Hepatitis B virus polymerase suppresses translation of pregenomic RNA via a mechanism involving its interaction with 5' stem-loop structure

The pregenomic RNA (pgRNA) of hepadnaviruses serves a dual role: as mRNA for the core (C) and polymerase (P) synthesis and as an RNA template for viral genome replication. A question arises as to how these two roles are regulated. We hypothesized that the P protein could suppress translation of the pgRNA via its interaction with 5' stem-loop structure (epsilon or encapsidation signal). Consistent with the hypothesis, we observed up-regulation of the C protein level in the absence of the P protein expression in a physiological context. Importantly, translational suppression depended on the 5' epsilon sequence. Furthermore, the impact of the P protein on ongoing translation of the C ORF was directly demonstrated by polysome distribution analysis. We conclude that the P protein suppresses translation of the pgRNA via a mechanism involving its interaction with the 5' epsilon sequence, a finding that implicates the coordinated switch from translation to genome replication.     

NSP5 phosphorylation regulates the fate of viral mRNA in rotavirus infected cells

Summary.  Elucidation of the function of the non-structural rotavirus proteins during infection is difficult in the absence of a reverse genetic system. To study the role of NSP5, nonstructural phosphoprotein NSP5, we constructed a reassortant strain (SACC11) in the SA11 background that harbours a heterologous segment 11 encoding a variant protein (h-NSP5). Cells infected by SACC11 produced viral polypeptides at earlier times than SA11 infected cells while showing less accumulation of genomic dsRNA. These changes suggested that NSP5 might direct viral messenger RNA to protein synthesis or genome replication. Distinct patterns of proteins were shown to form complexes with NSP5 in co-immunoprecipitation studies with SA11 and SACC11 infected cells. Recombinant h-NSP5 from either bacteria or eucaryotic cells migrated faster in PAGE suggesting that it was hypophosphorylated. Indeed, the kinase inhibitor H-7 enhanced translation of viral proteins in SA11 but not SACC11 infected cells suggesting that NSP5 function in the regulation of the fate of viral positive strand RNA is mediated by phosphorylation. Present address: LaboRetro, INSERM U412, Ecole Normale Supérieure de Lyon, 46 allée d’Italie 46, F-69364, Lyon, France. Received October 15, 2001; accepted May 20, 2002     

Control of vesicular stomatitis virus protein synthesis.

The five structural polypeptides of vesicular stomatitis virus are synthesized in infected cells at nonequimolar rates which correspond to their relative amounts in the virus particle. These experiments are concerned with the mechanism(s) responsible for the relative rates of viral polypeptide synthesis.Treatment of infected cells with amino acid analogs in order to prevent formation of functional viral proteins resulted in inhibition of viral genome replication and virion assembly. This treatment, however, did not alter the relative rates of viral polypeptide synthesis. This suggested that the mechanisms determining these rates do not require the function of newly-synthesized viral proteins and do not involve direct coupling between protein synthesis and virion assembly.Relative translation frequencies of viral messenger RNA molecules coding for each polypeptide were compared by treating cells with low levels of cycloheximide and anisomycin, inhibitors of protein synthesis that interfere preferentially with polypeptide chain elongation. Under these conditions the rate of synthesis of each viral polypeptide should be proportional only to the amount of its messenger RNA available for translation. The relative rates of viral polypeptide synthesis were not altered by this treatment, suggesting that viral messenger RNA molecules coding for each polypeptide are translated with similar average frequencies.Rates of peptide chain growth were compared by following the incorporation of ³H-amino acids into completed viral polypeptides following a pulse-label. The time necessary to achieve maximum incorporation of isotope into each completed viral polypeptide was taken as its translation time, the time necessary to synthesize and release a completed molecule. We were able to determine translation times for the M and G polypeptides by this method and found that they correlated directly with their molecular weights, suggesting that nascent chains of these two polypeptides are propagated at similar rates.     

Pseudosubstrate inhibition of protein kinase PKR by swine pox virus C8L gene product

The interferon-induced protein kinase PKR is activated upon binding double-stranded RNA and phosphorylates the translation initiation factor eIF2alpha on Ser-51 to inhibit protein synthesis in virally infected cells. Swinepox virus C8L and vaccinia virus K3L gene products structurally resemble the amino-terminal third of eIF2alpha. We demonstrate that the C8L protein, like the K3L protein, can reverse the toxic effects caused by high level expression of human PKR in yeast cells. In addition, expression of either the K3L or C8L gene product was found to reverse the inhibition of reporter gene translation caused by PKR expression in mammalian cells. The inhibitory function of the K3L and C8L gene products in these assays was found to be critically dependent on residues near the carboxyl-termini of the proteins including a sequence motif shared among eIF2alpha and the C8L and K3L gene products. Thus, despite significant sequence differences both the C8L and K3L proteins function as pseudosubstrate inhibitors of PKR.     

