Timothy W. Nilsen

Unequal distribution of N6-methyladenosine in influenza virus mRNAs.

Influenza virus mRNA is posttranscriptionally methylated at internal adenosine residues to form N6-methyladenosine (m6A). It has been previously shown that there is an average of three m6A residues per influenza virus mRNA (R. M. Krug, M. A. Morgan, and A. J. Shatkin, J. Virol. 20:45-53, 1976). To determine the distribution of m6A in the different influenza virus mRNAs, we purified six of the mRNAs by hybrid selection, digested them with nuclease, and determined their methylation patterns by high-pressure liquid chromatography. The amount of m6A in the different mRNAs varied from one in matrix to eight in hemagglutinin.

Unusual base-pairing of newly synthesized DNA in HeLa cells

A fraction of DNA in which both newly replicated strands are base-paired to each other has been detected in DNA extracted from HeLa cells synchronized in S phase. This fraction was observed by neutral CsCl gradient analysis of sheared DNA labeled with BrdUrd and [3H]dC. A significant proportion of the label was found in DNA substituted in both strands with BrdUrd. The formation of this HH DNA† is not the result of synchronization artifacts. HH DNA is found in highest proportion in early S phase, but small amounts can be detected throughout S phase. The proportion of HH DNA averages 48% of the extracted radioactive DNA when cells are briefly labeled in the presence of 1 mm-hydroxyurea, an inhibitor of DNA synthesis. Other inhibitors, like cycloheximide or arabinosyl cytosine, do not enhance the proportion of HH DNA. Pulse-chase experiments show that HH DNA is quantitatively chased into singly substituted DNA. HH DNA has a size of between 5500 and 9000 base-pairs and does not contain a high proportion of either palindromic or reiterated sequences. In vivo crosslinking experiments with trimethylpsoralen indicate that HH DNA is formed by branch migration during DNA isolation. This branch migration does not occur at replication forks and is not due to the presence of BrdUrd in newly synthesized DNA. Furthermore, HH DNA is not formed as a result of extrachromosomal replication or strand displacement from the replication bubble. We suggest that HH DNA is formed from DNA involved in the initiation of synthesis at the individual replicon level and suggest a possible model for its formation.

Cross-linking of viral RNA by 4′-aminomethyl-4,5′,8-trimethylpsoralen in HeLa cells infected with encephalomyocarditis virus and the tsG114 mutant of vesicular stomatitis virus

Abstract The presence of double-stranded RNA (dsRNA) of viral origin in infected cells is controversial. We established that in intact HeLa cells infected with encephalomyocarditis virus (EMCV) viral RNA can be cross-linked with 4′-aminomethyl-4,5′,8-trimethylpsoralen (AMT). This compound intercalates into dsRNA and forms cross-links upon irradiation with uv light. Addition of AMT to EMCV-infected cells, followed by irradiation, resulted in a dose-dependent inhibition of viral RNA synthesis, This inhibition was correlated with the cross-linking of nascent RNA strands to template strands of the viral replicative intermediate of EMCV. In contrast, treatment with AMT of vesicular stomatitis virus (VSV)-infected cells had no effect on viral mRNA synthesis and no cross-linking of viral RNA could be detected. The synthesis of viral RNA by the ts G114 mutant of VSV at the nonpermissive temperature, however, was inhibited by AMT. In cells infected with this mutant, cross-linked viral RNA could be detected. These results are discussed with regard to the role of dsRNA in cellular responses to viral infection mediated by interferon.

