Syncytial Hepatitis of Tilapia ( Oreochromis niloticus L.) is Associated With Orthomyxovirus-Like Virions in Hepatocytes.

Using transmission electron microscopy (TEM), the presented work expands on the ultrastructural findings of an earlier report on "syncytial hepatitis," a novel disease of tilapia (SHT). Briefly, TEM confirmed the presence of an orthomyxovirus-like virus within the diseased hepatocytes but not within the endothelium. This was supported by observing extracellular and intracellular (mostly intraendosomal), 60-100 nm round virions with a trilaminar capsid containing up to 7 electron-dense aggregates. Other patterns noted included enveloped or filamentous virions and virion-containing cytoplasmic membrane folds, suggestive of endocytosis. Patterns atypical for orthymyxovirus included the formation of syncytia and the presence of virions within the perinuclear cisternae (suspected to be the Golgi apparatus). The ultrastructural morphology of SHT-associated virions is similar to that previously reported for tilapia lake virus (TiLV). A genetic homology was investigated using the available reverse transcriptase polymerase chain reaction (RT-PCR) probes for TiLV and comparing clinically sick with clinically normal fish and negative controls. By RT-PCR analysis, viral nucleic acid was detected only in diseased fish. Taken together, these findings strongly suggest that a virus is causally associated with SHT, that this virus shares ultrastructural features with orthomyxoviruses, and it presents with partial genetic homology with TiLV (190 nucleotides).

The Thr205 phosphorylation site within respiratory syncytial virus matrix (M) protein modulates M oligomerization and virus production.

UNLABELLED: Human respiratory syncytial virus (RSV) is the most common cause of bronchiolitis and pneumonia in infants and the elderly worldwide; however, there is no licensed RSV vaccine or effective drug treatment available. The RSV matrix (M) protein plays key roles in virus assembly and budding, but the protein interactions that govern budding of infectious virus are not known. In this study, we focus on M protein and identify a key phosphorylation site (Thr205) in M that is critical for RSV infectious virus production. Recombinant virus with a nonphosphorylatable alanine (Ala) residue at the site was markedly attenuated, whereas virus with a phosphomimetic aspartate (Asp) resulted in a nonviable virus which could only be recovered with an additional mutation in M (serine to asparagine at position 220), strongly implying that Thr205 is critical for viral infectivity. Experiments in vitro showed that mutation of Thr205 does not affect M stability or the ability to form dimers but implicate an effect on higher-order oligomer assembly. In transfected and infected cells, Asp substitution of Thr205 appeared to impair M oligomerization; typical filamentous structures still formed at the plasma membrane, but M assembly during the ensuing elongation process seemed to be impaired, resulting in shorter and more branched filaments as observed using electron microscopy (EM). Our data thus imply for the first time that M oligomerization, regulated by a negative charge at Thr205, may be critical to production of infectious RSV. IMPORTANCE: We show here for the first time that RSV M's role in virus assembly/release is strongly dependent on threonine 205 (Thr205), a consensus site for CK2, which appears to play a key regulatory role in modulating M oligomerization and association with virus filaments. Our analysis indicates that T205 mutations do not impair M dimerization or viruslike filament formation per se but rather the ability of M to assemble in ordered fashion on the viral filaments themselves. This appears to impact in turn upon the infectivity of released virus rather than on virus production or release itself. Thus, M oligomerization would appear to be a target of interest for the development of anti-RSV agents; further, the recombinant T205-substituted mutant viruses described here would appear to be the first RSV mutants affected in viral maturation to our knowledge and hence of considerable interest for vaccine approaches in the future.

Mouse major satellite DNA is prone to eccDNA formation via DNA Ligase IV-dependent pathway.

Elevated levels of extrachromosomal circular DNA (eccDNA or spcDNA) are closely associated with genomic instability and aging. Despite extensive studies, the mechanism of its generation in mammalian cells is unknown. We report here that mouse major satellite DNA (MSD) is prone to eccDNA formation and that the resulting molecules are multimeres of the basic repeat. Extrachromosomal circular major satellite (ECMS) DNA constitutes the majority of eccDNA in B16 mouse melanoma cells and is highly abundant in other mouse cells. Production of these molecules is enhanced in proliferating cells, suggesting that processes associated with DNA replication are involved in their appearance. Using siRNA technique we show that DNA Ligase IV is engaged in ECMS synthesis. Based on our findings we propose a novel two-step model for eccDNA formation in mammalian cells.

Identification of residues of the Moloney murine leukemia virus nucleocapsid critical for viral DNA synthesis in vivo.

