Replication of Sindbis virus. VII. Location of 5-methyl cytidine residues in virus-specific RNA

Sindbis virus intracellular 26 S and 49 S RNA contain internal 5-methyl cytidine (m⁵C) residues. In [methyl-³H]methionine-labeled Sindbis virus RNA, an average of 19% of the methyl-³H label in 26 S RNA is in m ⁵C, whereas in intracellular 49 S RNA, an average of 8% of the methyl-³H label is in m⁵C, the remainder of the label being in the cap. Sindbis virion 49 S RNA contains much less m⁵C than intracellular 49 S RNA extracted from the same cells. In 26 S RNA, m⁵C residues occur in five oligonucleotides, four of which have been identified as having base compositions of C₄A₄UG, C₆A₂UG, C₃A₂G, and C₂AG, which are found distributed between two major locations, one approximately 4000 nucleotides from the 3′ end and the other about 1200 nucleotides from the 3′ end (out of a total length of 5000 nucleotides). The m⁵C containing nucleotide C₂AG is found only at the former location and the oligonucleotide of composition C₄A₄UG (which contains 40–50%. of the total m⁵C in 26 S RNA) is restricted to the latter location; the distribution of methyl label between the two locations suggests that each region contains at least two of the methylated sequences. Although there appear to be five specific sites for methylation on the 26 S RNA, only a minority of these sites are modified, as each 26 S RNA molecule contains an average of less than one m⁵C. Sindbis virus 26 S RNA isolated from both polysomes and nonpolysomal ribonucleoproteins contain equal amounts of m⁵C. 49 S RNA isolated from polysomes contains 60–80% more m⁵C than does 49 S RNA which is isolated primarily from nucleocapsids. The m⁵C residues thus may be somehow involved in translating the viral RNA.     

Selective detection of adenosine A1 receptor-dependent G-protein activity in basal and stimulated conditions of rat brain [35S]guanosine 5′-(γ-thio)triphosphate autoradiography

[³⁵S]Guanosine 5′-(γ-thio)triphosphate autoradiography is a novel technique to detect receptor-dependent activation of G-proteins in brain tissue sections. While an increasing number of reports using this approach are beginning to appear, little effort has been directed to the identification of factors responsible for the heterogeneously distributed [³⁵S]guanosine 5′-(γ-thio)triphosphate signal in basal conditions. The present study demonstrates that endogenously formed adenosine generates a widespread and prominent adenosine A₁ receptor-dependent signal in basal conditions using this technique. Treatment of rat brain tissue sections with the A₁-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine dose-dependently (ec₅₀<10 nM) suppressed basal [³⁵S]guanosine 5′-(γ-thio)triphosphate binding in a region-specific manner, an effect fully mimicked by the adenosine-depleting enzyme adenosine deaminase, and less so by the A₁ antagonist cirsimarin and by caffeine. That adenosine was continuously formed during the incubation is supported by the constant requirements of adenosine deaminase in order to suppress basal radioligand binding and further by the fact that low micromolar concentrations of adenine nucleotides evoked only adenosine-mimicking and fully 8-cyclopentyl-1,3-dipropylxanthine-sensitive binding responses. In the presence of adenosine deaminase, all responses to adenine nucleotides were abolished, indicating that prior conversion to adenosine was required. Upon stimulation, this technique selectively detected A₁ receptor-activated G-proteins, as the non-selective agonists adenosine and 2-chloroadenosine and the A₁-selective agonist N⁶-p-sulfophenyladenosine all evoked only 8-cyclopentyl-1,3-dipropylxanthine-sensitive responses in identical gray matter areas, and also in several white matter areas such as the corpus callosum, anterior commissure, optic tract and cerebellar white matter. Dose–response studies revealed region-specific differences in the magnitude of A₁ receptor-stimulated G-protein activation, with the highest response (nine-fold over basal) detectable in the hippocampus. No response to the A_2A-selective agonist 2-[(2-aminoethylamino)carbonylethylphenylethylamino]-5′-N-ethylcarboxamidoadenosine or the A₃-selective agonist 2-chloro-N⁶-(3-iodobenzyl)-adenosine-5′-N-methyluronamide was detected in any region.These data reveal that a significant amount of noise inherent to [³⁵S]guanosine 5′-(γ-thio)triphosphate autoradiography can be eliminated by removal of the adenosine signal, a step likely facilitating detection of responses to other receptors. Furthermore, the data establish [³⁵S]guanosine 5′-(γ-thio)triphosphate autoradiography as a novel and selective approach to directly assess A₁ receptor–G-protein coupling in anatomically defined regions of the central nervous system.     

