Molecular determinants of infectious pancreatic necrosis virus virulence and cell culture adaptation

Infectious pancreatic necrosis viruses (IPNVs) exhibit a wide range of virulence in salmonid species. In previous studies, we have shown that the amino acid residues at positions 217 and 221 in VP2 are implicated in virulence. To pinpoint the molecular determinants of virulence in IPNV, we generated recombinant IPNV strains using the cRNA-based reverse-genetics system. In two virulent strains, residues at positions 217 and 247 were replaced by the corresponding amino acids of a low-virulence strain. The growth characteristics of the recovered chimeric strains in cell culture were similar to the low-virulence strains, and these viruses induced significantly lower mortality in Atlantic salmon fry than the parent strains did in in vivo challenge studies. Furthermore, the virulent strain was serially passaged in CHSE-214 cells 10 times and was completely characterized by nucleotide sequencing. Deduced amino acid sequence analyses revealed a single amino acid substitution of Ala to Thr at position 221 in VP2 of this virus, which became highly attenuated and induced 15% cumulative mortality in Atlantic salmon fry, compared to 68% mortality induced by the virulent parent strain. The attenuated strain grows to higher titers in CHSE cells and can be distinguished antigenically from the wild-type virus by use of a monoclonal antibody. However, the virulent strain passaged 10 times in RTG-2 cells was stable, and it retained its antigenicity and virulence. Our results indicate that residues Thr at position 217 (Thr217) and Ala221 of VP2 are the major determinants of virulence in IPNV of the Sp serotype. Highly virulent isolates possess residues Thr217 and Ala221; moderate- to low-virulence strains have Pro217 and Ala221; and strains containing Thr221 are almost avirulent, irrespective of the residue at position 217.     

Ghrelin is dispensable for embryonic pancreatic islet development and differentiation

Ghrelin is a peptide hormone that has been implicated in the regulation of food intake and energy homeostasis. Ghrelin is predominantly produced in the stomach, but is also expressed in many other tissues where its functions are not well characterized. In the rodent and human pancreas, ghrelin levels peak at late gestation and gradually decline postnatally. Several studies have suggested that ghrelin regulates beta cell function during embryonic development and in the adult. In addition, in a number of mouse models, ghrelin cells appear to replace insulin- and glucagon-producing cells in the islet. In this analysis, we investigated whether the absence or overexpression of ghrelin influenced the development and differentiation of the pancreatic islet during embryonic development. These studies revealed that ghrelin is dispensable for normal pancreas development during gestation. Conversely, we demonstrated that elevated ghrelin in the Nkx2.2 null islets is not responsible for the absence of insulin- and glucagon-producing cells. Finally, we have also determined that in the absence of insulin, ghrelin cells form in their normal numbers and ghrelin is expressed at wild type levels.     

Immunogenicity of Lactobacillus-expressing VP2 and VP3 of the infectious pancreatic necrosis virus (IPNV) in rainbow trout

Infectious pancreatic necrosis virus (IPNV) infects salmonid fish with high mortality and causes serious economic losses to salmonid aquaculture. Lactobacillus strains have a number of properties that make them attractive candidates as delivery vehicles for the presentation to the mucosa of compounds with pharmaceutical interest, in particular vaccines. Here, Lactobacilli/Escherichia coli shuttle vector pPG1 (surface-displayed) or pPG2 (secretory) with the capsid VP2 gene inserted was transformed into Lactobacillus casei to yield two recombinant strains: Lc:PG1-VP2 and Lc:PG2-VP2, respectively. Rainbow trout immunized respectively with Lc:PG1-VP2, Lc:PG2-VP2, Lc:PG1-VP3 and Lc:PG2-VP3 elicited anti-IPNV immune responses (serum IgM) via oral route. Statistical results of serum IgM titer with neutralizing activity showed that immunogenicity of Lc:PG2-VP2 was more powerful than that of Lc:PG1-VP2 (P < 0.001), Lc:PG1-VP3 (P < 0.001) and Lc:PG2-VP3 (P < 0.001), which was confirmed by viral loads reduction analyzed by real-time RT-PCR in orally immunized rainbow trout after virus challenge. Comparing with negative control, rainbow trout orally dosed with Lc:PG2-VP2 resulted in ∼46-fold reduction in virus load on days 10 post viral challenge as well as Lc:PG1-VP2(∼20-fold), Lc:PG2-VP3(∼6-fold) and Lc:PG1-VP3(∼3-fold). Taken together, Lc:PG2-VP2 exhibited a more appropriate candidate as live bacteria vaccine against IPNV infection in rainbow trout.     

