Activation of extracellular signal-regulated kinase ERK after hypo-osmotic stress in renal epithelial A6 cells

Activation of mitogen-activated protein (MAP) kinases has been reported to occur after a hypo-osmotic cell swelling in various types of cells. In renal epithelial A6 cells, the hypo-osmotic shock induced a rapid increase in the phosphorylation of an extracellular signal-regulated kinase (ERK)-like protein that was maximal 10 min after osmotic stress. Activation of ERK was significantly increased when hypo-osmotic stress was performed in the absence of extracellular Ca2+, a condition that inhibits regulatory volume decrease (RVD). Exposure of cells to PD98059, an inhibitor of the MAP kinase kinase MEK, at a concentration that fully cancelled ERK activation, did not inhibit RVD. On the contrary, RVD was abolished when osmotic shock was induced in the presence of SB203580, an inhibitor of stress-activated protein kinases (SAPKs). These results suggest that different MAP kinases are activated after hypo-osmotic stress in A6 cells. SAPKs would be involved in the control of RVD, while ERK would lead to later events, such as gene expression or energy metabolism.     

Membrane requirements for uridylylation of the poliovirus VPg protein and viral RNA synthesis in vitro

Efficient translation of poliovirus (PV) RNA in uninfected HeLa cell extracts generates all of the viral proteins required to carry out viral RNA replication and encapsidation and to produce infectious virus in vitro. In infected cells, viral RNA replication occurs in ribonucleoprotein complexes associated with clusters of vesicles that are formed from preexisting intracellular organelles, which serve as a scaffold for the viral RNA replication complex. In this study, we have examined the role of membranes in viral RNA replication in vitro. Electron microscopic and biochemical examination of extracts actively engaged in viral RNA replication failed to reveal a significant increase in vesicular membrane structures or the protective aggregation of vesicles observed in PV-infected cells. Viral, nonstructural replication proteins, however, bind to heterogeneous membrane fragments in the extract. Treatment of the extracts with nonionic detergents, a membrane-altering inhibitor of fatty acid synthesis (cerulenin), or an inhibitor of intracellular membrane trafficking (brefeldin A) prevents the formation of active replication complexes in vitro, under conditions in which polyprotein synthesis and processing occur normally. Under all three of these conditions, synthesis of uridylylated VPg to form the primer for initiation of viral RNA synthesis, as well as subsequent viral RNA replication, was inhibited. Thus, although organized membranous structures morphologically similar to the vesicles observed in infected cells do not appear to form in vitro, intact membranes are required for viral RNA synthesis, including the first step of forming the uridylylated VPg primer for RNA chain elongation.     

Strand-specific RNA synthesis defects in a poliovirus with a mutation in protein 3A

Substitution of a methionine residue at position 79 in poliovirus protein 3A with valine or threonine caused defective viral RNA synthesis, manifested as delayed onset and reduced yield of viral RNA, in HeLa cells transfected with a luciferase-containing replicon. Viruses containing these same mutations produced small or minute plaques that generated revertants upon further passage, with either wild-type 3A sequences or additional nearby compensating mutations. Translation and polyprotein processing were not affected by the mutations, and 3AB proteins containing the altered amino acids at position 79 showed no detectable loss of membrane-binding activity. Analysis of individual steps of viral RNA synthesis in HeLa cell extracts that support translation and replication of viral RNA showed that VPg uridylylation and negative-strand RNA synthesis occurred normally from mutant viral RNA; however, positive-strand RNA synthesis was specifically reduced. The data suggest that a function of viral protein 3A is required for positive-strand RNA synthesis but not for production of negative strands.     