Methylated 5′-terminal sequences of vaccinia virus mRNA species made in Vivo at early and late times after infection

The 5′ terminals of vaccinia virus mRNAs synthesized in vivo contain additional sites of methylation not found in vaccinia virus mRNAs synthesized in vitro, suggesting the possibility of host cell modifications. mRNAs synthesized in vitro by enzymes present in purified vaccinia virions contain 7-methylguanosine (m⁶G) at their 5′ terminals in structures of the type m⁷G(5′)pppN₁^m-N₂ in which N₁^m is exclusively 2′-O-methyladenosine(A^m) or 2′-O-methylguanosine (G^m) and N₂ is not methylated. Two additional sites of methylation were found in the 5′ terminals of vaccinia virus-specific mRNAs synthesized in HeLa cells. The adenosine residue in the penultimate position is frequently doubly methylated to form N⁶,2′-O-dimethyladenosine (m⁶A^m) and N₂ is often methylated at the 2′ position to form structures of the type m⁷G(5′)pppN₁^m-N₂^m. Both of these modifications also occur in the mRNA of the uninfected host cell. In virus-specific mRNAs synthesized at early times after infection, N₁^m is either m⁶A^m,A^m, or G^m, and N₂^m is A^m, U^m, C^m, G^m, or m⁶A^m. Late vaccinia virus-specific RNA has a much higher proportion of m⁶A^m and A^m and correspondingly less G^m in the penultimate (N₁^m) position compared to early mRNA. In addition, late mRNA has relatively little methylation at the third (N₂) position of which a very high proportion is A_m. Both temporal classes of viral mRNA differ from the mRNAs of uninfected HeLa cells in lacking U^m or C^m in the penultimate position and having far more A^m in the third position. In early and late virus-specific mRNA species there were approximately one 5′ terminal per 1000 nucleotides and one 5′ terminal per 1250 nucleotides, respectively.     

Deletions and insertions in the immunity region of coliphage lambda: revised measurement of the promoter-startpoint distance

Plaque-forming λcI^? mutants which carry internal deletions in the immunity region, including genes cI and rex, were isolated. Among seven independent isolates, only three demonstrably different deletions were obtained. The largest deletion (KH54 or KH115), which eliminates almost all of the DNA between promoters p_L and p_R, spans the distance from 74.1 to 78.4 %λ, as measured in relation to the left (0 %λ) and right (100 %λ) termini of the mature λ DNA molecule. There were four representatives of a second (74.7–77.7%λ) and one of a third (75.2–78.1%λ) deletion. In addition, a mutant carrying a 1400-nucleotide pair-long insertion in gene cI was isolated.The cI-rex deletions served as markers for more accurate measurements of the physical positions of the endpoints of several bio insertions, a dy plasmid, and the endonucleolytic cleavage sites for the restriction nucleases by electron micrography of DNA heteroduplexes. This led to a reevaluation of our earlier estimate of the interval length between the p_L promoter mutation sex1 and the s_L startpoint for the major leftward operon of λ. In agreement with the independent results of others, we find that the p_L to s_L distance is within the limits of 24–62 nucleotide pairs (s_L-sex1) or 11 to 45 nucleotide pairs (s_L-HinII cut in o_L).     