Selective inhibition of viral protein accumulation in interferon-treated cells; nondiscriminate inhibition of the translation of added viral and cellular messenger RNAs in their extracts

Treatment of monolayers of mouse L cells with 7.5 vesicular stomatitis virus plaque reduction units/ml of mouse interferon (specific activity 2 × 10⁷ NIH mouse reference standard units/mg protein) results in a 95% decrease in the reovirus yield and, as shown earlier, an about 80% decrease in double-stranded and an about 60% decrease in single-stranded reo RNA accumulation in cells infected with 10 plaque-forming units of virus/cell. Interferon at this concentration does not affect the rate of accumulation of host proteins in uninfected or reovirus-infected cells. It inhibits reo virion protein accumulation in infected cells. In control cells the rate of reo virion protein accumulation increases about 2-fold between 10 and 14 hr after infection. In cells treated with interferon it remains at the same low inhibited level. Reo virion proteins were assayed in cell extracts by immunoprecipitation with antisera specific for various size classes or by gel electrophoresis. These results reveal the selective nature of the inhibition of protein accumulation in interferon-treated, virus-infected cells. The effect of treating L cells with interferon was also tested on the capacity of their extracts to translate various mRNAs. The translation of endogenous mRNA, of mRNA in L cell polysomes (added as such) and of polyuridylic acid is at most only slightly inhibited whereas that of added natural mRNAs (including reovirus and encephalomyocarditis virus mRNA, rabbit globin mRNA, and a mixture of L cell mRNAs) is strongly inhibited. The dominant nature of the inhibitor(s) is indicated by the fact that translation of added mRNA is impaired in a mixture of extracts from interferon-treated and from control cells. Thus an effect of interferon treatment of cells is manifested in subcellular systems. However, the selectivity of the effect in vivo, is not well reflected in vitro.     

Immunotargeting with CD154 (CD40 ligand) enhances DNA vaccine responses in ducks

Engagement of CD154 on activated T cells with CD40 on antigen-presenting cells (APCs) potentiates adaptive immune responses in mammals. Soluble multimeric forms of CD154 have been used as an adjuvant or in immunotargeting strategies to enhance vaccine responses. The objective of our study was to examine the ability of duck CD154 (DuCD154) to enhance DNA vaccine responses in the duck hepatitis B model. Constructs were generated to express the functional domain of DuCD154 (tCD154), truncated duck hepatitis B virus (DHBV) core antigen (tcore) and chimera of tcore fused to tCD154 (tcore-tCD154). Expression in LMH cells demonstrated that all proteins were secreted and that tCD154 and tcore-tCD154 formed multimers. Ducks immunized with the plasmid ptcore-tCD154 developed accelerated and enhanced core-specific antibody responses compared to ducks immunized with ptcore or ptcore plus ptCD154. Antibody responses were better sustained in both ptcore-tCD154- and ptcore plus ptCD154-immunized ducks. Core-specific proliferative responses of duck peripheral blood mononuclear cells were enhanced in ducks immunized with ptcore-tCD154 or ptcore alone. This study suggests that the role of CD154 in the regulation of adaptive immune responses had already evolved before the divergence of birds and mammals. Thus, targeting of antigens to APCs with CD154 is an effective strategy to enhance DNA vaccine responses not only in mammalian species but also in avian species.     

Expression of capsid proteins and non-structural proteins of waterfowl parvoviruses in Escherichia coli and their use in serological assays.

While there are a number of methods available for detection of antibodies against waterfowl parvoviruses, none is able to differentiate responses against the capsid and non-structural proteins. To enable this, the capsid and non-structural proteins of goose parvovirus (GPV) and Muscovy duck parvovirus (MDPV) were expressed in Escherichia coli. These proteins were purified and used as antigens in western blotting assays of antibodies against GPV and MDPV. The results showed that 94.7% of the goose and 90.0% of the duck sera collected from the field contained antibodies against GPV or MDPV. Moreover, these sera could be classified into distinct groups based on differences in patterns of western blot reactivity. These different patterns might indicate different stages in infection. Western blotting assays of sera collected from experimentally infected ducks showed that antibodies against the non-structural protein appeared first after infection, followed by antibodies against the capsid protein. It was concluded that the recombinant capsid and non-structural proteins might serve as useful antigens for assays for antibodies against GPV and MDPV. Moreover, because these assays could discriminate between antibodies against the non-structural protein and those against the capsid protein, they may be useful in differentiating vaccinated from infected birds when recombinant capsid protein is used as the vaccine.     