The nucleocapsid (NC) protein of retroviruses is a small nucleic acid-binding protein important in virion assembly and in the encapsidation of the viral RNA genome into the virion particle. Multiple single-amino-acid substitutions were introduced into the NC of Moloney murine leukemia virus to examine further its role in viral replication. Two residues were shown to play important roles in the early events of replication. Unlike viruses with previously characterized NC mutations, these viruses showed no impairment in the late events of replication. Viruses containing the substitutions L21A and K30A expressed the normal complement of properly processed viral Gag proteins. Analysis of the RNA content of mutant virions revealed normal levels of unspliced and spliced viral RNA, and the tRNA(Pro) primer was properly annealed to the primer binding site on the viral genome. The virions demonstrated no defect in initiation of reverse transcription using the endogenous tRNA primer or in the synthesis of long viral DNA products in vitro. Nonetheless, viruses possessing these NC mutations demonstrated significant defects in the synthesis and accumulation of viral DNA products in vivo.

The carboxy-terminal fragment of nucleolin interacts with the nucleocapsid domain of retroviral gag proteins and inhibits virion assembly.

A yeast two-hybrid screen for cellular proteins that interact with the murine leukemia virus (MuLV) Gag protein resulted in the identification of nucleolin, a host protein known to function in ribosome assembly. The interacting fusions contained the carboxy-terminal 212 amino acids of nucleolin [Nuc(212)]. The nucleocapsid (NC) portion of Gag was necessary and sufficient to mediate the binding to Nuc(212). The interaction of Gag with Nuc(212) could be demonstrated in vitro and was manifested in vivo by the NC-dependent incorporation of Nuc(212) inside MuLV virions. Overexpression of Nuc(212), but not full-length nucleolin, potently and specifically blocked MuLV virion assembly and/or release. A mutant of MuLV, selected to specifically disrupt the binding to Nuc(212), was found to be severely defective for virion assembly. This mutant harbors a single point mutation in capsid (CA) adjacent to the CA-NC junction, suggesting a role for this region in Moloney MuLV assembly. These experiments demonstrate that selection for proteins that bind assembly domain(s) can yield potent inhibitors of virion assembly. These experiments also raise the possibility that a nucleolin-Gag interaction may be involved in virion assembly.

Infectivity of Moloney murine leukemia virus defective in late assembly events is restored by late assembly domains of other retroviruses.

The p12 region of the Moloney murine leukemia virus (M-MuLV) Gag protein contains a PPPY motif important for efficient virion assembly and release. To probe the function of the PPPY motif, a series of insertions of homologous and heterologous motifs from other retroviruses were introduced at various positions in a mutant gag gene lacking the PPPY motif. The assembly defects of the PPPY deletion mutant could be rescued by insertion of a wild-type PPPY motif and flanking sequences at several ectopic positions in the Gag protein. The late assembly domain (L-domain) of Rous sarcoma virus (RSV) or human immunodeficiency virus type 1 (HIV-1) could also fully or partially restore M-MuLV assembly when introduced into matrix, p12, or nucleocapsid domains of the mutant M-MuLV Gag protein lacking the PPPY motif. Strikingly, mutant viruses carrying the RSV or the HIV-1 L-domain at the original location of the deleted PPPY motif were replication competent in rodent cells. These data suggest that the PPPY motif of M-MuLV acts in a partially position-independent manner and is functionally interchangeable with L-domains of other retroviruses. Electron microscopy studies revealed that deletion of the entire p12 region resulted in the formation of tube-like rather than spherical particles. Remarkably, the PPPY deletion mutant formed chain structures composed of multiple viral particles linked on the cell surface. Many of the mutants with heterologous L-domains released virions with wild-type morphology.

Deletion of a short, untranslated region adjacent to the polypurine tract in Moloney murine leukemia virus leads to formation of aberrant 5' plus-strand DNA ends in vivo.

Experiments were performed to determine the function of a 28-nucleotide untranslated sequence lying between the envelope gene and the polypurine tract (PPT) sequence in the Moloney murine leukemia virus (Mo-MuLV) genome. A mutant virus carrying a deletion of this sequence (Mo-MuLVDelta28) replicated more slowly than wild-type (wt) virus and reverted by recombination with endogenous sequences during growth in NIH 3T3 cells. We show that this deletion did not affect the level of viral protein expression or genomic RNA packaging. Mo-MuLVDelta28 served as a helper virus as efficiently as the wt virus; in contrast, a retroviral vector harboring this mutation exhibited reduced transduction efficiency, indicating that the mutation acts not in trans but in cis. Analysis of acutely infected cells revealed that reduced levels of viral DNA were generated by reverse transcription of the Mo-MuLVDelta28 RNA as compared to the wt RNA. Analysis of DNA circle junctions revealed that plus-strand DNA of Mo-MuLVDelta28 but not wt virus often retained the PPT and additional upstream sequences. These structures suggest that aberrant 5' ends of plus-strand DNA were generated by a failure to remove the PPT RNA primer and/or by mispriming at sites upstream of the PPT. These data demonstrate that the major role of the sequences immediately upstream of the PPT is specifying efficient and accurate plus-strand DNA synthesis.