Molecular cloning of African swine fever virus DNA

African swine fever virus DNA (about 170 kbp) was cleaved with the restriction endonuclease EcoRI and most of the resulting 31 fragments were cloned in either the phage vector λWES.λB or the plasmid pBR325. Three fragments were not cloned in those vectors, the largest fragment EcoRI-A (21.2 kbp) and the two crosslinked terminal fragments, EcoRI-K′ and D′. Endonuclease SalI cut fragment EcoRI-A into three pieces which were cloned in plasmid pBR322. The two terminal EcoRI fragments were cloned after removal of the crosslinks with nuclease S1 and addition of EcoRI linkers to the fragment ends. The complete library of the cloned fragments accounted for about 98% of ASF virus genome, the missing sequences being those removed by the nuclease S1 in the process of cloning the terminal fragments.     

Isolation of foot-and-mouth disease virus messenger RNA from membrane-bound polyribosomes and characterization of its 5' and 3' termini.

A membrane-bound fraction prepared from foot-and-mouth disease virus (FMDV)-infected baby hamster kidney cells included [³H]uridine-labeled 37 S viral messenger RNA (mRNA) and a 20 S RNA species that is partly ribonuclease resistant. The mRNA appeared to contain both a polyadenylic acid (poly (A)) and a polycytidylic acid (poly (C)) tract as demonstrated by its binding to oligo(dT)- and oligo(dG)-cellulose, respectively. Paper chromatography of an RNase T₂ digest of the [³H]adenosine-labeled poly(A) fragment from mRNA showed that the poly(A) is at the 3′ end of the mRNA and is approximately 45 nucleotides long. Digestion of ³²P-labeled FMDV mRNA with RNase A and T₂ yielded pUp as the 5′ terminus; RNases A and T₂ digested FMD virion RNA lacked this structure.     

The extreme 5'-terminal sequences of sindbis virus 26 and 42 S RNA.

The messenger RNA species specified by Sindbis virus in infected cells, 26 and 42 S RNA, have been characterized with regard to their extreme 5′-terminal sequences. (Methyl-³H)-Labeled m⁷G in “caps” was used to detect cap-containing, and hence 5′-terminal, oligonucleotides after digestion with ribonuclease A, or ribonuclease T₁, plus phosphatase. Digests were fractionated by DEAE column chromatography, by cellulose acetate electrophoresis, and by DEAE paper electrophoresis. The latter two systems were also applied sequentially to ³²P-labeled samples to generate oligonucleotide fingerprints. The cap-containing oligonucleotide from T₁-plus-phosphatase digests of 26 S RNA was isolated and shown to be m⁷ GpppApUpG. The homologous oligonucleotide from such a digest of 42 S RNA behaved as if it were one pyrimidine residue larger, whereas cap-containing oligonucleotides from ribonuclease A-plus-phosphatase digests of the two RNA species had identical chromatographic and electrophoretic properties. We infer from these and earlier findings that Sindbis virus-specified 42 S RNA terminates in m⁷GpppApUpYpG. These results support the idea that 26 S RNA and 42 S RNA arise from two distinct and different initiation sites.     

Isolation and characterization of a large "hairpin" segment from avian retrovirus RNA.

Avian retrovirus RNA (both 70 S and 35 S) possesses several attributes of double-stranded (ds) RNAs. Among them are an affinity for hydroxyapatite equal to that of authentic ds RNAs and exceeding that of any other single-stranded RNA tested except hnRNA, and an unexpectedly high melting temperature of some of its sequences, indicating the presence of intramolecular base-paired regions. Limited digestion with pancreatic RNase A of Prague C strain of Rous sarcoma virus as well as B77 and RAV-2 35 S RNA yielded a product that accounted for about 7% of the total viral RNA, behaved like reovirus ds RNA when chromatographed on hydroxyapatite, possessed a T_m that was similar to that of reovirus ds RNA, was almost as susceptible to RNase III as reovirus ds RNA under conditions when reovirus messenger RNA was completely resistant, and could be isolated as a relatively homogenous component following centrifugation in sucrose density gradients or electrophoresis in formamide-containing polyacrylamide gels. Its properties were consistent with the interpretation that it is a highly (but not perfectly) base-paired hairpin about 350 base pairs long. The was mapped by determining which of various size classes of poly(A)-containing fragments of viral RNA contained it and found to be located in the region between 5000 and 6000 nucleotides from the 3′-terminus of nondefective viral RNA; this region is at, or close to, the junction of the pol and env genes. The fact that the RNA of the helper virus free Bryan high-titer strain of RSV, which lacks most of the env gene, did not yield such a hairpin fragment agrees with this conclusion.     

