3’ UTR/ polyA respectively. Even with these similarities, coronaviruses are adept at selectively packaging gRNA over sgRNA. This selective packaging is determined by the P/S, a 95 base pair stem-loop structure in the nsp15 locus. This RNA motif is sufficient for packaging of nonviral RNAs and has been shown to interact with the M protein from MHV. Moreover, when this RNA motif is deleted from MHV, (MHVPS-) selective packaging is lost during infection as sgRNAs become a large percentage of packaged viral RNA. In chapter IV I determined the effect of selective packaging on pathogenicity in vivo. Immunocompetent mice infected with MHVPS- had significantly better outcomes compared to MHV wild-type (MHVWT) infected mice. Peak viral loads were decreased in MHVPS- compared to MHVWT. Strikingly I found MHVPS- infected bone marrow derived macrophages had significant increases in type-I interferons (IFNs) and pathogenesis of MHVPS- was restored in mice deficient in IFN signaling. These data indicate that the P/S of MHV is an uncharacterized MHV virulence factor, which acts by preventing an increased IFN response during infection. In MHV, the nsp15 locus is translated into a functional protein and contains functional cis acting RNA elements both of which play a role in MHV replication. This work provides understanding of nsp15 localization and interactions which educate our understanding of the function of this conserved endoribonuclease. Additionally, this work demonstrates a unique function for the P/S not previously described. This work informs future studies of nsp15 protein function and the function of selective packaging during coronavirus infection.

Orientadores: Iscia Teresinha Lopes-Cendes, Rovilson Gilioli / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-07T01:47:49Z (GMT). No. of bitstreams: 1 Grippo_MariangelaCarnivalli_D.pdf: 1241429 bytes, checksum: 5f05623ad1e884a0014d2eca9109fb9a (MD5) Previous issue date: 2006 / Resumo: A interferência do RNA (RNAi) pode ser usada como uma ferramenta eficaz no silenciamento gênico específico mediado por moléculas de dupla fita de RNA (dsRNAs). Nesse contexto possui uma variedade de aplicações biológicas, incluindo o combate a patógenos infecciosos de importância biomédica. O objetivo do estudo foi determinar a eficiência e a especificidade da técnica de RNAi em eliminar o vírus da hepatite murina tipo 3 (MIN-3) in vitro. MHVs são vírus envelopados, cujo genoma é formado por uma cadeia de RNA fita simples (+) pertecentes a família Coronaviridae. Seu genoma codifica quatro proteínas estruturais: S (proteína da espícula); M (glicoproteína da transmembrana), N (proteína do nucleocapsídeo) e E (proteína associada à membrana) . Neste trabalho foi escolhido como alvo para o silenciamento gênico a proteína N, tendo sido produzidas moléculas de dsRNA complementares a sua seqüência genômica (GenBank AF 201929). Foram obtidas duas moléculas siRNAs transcritas por T7 RNA polimerase e uma terceira molécula interferente sintetizada comercialmente. Foi observado que os siRNAs produzidos pela transcrição in vitro, induziram uma resposta antiviral não específica. Além disso demonstrou-se que este efeito foi mediado através de substâncias secretadas no meio de cultura celular, provavelmente interferons (IFNs). Este efeito foi eficientemente eliminado após tratamento dos siRNAs com fosfatase alcalina. Observou-se também que a técnica de RNAi in vitro, tendo como alvo a proteína N de MHV-3, foi um tratamento eficaz e específico na infecção viral, confirmados através de estudos fenotípicos e moleculares. Desse modo, concluímos que experiências que utilizam RNAi contra alvos virais devem ser cuidadosamente monitoradas devido aos efeitos não específicos que podem ser induzidos por moléculas de dsRNA / Abstract: RNA Interference (RNAi) can be used as a powerful tool for post transcriptional gene-silencing mediated by double stranded RNA (dsRNAs) molecules. RNAi has a variety of biological applications including the combat against pathogens of biomedical importance. The objective of our study was to determine the efficiency and specificity of this new technique in eliminating mouse hepatitis virus type 3 (MIN-3) in vitro. MIN-3 is a subtype of enveloped viroses with a large plus-stranded RNA genome belonging to the Coronavirus family. Its genome codifies four structural proteins: S (spike protein); M (membrane protein); E (transmembrane glycoprotein); N (nucleocapsid protein). In the present study we target protein N by designing and producing dsRNA molecules complementary to its genomic sequence (GenBank AF 201929). We obtained three small interfering RNAs (siRNA) by in house T7 polymerase in vitro transcription and a fourth siRNA molecule that was commercially synthetized. We identified that siRNAs produced by in vitro transcription triggered a potent and sequence-unspecificied antiviral response. In addition, we demonstrated that this antiviral effect was mediated through molecules that were secreted in medium culture, probably interferons (IFNs). This unspecific effect was efficient1y suppressed when siRNAs were treated with aIkaline phosphatase prior to in vitro experiments. We also observed that RNAi targeting the N protein ofMIN-3 was a potent and specific treatment against in vitro infection, showing significant phenotypic protection and molecular evidence of specific gene-silencing. We concluded that experiments using RNAi against viral targets, although efficient, must be carefully controlled and monitored against possible sequence-unspecific effects triggered by dsRNA molecules / Doutorado / Genetica Animal e Evolução / Doutor em Genetica e Biologia Molecular

Mice infected with neurotropic variants of the murine coronavirus, mouse hepatitis virus, (strains JHMV or J2.2–V–1) develop acute and chronic CNS infections, and provide a model system to study the pathogenesis of virus–induced neuroinflammation, mechanisms of virus persistence, and anti–viral immune responses in the CNS. Using the J2.2–V–1 model of CNS infection, we addressed the role of sustained CCL2 production during viral infection using mice in which CCL2 was expressed transgenically in oligodendrocytes. Tonic CCL2 expression in the CNS resulted in delayed kinetics of virus clearance, and converted what is typically a mild, nonlethal disease to acutely lethal encephalitis, with the majority of mice succumbing to the infection. CCL2 induced a rapid and dysregulated inflammatory response that was no longer protective and was unable to efficiently clear virus from the CNS. Infected CCL2 Tg mice had increased numbers of Foxp3–expressing CD4 T cells (Tregs) and of macrophages and microglia expressing elevated levels of YM–1, a marker for alternatively activated macrophages, and nitric oxide. Our results showed that CCL2 has effects beyond serving as a chemoattractant for leukocytes, and has effects on the composition and function of inflammatory cells at sites of infection. In a separate set of experiments, I identified and characterized two additional heteroclitic variants of the JHMV epitope S598 that induced CD8 T cells with greater antigen sensitivity to the native S598 determinant relative to the cells primed by the native epitope. One of these heteroclitic epitopes elicited a T cell response with nearly complete cross–reactivity towards the native peptide. The structural data show that these heteroclitic epitopes induced modest conformational changes in the local environment of the peptide–MHCI complex. I also provide data to support the notion that heteroclitic determinants augment functional avidity by increasing surface epitope density. Collectively, these data will help guide the design of heteroclitic epitopes in the setting of vaccine development. Lastly, I examined the consequences of oxidative stress induced by viral infection on antigen presentation. The brains of JHMV–infected mice were found to have signs of oxidative stress, with significantly decreased ratios of reduced (GSH) to oxidized (GSSG) glutathione, suggesting that there is an environment that is conducive for cysteine modification with oxidized glutathione. We found that virus–induced oxidative stress resulted in the presentation of both native and S–glutathionylated forms of the JHMV epitope S510 by infected cells. A subset of the S510–specific CD8 T cells failed to recognize the modified form of the epitope, suggesting that GSH–modification of a cysteine–containing viral epitope might interfere with T cell recognition. Further, GSH-modified peptides were identified in stressed human cells, including herpes virus–transformed B cells, suggesting that the modification is not limited to mouse cells. Collectively these findings have implications for both anti–viral immunity and anti–tumor immunity, where oxidative stress has been shown to play a role during infection and tumorgenesis.