The deubiquitinase activity of the Salmonella pathogenicity island 2 effector, SseL, prevents accumulation of cellular lipid droplets

To cause disease, Salmonella enterica serovar Typhimurium requires two type III secretion systems that are encoded by Salmonella pathogenicity islands 1 and 2 (SPI-1 and -2). These secretion systems serve to deliver specialized proteins (effectors) into the host cell cytosol. While the importance of these effectors to promote colonization and replication within the host has been established, the specific roles of individual secreted effectors in the disease process are not well understood. In this study, we used an in vivo gallbladder epithelial cell infection model to study the function of the SPI-2-encoded type III effector, SseL. The deletion of the sseL gene resulted in bacterial filamentation and elongation and the unusual localization of Salmonella within infected epithelial cells. Infection with the ΔsseL strain also caused dramatic changes in host cell lipid metabolism and led to the massive accumulation of lipid droplets in infected cells. This phenotype was directly attributable to the deubiquitinase activity of SseL, as a Salmonella strain carrying a single point mutation in the catalytic cysteine also resulted in extensive lipid droplet accumulation. The excessive buildup of lipids due to the absence of a functional sseL gene also was observed in murine livers during S. Typhimurium infection. These results suggest that SseL alters host lipid metabolism in infected epithelial cells by modifying the ubiquitination patterns of cellular targets.     

Pregenomic RNA: How to assist the management of chronic hepatitis B?

Pregenomic RNA (pgRNA) is an emerging serological marker for chronic hepatitis B virus (HBV) infection. While pgRNA is principally the template for viral proteins and viral DNAs, additional novel functions for the serum pgRNA have recently been described. These results extend for pgRNA a regulatory function in the viral life cycle and potentially a role in pathogenesis. Here, we review the diverse roles of pgRNA in HBV replication and pathogenesis, emphasizing how the unique structure of this RNA is key to its various functions. We focus in particular on the role of HBV pgRNA in guiding antiviral therapy including nucleot(s)ide analog interruption and role as a marker of cure with new curative therapies. We also briefly allude to the emerging niche for new direct‐acting or indirect‐acting antivirals targeting pgRNA.     

The importance of tRNA for the in vitro cell-free translation of messenger RNA isolated from the malaria parasite Plasmodium lophurae

Preliminary studies had indicated the inadequacy of the wheat germ and rabbit reticulocyte cell-free translation systems for the in vitro translation of mRNA isolated from Plasmodium lophurae. To identify the factors which are important for the efficient translation of parasite proteins, an homologous system was established using polysomes, the pH 5 fraction, and tRNA prepared from P. lophurae. For comparison, the same components were isolated from the host duck reticulocytes and tested. The effect of each of these factors was evaluated by analysis of the translation products and by comparison with products synthesized in vivo. The results indicated that P. lophurae tRNA had a marked stimulatory effect on the synthesis of parasite proteins while it inhibited the synthesis of host proteins. Duck reticulocyte tRNA could not be used as a substitute for the parasite tRNA. Based on these findings, a commercially available rabbit reticulocyte system was supplemented with P. lophurae tRNA, which markedly increased the efficiency of translation of P. lophurae proteins by this system.     

Alternate poliovirus nonstructural protein processing cascades generated by primary sites of 3C proteinase cleavage.

The post-translational regulation of picornavirus gene expression mediated by the cascade processing of viral proteins is not well understood. Both pulse-chase studies of infected cells and in vitro studies of the translation of poliovirus type 1 RNA transcribed from genomic cDNA clones indicate a specific cascade of polyprotein processing in which the P1, P2, and P3 precursor proteins are primary products of viral proteinase cleavage. We report the results of a short-time kinetic analysis of poliovirus type 1 protein processing in an in vitro translation system and in infected HeLa cells which indicate the existence of another, rapid pathway of polyprotein processing mediated by the activity of the 3C proteinase. The observed pathway is distinct from and in addition to the one previously known. The potential role of this alternative pathway of processing in the post-translational regulation of viral gene expression is discussed.     

Recombinant avian adenovirus CELO expressing the human interleukin-2: characterization in vitro, in ovo and in vivo

In our study, a recombinant adenovirus based on the avian adenovirus CELO genome, has been constructed that contains the human interleukin-2 gene. We have shown the production of biologically active recombinant interleukin-2 in vitro (LMH and 293 cells) and in ovo (chicken embryos) infected with recombinant virus CELO-IL2. An increase in the median survival time of C57BL/6 mice carrying B16 melanoma tumors has been demonstrated after multiple intra-tumors injections of the recombinant adenovirus CELO-IL2.     