Terminal sequences of the genome and replicatioe-form RNA of the flavivirus west nile virus: absence of poly(A) and possible role in RNA replication

The structures of the infectious 42 S genome RNA of the flavivirus West Nile (WN) virus and of the replicative-form (RF) RNA containing 42 S RNA of positive and negative polarity have been investigated. The RF RNA has been labeled in vitro at the 3′ and 5′ termini and the terminal sequences have been determined by the mobility shift method. The results obtained indicate that both RNA molecules are exact complements of each other and that the 3′ terminus of the 42 S plus-strand RNA component of the RF RNA does not contain a poly(A) sequence but terminates with a heteropolymeric AACACAGGAUCU_OH sequence. The 3′ terminus of the 42 S minus-strand RNA has the sequence CUCACACAGGCGAACUACU_OH. Comparison of these sequences shows that both molecules contain the 3′-terminal dinucleotide CUOH and the heptanucleotide ACACAGG which is separated from the 3′-terminal dinucleotide by two and seven nucleotides in 42 S plus- and minus-strand RNA, respectively. The 42 S viral genome RNA also does not contain a 3′-terminal poly(A) sequence but terminates with the 3′-terminal sequence identified in the 42 S plus-strand RNA of the RF. Analysis of the nucleotides adjacent to the cap at the 5′ terminus of the viral genome RNA together with the 3′-terminal sequence analysis indicates that the nucleotide sequence of the viral genome RNA is identical to that of the 42 S plus-strand RNA component of the virus-specific RF RNA.     

Analyses of the genomes of prototype pichinde arenavirus and a virulent derivative of Pichinde Munchique: evidence for sequence conservation at the 3' termini of their viral RNA species

Sequence analyses of 3′-[³²P]pCp end-labeled large (L) and small (S) viral RNA species of prototype Pichinde (PIC) arenavirus and a virulent derivative of Pichinde Munchique (MUC) virus have shown that the two viral RNA species of each virus have similar 3′-terminal RNA sequences. The 3′-terminal sequence of the first 19 nucleotides of the S RNA of either virus is: 5′ … GCCUAGGAUCCACUGUGCG_OH3′. By comparison, the sequence of the first 19 nucleotides of the L RNA species of either virus is identical, except for a position 6 G residue (from the 3′ end), and a position 8 U residue. The first UAC triplet on S RNA occurs at nucleotide position 84 from the 3′ end; for the L RNA it occurs at nucleotide position 31. Whether these UAC triplets represent the initiation points of translation on viral complementary mRNA species remains to be determined.     

RNA-nascent DNA complexes in Ehrlich ascites tumor cells in vivo

When mice bearing Ehrlich ascites tumors were given a single intraperitoneal injection of [5-³H]uridine, the ascites cells incroporated radioactivity into DNA for 4 hr, while incorporation into total cellular RNA ceased after 20–30 min. Nascent DNA, isolated by Cs₂SO₄ isopycnic centrifugation 30 min, 4 hr or 50 hr after [5-³H]uridine injection contained RNA fragments. Treatments with hydroxyurea, an inhibiton hibitor of DNA synthesis, reduced the incorporation of [5-³H]uridine into the DNA-RNA complex to 43% of controls. Treatment with actinomycin D, an inhibitor of DNA-dependent RNA synthesis, abolished the incorporation of [5-³H]uridine into the DNA-RNA complex. Finally, when DNA was pulse-labeled with [¹⁴C]deoxythymidine and then denatured by heat-treatment, it banded in a region with a higher density than that of reference DNA. However, if treated with alkali, it banded at the density of reference DNA, again suggesting the presence of RNA fragments in nascent DNA. The length of the RNA chains in this DNA-RNA complex was estimated to be in the order of 70 nucleotides.     

In vitro translation of 42 S virus-specific RNA from cells infected with the flavivirus West Nile virus.