Mint3 is dispensable for pancreatic and kidney functions in mice

Munc-18 interacting protein 3 (Mint3) is an activator of hypoxia-inducible factor-1 in cancer cells, macrophages, and cancer-associated fibroblasts under pathological conditions. However, exactly which cells highly express Mint3 in vivo and whether Mint3 depletion affects their physiological functions remain unclear. Here, we surveyed mouse tissues for specific expression of Mint3 by comparing Mint3 expression in wild-type and Mint3-knockout mice. Interestingly, immunohistochemical analyses revealed that Mint3 was highly expressed in islet cells of the pancreas, distal tubular epithelia of the kidney, choroid plexus ependymal cells of the cerebrum, medullary cells of the adrenal gland, and epithelial cells of the seminal gland. We also studied whether Mint3 depletion affects the physiological functions of the islets and kidneys. Mint3-knockout mice did not show any abnormalities in glucose-tolerance and urine-biochemical tests, indicating that Mint3 depletion was compensated for in these organs. Thus, loss of Mint3 might be compensated in the islets and kidneys under physiological conditions in mice.     

VP3, a structural protein of infectious pancreatic necrosis virus, interacts with RNA-dependent RNA polymerase VP1 and with double-stranded RNA

Infectious pancreatic necrosis virus (IPNV) is a bisegmented, double-stranded RNA (dsRNA) virus of the Birnaviridae family that causes widespread disease in salmonids. Its two genomic segments are encapsulated together with the viral RNA-dependent RNA polymerase, VP1, and the assumed internal protein, VP3, in a single-shell capsid composed of VP2. Major aspects of the molecular biology of IPNV, such as particle assembly and interference with host macromolecules, are as yet poorly understood. To understand the infection process, analysis of viral protein interactions is of crucial importance. In this study, we focus on the interaction properties of VP3, the suggested key organizer of particle assembly in birnaviruses. By applying the yeast two-hybrid system in combination with coimmunoprecipitation, VP3 was proven to bind to VP1 and to self-associate strongly. In addition, VP3 was shown to specifically bind to dsRNA in a sequence-independent manner by in vitro pull-down experiments. The binding between VP3 and VP1 was not dependent on the presence of dsRNA. Deletion analyses mapped the VP3 self-interaction domain within the 101 N-terminal amino acids and the VP1 interaction domain within the 62 C-terminal amino acids of VP3. The C-terminal end was also crucial but not sufficient for the dsRNA binding capacity of VP3. For VP1, the 90 C-terminal amino acids constituted the only dispensable part for maintaining VP3-binding ability. Kinetic analysis revealed the presence of VP1-VP3 complexes prior to the formation of mature virions in IPNV-infected CHSE-214 cells, which indicates a role in promoting the assembly process.     

Kaposi's sarcoma-associated herpesvirus glycoprotein K8.1 is dispensable for virus entry