Non-infectious morphologically altered nucleocapsids of measles virus from persistently infected cells

Summary We have shown that a latent infection of herpes simplex virus type 2 (HSV-2) can be established in a human neuroblastoma cell line IMR-32 if the infected cells are cultured at 40°C. In the present study, viral polypeptides and cellular heat-shock proteins which were synthesized in HSV-2 infected IMR-32 cells cultured at 40°C were analyzed by polyacrylamide gel electrophoresis. It was found that the synthesis of late viral polypeptide ICP 5 was markedly reduced in the infected cells at 40°C as compared with those at 37°C. Although infection of IMR-32 cells with HSV-2 at 40°C resulted in shutoff of cellular protein synthesis, it was found that some cellular heat-shock proteins (90, 72 and 70 kd polypeptides) were synthesized and accumulated intracellularly. These findings suggest that modification of cascade regulation of HSV-2 polypeptide synthesis and/or accumulation of heat-shock proteins may be involved in the incomplete arrest of virus growth and in survival of the infected cells, leading to the establishment of HSV-2 latency in IMR-32 cells.     

Avian oncovirus proteins expressed on the surface of infected cells

Lactoperoxidase-catalyzed iodination and anti-AMV immunoprecipitation showed that both chicken and duck fibroblasts infected with a sarcoma virus (Rous sarcoma virus PrC) or a leukemia virus (avian myeloblastosis virus; AMV) had on their surface a protein of approximately 120 kilodaltons molecular weight (120K), as well as envelope glycoprotein precursors of 90–92 kd. Uninfected chicken fibroblasts of the gs^?, chf^? phenotype had much lower, but detectable amounts of surface 120K, whereas uninfected duck fibroblasts did not have any, suggesting a relationship between surface 120K and expression of chicken virus information in the cell. 120K is a glycoprotein, since it could be labeled with [³H]mannose and contained a component that bound to a concanavalin A affinity column. The 120K protein was characterized by tryptic fingerprinting after reiodination with chloramine-T. Total and Con A-selected 120K from infected chicken cells and total 120K from infected duck cells had essentially identical fingerprints. Moreover, they were extensively related to the iodinated fingerprint of Pr76^gag, the intracellular precursor of viral core proteins. These results indicate that expression on the cell surface of glycosylated forms of gag polyproteins occurs also in avian oncornavirus infections, similarly to findings in the murine leukemia virus system.     

A new avian hepadnavirus infecting snow geese (Anser caerulescens) produces a significant fraction of virions containing single-stranded DNA.

We describe the identification and functional analysis of an evolutionary distinct new avian hepadnavirus. Infection of snow geese (Anser caerulescens) with a duck hepatitis B virus (DHBV)-related virus, designated SGHBV, was demonstrated by detection of envelope proteins in sera with anti-DHBV preS and S antibodies. Comparative sequence analysis of the PCR-amplified SGHBV genomes revealed unique SGHBV sequence features compared with other avian hepadnaviruses. Unlike DHBV, SGHBV shows an open reading frame in an analogous position to orthohepadnavirus X genes. Four of five cloned genomes were competent in replication, gene expression, and virus particle secretion in chicken hepatoma cells. Primary duck hepatocytes were permissive for infection with SGHBV, suggesting a similar or identical host range. SGHBV was found to secrete a significant fraction of virion-like particles containing single-stranded viral DNA. This was observed both in cell culture medium of SGHBV DNA-transfected LMH cells and in viremic sera of several birds, suggesting that it is a stable trait of SGHBV. Taken together, SGHBV has several unique features that expand the knowledge of the functional and evolutionary diversity of hepadnaviruses and offers new experimental opportunities for studies on the life cycle of hepadnaviruses.     

Proteins of murine cytomegalovirus: identification of structural and nonstructural antigens in infected cells.

The synthesis of viral proteins in tertiary mouse embryo cells infected with murine cytomegalovirus (MCMV) has been studied by polyacrylamide gel electrophoresis. Three immediate-early proteins were detected by 4 hr postinfection (p.i.), but between 4 hr and the onset of viral DNA synthesis at 8–10 hr, no further viral proteins could be distinguished. The major structural proteins appeared after viral DNA synthesis had commenced and continued to be made until 36 hr p.i. or later. Host translation occurred until 24 hr p.i. but was inhibited between then and 30 hr p.i. at which time viral protein synthesis predominated. At this time 52 proteins could be labeled in infected cells and, of these, 30 could be precipitated with viral-specific antisera. The proteins precipitated included 22 with electrophoretic mobilities similar to structural viral proteins and a further 8 which were not present in purified MCMV. These, together with the three immediate-early proteins, were the only nonstructural proteins which were detected in the infected cell.