Interaction of Poly(rC) Binding Protein 2 with the 5′ Noncoding Region of Hepatitis A Virus RNA and Its Effects on Translation

Utilization of internal ribosome entry segment (IRES) structures in the 5′ noncoding region (5′NCR) of picornavirus RNAs for initiation of translation requires a number of host cell factors whose distribution may vary in different cells and whose requirement may vary for different picornaviruses. We have examined the requirement of the cellular protein poly(rC) binding protein 2 (PCBP2) for hepatitis A virus (HAV) RNA translation. PCBP2 has recently been identified as a factor required for translation and replication of poliovirus (PV) RNA. PCBP2 was shown to be present in FRhK-4 cells, which are permissive for growth of HAV, as it is in HeLa cells, which support translation of HAV RNA but which have not been reported to host replication of the virus. Competition RNA mobility shift assays showed that the 5′NCR of HAV RNA competed for binding of PCBP2 with a probe representing stem-loop IV of the PV 5′NCR. The binding site on HAV RNA was mapped to nucleotides 1 to 157, which includes a pyrimidine-rich sequence. HeLa cell extracts that had been depleted of PCBP2 by passage over a PV stem-loop IV RNA affinity column supported only low levels of HAV RNA translation. Translation activity was restored upon addition of recombinant PCBP2 to the depleted extract. Removal of the 5′-terminal 138 nucleotides of the HAV RNA, or removal of the entire IRES, eliminated the dependence of HAV RNA translation on PCBP2.     

Assessing the Impact of Disease Vectors on Animal Populations

Many studies have attempted to assess the relative effects of different vectors of a disease on animal populations. To this end, three measures have been proposed: Vectorial efficiency, Vectorial capacity and recently Vectorial effectiveness (or Vectorial impact). In this study we relate these measures to derive some of their properties emphasising in the vectorial impact for its importance in both, population performance of parasites and the proportion of the prevalence of one parasite due to a given vector. We applied the quantitative expressions advanced in this study to a simple Chilean example with one parasite (Trypanosoma cruzi), two vectors (Triatoma infestans and Mepraia spinolai) and one animal population (humans: Chagas's disease).     

Identification of terminal adenylyl transferase activity of the poliovirus polymerase 3Dpol.

A terminal adenylyl transferase (TATase) activity has been identified in preparations of purified poliovirus RNA-dependent RNA polymerase (3Dpol). Highly purified 3Dpol is capable of adding [32P]AMP to the 3' ends of chemically synthesized 12-nucleotide (nt)-long RNAs. The purified 52-kDa polypeptide, isolated after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and renatured, retained the TATase activity. Two 3Dpol mutants, purified from Escherichia coli expression systems, displayed no detectable polymerase activity and were unable to catalyze TATase activity. Likewise, extracts from the parental E. coli strain that harbored no expression plasmid were unable to catalyze formation of the TATase products. With the RNA oligonucleotide 5'-CCUGCUUUUGCA-3' used as an acceptor, the products formed by wild-type 3Dpol were 9 and 18 nt longer than the 12-nt oligomer. GTP, CTP, and UTP did not serve as substrates for transfer to this RNA, either by themselves or when all deoxynucleoside triphosphates were present in the reaction. Results from kinetic and stoichiometric analyses suggest that the reaction is catalytic and shows substrate and enzyme dependence. The 3'-terminal 13 nt of poliovirus minus-strand RNA also served as an acceptor for TATase activity, raising the possibility that this activity functions in poliovirus RNA replication. The efficiency of utilization and the nature of the products formed during the reaction were dependent on the acceptor RNA.     

Capsid protein precursor is one of two initiated products of translation of poliovirus RNA in vitro.

Previous studies in our laboratory have demonstrated that cell-free systems translating the Mahoney strain of poliovirus type I RNA utilize two unique initiation sites. In this study, defective-interfering particles of poliovirus, which contain deletions in the region encoding the capsid proteins, are shown to initiate translation of proteins in vitro at these same two sites. Both the standard virus and the defective-interfering virus RNA direct the synthesis of two polypeptides labeled with n-formyl-methionine (fmet) at their amino termini. The size of the smaller fmet polypeptide synthesized in vitro by the defective virus appears identical in size to that of the standard virus. However, the larger-molecular-weight fmet polypeptide is reduced in size from 115,000 to 69,000 daltons. This correlates exactly with the reduced size of the precursor to the capsid proteins synthesized by the defective virus in vivo and with the size of the deletion in the defective virus RNA (1,200 bases). This provides genetic evidence that the 115,000-dalton fmet polypeptide synthesized into vitro by the standard virus is NCVP1a, the precursor to the coat proteins. Although the identity of the small (5,000 to 10,000 daltons) fmet polypeptide is not clear, several lines of evidence enable us to exclude the possibility that it is VP4, the smallest viral capsid protein.