The effect of salt concentration on RNA synthesis by DNA-dependent RNA-polymerase from Escherichia coli has been investigated. For a double stranded template the following influences are observed: (1) Free enzyme and enzyme bound to DNA, but not synthesizing RNA, are inhibited above an ionic strength of about 0.15. (2) The rate of RNA-synthesis, by those enzyme molecules which have started RNA chains, is stimulated by increasing the salt concentration up to an optimum. Magnesium salts cause a larger stimulation than monovalent cations. However, the optimal effect always occurs at an ionic strength of about 0.36. (3) This stimulatory effect is distinguished from the cofactor function which requires only low concentrations of divalent cations (Mg2+, Co2+, Mn2+). (4) RNA synthesis stops at low ionic strength (< 0.2) due to a first order inactivation of the synthesizing enzyme. A t high ionic strength synthesis continues over many hours a t a slowly decreasing rate. Raising the ionic strength after RNA synthesis has stopped at low ionic strength causes an immediate resumption of synthesis. Mg2+ and spermidine are most effective in this action. However, Co2+ and Mn2+ are unable to reactivate the enzyme once it has stopped.With single stranded DNA as template neither a first order inactivation of synthesizing enzyme a t low ionic strength nor a stimulation of RNA synthesis by monovalent> salts is observed. The effect of divalent cations on enzyme activity can be due to their cofactor function.The DNA-dependent RNA-polymerase from Escherichia coli is strongly influenced by changes in the ionic strength. At low ionic strength (between 0.02 and 0.12) it has been shown to exist in a form sedimenting a t 24s [l-51; a t high ionic strength the enzyme reversibly dissociates into subunits of 13s [2,4-61. At low salt concentration the polymerase forms a complex with DNA [1,7--12j which by raising the ionic strength is dissociated into free DNA and the i3S form of the enzyme [Z, 13-15].The polymerization process itself has been reported to be inhibited only after some time of synthesis at high ionic strength [5,13]. However, the effect of ionic strength on this phase of the reaction has not been extensively investigated.This communication presents a detailed analysis of the effect of ionic strength on the kinetics of RNAsynthesis with double and single-stranded DNA. It is shown that increasing the ionic strength causes Non-Standard Abbreviations. PPO, 2,5 diphenyloxazol; POPOP, 1,4-bis-2-(4 methyl4 phenoxazoly1)-bcnzene; TMA, buffer, containing: 0.01 M Tris acetate, 0.022 M NH,CI, 0.01 M magnesium acetate, 1 mM 2-mercaptoethanol, pH 7.3; TCA, trichloroacetic acid.Enzymes. DNA-dependent RNA-polymerase or nucleosidetripliosphate : RNA nucleotidyltransferase (EC 2.7.7.6) ; Ribonuclease I (EC 2.7.7.17); Pyruvate kinase (EC 2.7.1.40). a striking stimulation of both the rate and extent of RNA synthesis. This eliminates the typical plateau in the kinetics [l6-211 and allows RNA-synthesis t o continue over many hours. Moreover, synthes...

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The viral infectivity factor (Vif) of human immunodeficiency virus type 1 (HIV-1) neutralizes an unidentified antiviral pathway that occurs only in nonpermissive (NP) cells. Using a yeast two-hybrid screen of a human lymphocyte cDNA library, we identified several potential Vif partners. One, the nuclear body protein Sp140, was found specifically in all NP cells (n ‫؍‬ 12 cell lines tested; P < 0.001), and HIV-1 infection induced its partial dispersal from nuclear bodies into cytosolic colocalization with Vif. Our results implicate Sp140 in a response to HIV-1 that may be related to or coordinated with the pathway that inactivates HIV-1 lacking vif.The Vif protein encoded by human immunodeficiency virus type 1 (HIV-1) is a 23,000-M r phosphoprotein that has been detected in the cytosol and nucleus in association with membranes, intermediate filaments, the viral Gag polyprotein, and the viral genomic RNA (8,10,17,23,25,27,43,55). It enhances the infectivity but not the quantity of HIV-1 virions released from nonpermissive (NP) cells, including T lymphocytes and macrophages and some leukemic T-cell lines, by 20-to 100-fold, but it is unnecessary in permissive (P) cells (7,13,22,39,44,53). Although the HIV-1 lacking vif that is made in P cells efficiently infects NP cells, the NP cells release noninfectious HIV-1 virions that appear to have a normal protein and RNA composition but that are irreversibly altered in a manner that inhibits reverse transcription in target cells (9,13,17,24,36,44,53).Two lines of evidence suggest that for Vif to function, the protein must interact with an unknown cellular factor that occurs in NP cells. Recently, we and others found that the NP phenotype is dominant in P ϫ NP heterokaryons (31,42). This implies that NP cells contain a pathway for inactivating HIV-1, that this pathway is absent or incomplete in P cells, and that Vif neutralizes this pathway. Additionally, it was reported that Vif proteins of primate lentiviruses function only in the lymphocytes of species closely related to the viral hosts (41). This finding implies that for Vif to function, rather than associating with a viral factor, the protein must bind to a cellular factor that changes during evolution (41). Consequently, we hypothesized that P cells lack this Vif-binding target factor and/or another component or components of the pathway that inactivates HIV-1 lacking vif.We used the L40 yeast system (26) with full-length HXB2 strain Vif as bait in a two-hybrid screen of a human leukocyte Matchmaker cDNA library (Clontech, Palo Alto, Calif.). This system provides high-level expression of LexA-B domain (BD)-bait and Ga14-A domain (AD)-prey proteins, thereby enhancing weak bait-prey associations. Although several prey were of potential interest, we focused principally on Sp140 because it is lymphocyte and macrophage specific and is induced by gamma interferon (3,4,15). Additionally, Sp140 is closely related to the widely expressed protein Sp100, which has been implicated in defenses against other viruses and associates...

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