RNA sequences and higher-order structures in the 5’ and 3’ untranslated regions (UTRs) of positive-strand RNA viruses are known to function as cis-acting elements for translation, replication, and transcription. In coronaviruses, these are best characterized in the group 2a bovine coronavirus (BCoV) and mouse hepatitis virus (MHV), yet their precise mechanistic features are largely undefined. Here, we use a reverse genetics system in MHV to exploit the ~30% nt sequence divergence between BCoV and MHV to establish structure/function relationships of 5’ UTR cis-replication elements. It had been previously shown that a precise replacement of the 391-nt MHV 3’ UTR with the 288-nt BCoV 3’ UTR yields wt-like MHV. Our attempts to replace the 209-nt MHV 5’ UTR with the 210-nt BCoV 5’ UTR, however, yielded a non-viable chimera. Therefore, a systematic analysis of individual 5’-terminal structures was made to identify compatible elements. By placing each of four putative cis-acting domains from the BCoV 5’ UTR into the MHV genome, we learned that (i) stem-loops (SLs) I & II and SLIII are functionally compatible, (ii) SLIV is compatible if it spans parts of the 5’ UTR and the nonstructural protein 1 (nsp1) cistron, thus identifying this part of ORF 1 as a component of the cis-replication signal, (iii) a relatively unstructured 32-nt region mapping between SLIII and SLIV defines a novel virus species-specific cis-replication element, (iv) spontaneous suppressor mutations within MHV SLI and nsp1 cistron compensated for growth defects arising from the BCoV 32-nt element in the MHV genome, (v) cross talk between the 32-nt element, SLI, and the nsp1 cistron appears essential for virus replication, (vi) the BCoV 5’ UTR and nsp1 cistron function together in the MHV genome to generate a wt-like MHV phenotype, and (vii) a functional 5’ UTR-nsp1 domain in group 2a coronaviruses cannot be substituted by the corresponding genomic element from the group 2b SARS-CoV. We postulate that the interaction between the 5’ UTR and nsp1 cistron (or possibly nsp1 protein) functions as a molecular switch between genome translation and ignition of negative-strand RNA synthesis.

abstract: Coronaviruses are the causative agents of SARS, MERS and the ongoing COVID-19 pandemic. Coronavirus envelope proteins have received increasing attention as drug targets, due to their multiple functional roles during the infection cycle. The murine coronavirus mouse hepatitis virus strain A59, a hepatic and neuronal tropic coronavirus, is considered a prototype of the betacoronaviruses. The envelope protein of the mouse hepatitis virus (MHV-E) was extensively screened with various membrane mimetics by solution state nuclear magnetic resonance spectroscopy to find a suitable mimetic, which allowed for assignment of ~97% of the backbone atoms in the transmembrane region. Following resonance assignments, the binding site of the ion channel inhibitor hexamethylene amiloride (HMA) was mapped to MHV-E using chemical shift perturbations in both amide and aromatic transverse relaxation optimized spectroscopy (TROSY) spectra, which indicated the inhibitor binding site is located at the N-terminal opening of the channel, in accord with one of the proposed HMA binding sites in the envelope protein from the related SARS (severe acute respiratory syndrome) betacoronavirus. Structure calculation of residues M1-K38 of MHV-E, encompassing the transmembrane region, is currently in progress using dihedral angle restraints obtained from isotropic chemical shifts and distance restraints obtained from manually assigned NOE cross-peaks, with the ultimate aim of generating a model of the MHV-E viroporin bound to the inhibitor HMA. This work outlines the first NMR studies on MHV-E, which have provided a foundation for structure based drug design and probing interactions, and the methods can be extended, with suitable modifications, to other coronavirus envelope proteins. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2020