Virus-specific proteins synthesized in BHK cells infected with the flavivirus West Nile (WN) virus and in vitro using the virus-specific infectious 42 S plus-strand RNA as messenger RNA, have been studied. Mapping of the tryptic peptides indicates that the viral core protein V2 and the viral glycoprotein V3 do not share common sequences. No tryptic peptides identifiable by mapping were obtained from the viral membrane-associated protein V1. Seven apparently virus-specific intracellular proteins were detected by comparative SDS-PAGE of mock-infected and virus-infected [³⁵S]methionine-labeled cell lysates: p_i 15, p_i 20, p_i 27, p_i 37, p_i 49, p_i 71, and p_i 100 (the index i indicates the intracellular origin of the proteins, the number gives the apparent molecular weight in 10³ daltons). The proteins p_i 15 and p_i 49 represent the intracellular forms of the viral structural proteins V2 and V3, respectively. p_i 15 and V2 are not identical but differ slightly from each other. V1 has not been detected in infected cells. Mapping has shown that the other intracellular proteins (with the possible exception of p_i 37, which has not been analyzed) are unrelated to either V2 or V3 and do not share common sequences. They represent nonstructural proteins. The total molecular weight of the apparently unrelated nonstructural and structural proteins is about 290,000 daltons. Data obtained in a number of laboratories have shown that a virus-specific 42 S RNA molecule, which is structurally indistinguishable from the viral genome, probably functions as mRNA for all virus-specific proteins in vivo. This RNA has been isolated from WN virus-infected cells and translated in vitro in the wheat germ and the rabbit reticulocyte lysate system. Digestion of the total [³⁵S]methionine-labeled proteins synthesized in vitro in either of both systems with trypsin followed by peptide mapping has shown that the great majority of the resulting peptides are present in the structural proteins V2 and V3 and vice versa. No evidence was obtained for the in vitro synthesis of nonstructural proteins. Proteins synthesized in the reticulocyte lysate were fractionated by SDS-PAGE and isolated polypeptides studied by peptide mapping. Polypeptides of molecular weights between 11,500 and 90,000 daltons were obtained. Their peptide maps indicate that all polypeptides are translated from a single initiation sequence. The map of the smallest in vitro synthesized polypeptide P_retic 11.5, having a molecular weight of 11,500 daltons, was almost identical to that of V2. The map of the largest protein synthesized in significant amounts in vitro, the protein P_retic 90, was very similar to the map of a mixture of the viral proteins V2 and V3. The analyses suggest the following gene order on the 42 S RNA: 5′-terminus-V2-V3-(V1, p_i 20, p_i 27, p_i 37, p_i 71, p_i 100)-3′-terminus. The order of genes indicated in brackets remains to be determined. Some implications of these results concerning the possible mode of translation of flavivirus-specific 42 S RNA are discussed.     

The complete genome sequence of polygonum ringspot virus

The complete genome sequence of polygonum ringspot virus (PolRSV), genus Tospovirus, family Bunyaviridae, was determined. This is the first report of the complete genome sequence for a European tospovirus isolate. The large RNA of PolRSV was 8893 nucleotides (nt) in size and contained a single open reading frame of 8628 nucleotides in the viral-complementary sense, coding for a predicted RNA-dependent RNA polymerase of 330.9 kDa. Two untranslated regions of 230 and 32 nucleotides were present at the 5′ and 3′ termini, respectively, which showed conserved terminal sequences, as commonly observed for tospovirus genomic RNAs. The medium and small (S) RNAs were 4710 and 2485 nucleotides in size, respectively, and showed 99 % homology to the corresponding genomic segment of a previously partially characterized PolRSV isolate, Plg3. Protein sequences for G_N/G_C, N and NSs were identical in length in the two PolRSV isolates, while an amino acid insertion was observed for the NSm protein of the newly characterized isolate. The noncoding intergenic region of the S RNA was very short (183 nt) and was not predicted to form a hairpin structure, confirming that this unique characteristic within tospoviruses, previously observed for Plg3, is not isolate specific.