INTERFERON‐INDUCED ENZYMES: ACTIVATION AND ROLE IN THE ANTIVIRAL STATE*

Two enzymes are induced in mammalian and avian cells by interferon (IF): an oligoadenylate polymerase and a protein kinase.’ Both enzymes are activated by double-stranded RNA (dsRNA) and utilize ATP as substrate. The oligoadenylate polymerase converts ATP into a series of oligonucleotides characterized by 2’5’phosphodiester bonds and designated 2’5’oligo(A) or 2,5A. The 2,5A in turn activates an endonuclease present in extracts of control and IF-treated cells, which degrades RNA.’ The protein kinase (PI&,) phosphorylates two polypeptides of M, 38.000 and about 70,000. The first is the a subunit of initiation factor eIF-2; this phosphorylation is thought to inhibit the initiation of protein synthesis. We have reported studies on the activation of the endonuclease by 2,5A in HeLa cell extracts’ and on the kinetics of induction by IF of the 2,5A polymerase and PKd:4; we have developed sensitive and quantitative assays for these enzymatic activitie~‘.~ and have proposed a mechanism that accounts for the preferential degradation of viral RNA at the level of the viral replicative complexes of RNA viruses.‘ More recently, we have investigated the structural requirements of dsRNA for the activation of 2,5A polymerase and PKd? and the substrate specificity of the 2,5A-activated endonuclea~e .~ These studies have allowed us to define some possible roles of these enzymatic activities in the antiviral state. We present here a concise description of this recent work from our laboratory. The limitation of space does not allow us to review this active field of research in a comprehensive way: only references to work directly related to our findings will be given. Other relevant references can be found in a recent review.’

Chapter 21. Isolation of Histone Messenger RNA and Its Translationin Vitro

Publisher Summary This chapter discusses: (1) the isolation of histone mRNA from HeLa cells and marine eggs and embryos; (2) its translation in cell-free proteinsynthesizing systems obtained from mouse ascites, rabbit reticulocytes, and wheat germ; and (3) the analysis of the products of in vitro translation. The procedures discussed in the chapter can be used to isolate and translate any mRNA present in discrete amounts in different cell types and tissues. The techniques involved are quite straightforward, and success in the isolation and translation of mRNA depends on the care with which RNA and protein synthesizing systems are prepared. The synthesis of histones in embryos of marine invertebrates and animal cells in culture is regulated both at the level of transcription and translation of messenger RNA (mRNA). Histone mRNA is present in the cytoplasm of sea urchin eggs but is not translated until after fertilization. Very little protein synthesis takes place prior to fertilization, however, and histone synthesis may be under the control of a general regulatory mechanism that prevents the translation of all mRNAs in unfertilized eggs. After fertilization, histone mRNA is actively synthesized and both newly synthesized and maternal histone mRNA are translated. A different type of regulation of histone synthesis occurs in Spisula solidissima eggs, as no histone mRNA is found among the maternal mRNAs and active histone synthesis upon fertilization seems uniquely dependent on de novo transcription of histone templates.

2,′5′-OLIGO(A): A MEDIATOR OF VIRAL RNA CLEAVAGE IN INTERFERON-TREATED CELLS?

The replicative complex (RC) of encephalomyocarditis virus has been isolated from infected HeLa cells by isopycnic centrifugation. The RC prepared in this way promotes the synthesis of 2′5′ oligo(A) in extracts of interferon-treated HeLa cells. The 2′5′ oligo(A) synthesized activates an endonuclease which degrades mRNA. These results are discussed with reference to the proposed localization of the 2′5′oligo(A) polymerase/endonuclease system on the RC formed in interferon-treated cells.

An Unusual Structure Implicated in Initiation of DNA Synthesis

Despite the fact that rapid advances have been made in deciphering the detailed molecular mechanism of DNA replication in prokaryotes (1), parallel progress has not been made in eukaryotic systems. This has been due in great part to the inherent complexity of eukaryotic cells, the lack of well characterized enzymes, and the lack of specific mutations, the availability of which has greatly facilitated investigations in prokaryotes. Since the publication in 1968 of the replicon model of eukaryotic DNA replication (2), very little has been established in terms of defining the replicon at the molecular level. In particular, there is almost no knowledge concerning either possible chromosomal determinants or mechanisms involved in the initiation of DNA synthesis at the level of individual replicons. In their 1968 paper (2) Huberman and Riggs state: “It is now well established that, at the level of resolution of whole chromosome auto-radiography, regions of initiation and termination of DNA replication are reproducibly and heritably controlled. However, neither whole chromosome autoradiographic studies nor the studies presented in this paper have been able to determine whether sites of initiation (origins) are reproducible at the atomic level. The