Kaposi's sarcoma-associated herpesvirus (KSHV) is considered the etiologic agent of Kaposi's sarcoma and several lymphoproliferative disorders. Recently, the KSHV genome was cloned into a bacterial artificial chromosome and used to construct a recombinant KSHV carrying a deletion of the viral interferon regulatory factor gene (F. C. Zhou, Y. J. Zhang, J. H. Deng, X. P. Wang, H. Y. Pan, E. Hettler, and S. J. Gao, J. Virol. 76:6185-6196, 2002). The K8.1 glycoprotein is a structural component of the KSHV particle and is thought to facilitate virus entry by binding to heparan sulfate moieties on cell surfaces. To further address the role of K8.1 in virus infectivity, a K8.1-null recombinant virus (BAC36DeltaK8.1) was constructed by deletion of most of the K8.1 open reading frame and insertion of a kanamycin resistance gene cassette within the K8.1 gene. Southern blotting and diagnostic PCR confirmed the presence of the engineered K8.1 gene deletion. Transfection of the wild-type genome (BAC36) and mutant genome (BAC36DeltaK8.1) DNAs into 293 cells in the presence or absence of the complementing plasmid (pCDNAK8.1A), transiently expressing the K8.1A gene, produced infectious virions in the supernatants of transfected cells. These results demonstrated that the K8.1 glycoprotein is not required for KSHV entry into 293 cells.     

Crystal structure of the VP4 protease from infectious pancreatic necrosis virus reveals the acyl-enzyme complex for an intermolecular self-cleavage reaction

Infectious pancreatic necrosis virus (IPNV), an aquatic birnavirus that infects salmonid fish, encodes a large polyprotein (NH(2)-pVP2-VP4-VP3-COOH) that is processed through the proteolytic activity of its own protease, VP4, to release the proteins pVP2 and VP3. pVP2 is further processed to give rise to the capsid protein VP2 and three peptides that are incorporated into the virion. Reported here are two crystal structures of the IPNV VP4 protease solved from two different crystal symmetries. The electron density at the active site in the triclinic crystal form, refined to 2.2-A resolution, reveals the acyl-enzyme complex formed with an internal VP4 cleavage site. The complex was generated using a truncated enzyme in which the general base lysine was substituted. Inside the complex, the nucleophilic Ser(633)Ogamma forms an ester bond with the main-chain carbonyl of the C-terminal residue, Ala(716), of a neighboring VP4. The structure of this substrate-VP4 complex allows us to identify the S1, S3, S5, and S6 substrate binding pockets as well as other substrate-VP4 interactions and therefore provides structural insights into the substrate specificity of this enzyme. The structure from the hexagonal crystal form, refined to 2.3-A resolution, reveals the free-binding site of the protease. Three-dimensional alignment with the VP4 of blotched snakehead virus, another birnavirus, shows that the overall structure of VP4 is conserved despite a low level of sequence identity ( approximately 19%). The structure determinations of IPNV VP4, the first of an acyl-enzyme complex for a Ser/Lys dyad protease, provide insights into the catalytic mechanism and substrate recognition of this type of protease.     

Capn5 is expressed in a subset of T cells and is dispensable for development

The Capn5 gene was inactivated by homologous recombination in ES cells that subsequently colonized the germ line of mice. The targeted mutation integrated a lacZ expression cassette into the Capn5 gene, allowing the expression of Capn5 mRNA to be examined in detail in heterozygous animals. Expression was observed in embryonic and newborn thymuses, in various epithelial tissues, and in tissues of the central nervous system. In the thymus, Capn5 was expressed mainly in relatively immature CD25(+) embryonic thymocytes. Despite the numerous expression sites of Capn5, the majority of Capn5-null mice were viable and fertile and appeared healthy. Histopathological analysis did not reveal any differences between Capn5-null and wild-type mice. There were no defects in the major T- or B-cell populations in the thymus, spleen, bone marrow, or peritoneum, nor did apoptosis appear abnormal in Capn5-null T cells. There was no evidence for the development of autoimmune disease in Capn5-null animals. However, a small proportion of homozygous null offspring from heterozygous matings were runted and most often did not survive to adulthood.     