Binding of the human immunodeficiency virus type 1 Gag protein to the viral RNA encapsidation signal in the yeast three-hybrid system.

We have used the yeast three-hybrid system (D. J. SenGupta, B. Zhang, B. Kraemer, P. Pochart, S. Fields, and M. Wickens, Proc. Natl. Acad. Sci. USA 93:8496-8501, 1996) to study binding of the human immunodeficiency virus type 1 (HIV-1) Gag protein to the HIV-1 RNA encapsidation signal (HIVPsi). Interaction of these elements results in the activation of a reporter gene in the yeast Saccharomyces cerevisiae. Using this system, we have shown that the HIV-1 Gag protein binds specifically to a 139-nucleotide fragment of the HIVPsi signal containing four stem-loop structures. Mutations in either the Gag protein or the encapsidation signal that have been shown previously to impair this interaction reduced the activation of the reporter gene. Interestingly, the nucleocapsid portion of Gag retained the RNA binding activity but lost its specificity compared to the full-length Gag. These results demonstrate the utility of this system and suggest that a variety of genetic analyses could be performed to study Gag-encapsidation signal interactions.

Apposition-dependent induction of plasminogen activator inhibitor type 1 expression: a mechanism for balancing pericellular proteolysis during angiogenesis.

Plasminogen-activator inhibitor type I (PAI-1), the primary inhibitor of urinary-type plasminogen activator, is thought to play an important role in the control of stroma invasion by both endothelial and tumor cells. Using an in vitro angiogenesis model of capillary extension through a preformed monolayer, in conjunction with in situ hybridization analysis, we showed that PAI-1 mRNA is specifically induced in cells juxtaposed next to elongating sprouts. To further establish that PAI-1 expression is induced as a consequence of a direct contact with endothelial cells, coculture experiments were performed. PAI-1 mRNA was induced exclusively in fibroblasts (L-cells) contacting endothelial cell (LE-II) colonies. Reporter gene constructs driven by a PAI-1 promoter and stably transfected into L-cells were used to establish that both mouse and rat PAI-1 promoters mediate apposition-dependent regulation. This mode of PAI-1 regulation is not mediated by plasmin, as an identical spatial pattern of expression was detected in cocultures treated with plasmin inhibitors. Because endothelial cells may establish direct contacts with fibroblasts only during angiogenesis, we propose that focal induction of PAI-1 at the site of heterotypic cell contacts provides a mechanism to negate excessive pericellular proteolysis associated with endothelial cell invasion.

In vivo patterns of expression of urokinase and its inhibitor PAI-1 suggest a concerted role in regulating physiological angiogenesis.

To evaluate the role of plasminogen activators (PAs) in physiological angiogenesis, we have investigated the in vivo patterns of expression of urokinase-type PA (uPA) and PA-inhibitor type 1 (PAI-1) during neovascularization of ovarian follicles, the corpus luteum, and the maternal decidua. Using in situ hybridization, we detected uPA mRNA in the ovary along the route of capillary extension, originating at the existing ovarian vasculature, extending toward growing follicles, and terminating at the newly formed capillary sheaths surrounding each growing follicle. Following ovulation, uPA mRNA was expressed in capillary sprouts within the developing corpus luteum. During the process of decidual neovascularization, uPA expression was detected in endothelial cell cords traversing the maternal decidua in the direction of the newly implanted embryo. uPA mRNA was not detected in endothelial cells upon completion of neovascularization, suggesting that uPA expression is a part of the angiogenic response. During in vitro "angiogenesis" of cultured aortic explants, uPA was expressed in capillary sprouts but not in underlying endothelial cell sheets, suggesting that the expression of uPA depends on the histological context of the endothelial cell. Interestingly, during corpus luteum development and decidual neovascularization, and in aortic explants, PAI-1 expression was preferentially activated in cells in the vicinity of uPA-expressing capillary-like structures. These findings suggest a functional interplay between uPA- and PAI-1-expressing cells and support the idea that natural PA inhibitors function during angiogenesis to protect neovascularized tissues from excessive proteolysis.