The type B leukemogenic virus truncated superantigen is dispensable for T-cell lymphomagenesis

Type B leukemogenic virus (TBLV) is a variant of mouse mammary tumor virus (MMTV) that causes T-cell lymphomas in mice. We have constructed a TBLV-MMTV hybrid, pHYB-TBLV, in which 756 bp of the C3H MMTV long terminal repeat (LTR) was replaced with 438 bp of the TBLV LTR. Intraperitoneal injection of pHYB-TBLV transfectants consistently resulted in T-cell lymphomas in 50% of injected weanling BALB/c mice with an average latency period of 5.7 (+/- 1.5) months. Transfectants of pHYB-TBLV containing a double-frameshift mutation in the truncated superantigen gene (sag) induced T-cell lymphomas with similar incidences, latency periods, and phenotypes, suggesting that cis-acting elements in the TBLV LTR determine disease specificity.     

VP5 of infectious bursal disease virus is not essential for viral replication in cell culture.

Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, encodes in its bisegmented double-stranded RNA genome four structural virion proteins, VP1, VP2, VP3, and VP4, as well as a nonstructural protein, VP5. Recently, the establishment of an infectious cRNA system for IBDV has been described (E. Mundt and V. N. Vakharia, Proc. Natl. Acad. Sci. USA 93:11131-11136, 1996). Here, we report the isolation of a VP5- IBDV mutant constructed by site-directed mutagenesis of the methionine start codon of VP5, followed by cRNA transfection. The resulting virus mutant was replication competent in cell culture, which indicates that VP5 is not required for productive replication of IBDV. Absence of VP5 expression was verified by lack of reactivity with newly established anti-VP5 monoclonal antibodies and polyclonal sera. VP5- IBDV exhibited a delay in replication in chicken embryo cells compared to the VP5+ parental virus. However, final yields were similar. Our results thus show that VP5 is nonessential for IBDV replication, which makes it a prime candidate for the construction of deleted, marked vaccines.     

Regulatory networks for virulence and persistence of Bacillus anthracis

Bacillus anthracis, the etiological agent of anthrax, is a Gram-positive sporulating bacterium. Its life-cycle can be divided schematically into two phases: multiplication in the mammalian host and persistence in the soil. A central regulator AtxA interferes with expression of more than 70 genes in vitro and an undefined number ex vivo. The exact molecular mechanism of action of AtxA is unknown, but the involvement of cascades of relay regulators has been described. Other regulators have also been implicated in the regulatory networks; these are mainly transition state regulators, which have been studied in other Bacillus species. They contribute to the regulation of expression of virulence- and persistence-factor genes, and to the regulation of atxA itself.     

Biophysical studies of infectious pancreatic necrosis virus.

The molecular weight of infectious pancreatic necrosis virus (IPNV) has been determined by analytical ultracentrifugation and dynamic light scattering. The sedimentation coefficient of the virus was found to be 435S. The average value for molecular weight is (55 +/- 7) x 106. The virus genome consists of two segments of double-stranded RNA (molecular weights, 2.5 x 106 and 2.3 x 106), which represents 8.7% of the virion mass. The capsid protein moiety of IPNV consists of four species of polypeptides, as determined by polyacrylamide gel electrophoresis. The number of molecules of each polypeptide in the virion has been determined. There are 22 molecules of the internal polypeptide alpha (molecular weight, 90,000), 544 molecules of the outer capsid polypeptide beta (molecular weight, 57,000), and 550 and 122 molecules, respectively, of the internal polypeptides gamma1 (molecular weight, 29,000) and gamma2 (molecular weight, 27,000). IPNV top component contains only the beta polypeptide species, and its molecular weight is estimated to be 31 x 106. The hydrodynamic diameter and electron microscopic diameter (calculated by catalase crystal-calibrated electron microscopy) of IPNV was compared with those of reovirus and encephalomyocarditis virus. Due to the swelling of the outer capsid, reovirus particles were found to be much larger when hydrated (96-nm diameter) than when dehydrated (76-nm diameter), having a large water content content and low average density. In contrast, IPNV particles are more rigid, having nearly the same average diameter under hydrous (64 nm) as under anhydrous conditions (59.3 nm). Encephalomyocarditis virus has a very low water content and does not shrink at all when prepared for electron microscopy.