Early Cytoplasmic RNA Research Articles

Translational control of early protein synthesis at the late stage of vaccinia virus infection

The cytoplasmic RNA obtained at 1 hr (early) or 6 hr (late) after infection of Ehrlich ascites tumor (EAT) cells with vaccinia virus stimulated to similar extent the incorporation of [ 35S]methionine into proteins when translated in a micrococcal nuclease-treated lysate obtained also from EAT cells. Comparison on SDS-polyacrylamide gels of the in vitro translation products at nonsaturating RNA concentrations with those obtained in vivo by pulse labeling the infected cells showed that essentially all the early in vivo labeled proteins were made upon translation of the early RNA. Analysis of the cells pulse labeled in vivo at the late time demonstrated the presence of late proteins and of only residual amounts of some early proteins. The in vitro translation products of the late RNA, however, not only contained the late proteins but also appreciable amounts of several early proteins. This observation suggested that some early mRNA species were still present at the late time after infection but not translated in vivo, at least not to the same relative extent. In parallel, hybridization competition experiments revealed that the late cytoplasmic RNA contained almost twice as much early sequences as did the early cytoplasmic RNA. These results suggest that at the late stage of vaccinia virus infection the synthesis of the early proteins is subjected to translational control.

Host range deletion mutant of vaccinia virus defective in human cells

A vaccinia virus host range (hr) mutant unable to multiply in most human cell lines assayed has been isolated after nitrous acid mutagenesis. This mutant also displayed various alterations in plaque morphology and cytopathic effect on permissive cell lines. The block in multiplication in human cells was at an early stage of infection. Only early cytoplasmic RNA and early viral-induced polypeptides could be detected and there was no evidence of morphological events within viroplasms. Protein synthesis constantly declined as infection proceeded suggesting either that early viral mRNA was unstable or that the mutant virus was defective in a step necessary for the maintenance of translation. Restriction enzyme digestion of DNA from the hr mutant revealed that it was deleted of about 12.6 × 10 6 daltons in the left-hand end of the genome leaving intact a fragment containing the terminal crosslink. In both permissive and nonpermissive cells the mutant failed to induce the synthesis of an early 42K polypeptide which could thus be encoded within a region of the deleted sequence. The failure to segregate the various phenotypic and biochemical properties of the hr mutant from one another through recombination with a temperature sensitive mutant indicated that the pleiotropic characteristics of the mutant virus were due to the deletion.

Regulation of an early RNA transcribed from the transforming segment of the adenovirus 2 genome

Early cytoplasmic RNA transcribed from the left end of the adenovirus 2 genome (region 1) is composed of two major size classes which migrate as 22 and 13 S RNAs. Previous studies demonstrated that translational inhibitors (such as cycloheximide) stimulate a 10-fold accumulation of early viral RNAs labeled 3–5 hr after infection. One notable exception to the cycloheximide effect is the 22 S RNA transcribed from region 1. To clarify this differential effect of cycloheximide, the metabolism of region 1 RNA was compared in several experiments. The kinetics of accumulation of these RNAs in the absence of drug was analyzed beginning 3 hr after infection. The 13 S size class accumulated more rapidly than the 22 S RNA; after 2 hr of labeling, there was a 3-fold excess of 13 S RNA as compared with cultures labeled for 3 hr. Chase experiments using either actinomycin D in the absence of cycloheximide or cold uridine when cycloheximide was present demonstrated that the 22 S RNA was more stable than the 13 S size class. Thus, a 22 S species is unlikely to serve as precursor to a major smaller RNA. Early RNAs synthesized in the presence of the amino acid analogues canavanine or β-hydroxynorvaline also contained increased amounts of region 1 13 S RNA but not the 22 S size class. To determine the effect of delayed cycloheximide addition, infections were performed in the absence of inhibitors and cycloheximide was then added 4 hr later. Under these conditions, the 22 S RNA synthesized from 4.5–6.5 hr accumulated in increased amounts, comparable to the other early RNAs. The results suggest that a protein synthesized early in infection regulates the cytoplasmic concentration of 22 S RNA.

Transcription map for adenovirus type 12 DNA.

The regions of the adenovirus type 12 genome which encode l- and r-strand-specific cytoplasmic RNA were mapped by the following procedure. Radioactive, intact, separated complementary strands of the viral genome were hybridized to saturating amounts of unlabeled late cytoplasmic RNA. The segments of each DNA strand complementary to the RNA were then purified by S1 nuclease digestion of the hybrids. The arrangement of the coding regions of each strand was deduced from the pattern of hybridization of these probes to unlabeled viral DNA fragments produced by digestion with EcoRI, BamHI, and HindIII.. The resulting map is similar, if not identical, to that of adenovirus type 2. The subset of the late cytoplasmic RNA sequences which are expressed at early times were located on the map by hybridizing labeled, early cytoplasmic RNA to both unlabeled DNA fragments and unlabeled complementary strands of specific fragments. Early cytoplasmic RNA hybridized to the r-strand to EcoRI-C and BamHI-B and to the l-strand of BamHI-E. Hybridization to BamHI-C was also observed. The relative rates of accumulation of cytoplasmic RNA complementary to individual restriction fragments was measured at both early and late times. Early during infection, most of the viral RNA appearing in the cytoplasm was derived from the molecular ends of the genome. Later (24 to 26 h postinfection) the majority of the newly labeled cytoplasmic RNA was transcribed from DNA sequences mapping between 25 and 60 map units on the genome.

Loop structures in hybrids of early RNA and the separated strands of adenovirus DNA.

Separated strands of adenovirus DNA were annealed with early cytoplasmic RNA and visualized in the electron microscope. DNA-RNA duplex regions within the DNA filaments could be recognized by their heavy contour. This contour was often interrupted at distinct locations by loops of displaced, single-stranded DNA. Loops have been observed and mapped in all four early regions of the genome. The structures appear to signal hitherto unknown mechanisms of eukaryotic gene expression.

Species identification and genome mapping of cytoplasmic adenovirus type 2 RNAs synthesized late in infection.

Adenovirus type 2 cytoplasmic RNAs synthesized late in productive infection were resolved by electrophoresis on formamide gels. Regions of the adenovirus 2 genome specifying RNAs of distinct size were determined by hybridization to specific DNA fragments generated by cleavage with endo R.EcoRI and endo R.SmaI. From these studies 13 distinct viral RNA species were identified. A 26S to 28S size class and a 21S to 23S size class were each found to consist of four distinct RNA species. Three RNA species were identified in a 16S to 18S size class, and a fourth size class, 11S to 13S, was resolved into two components. The SmaI-D region (0.38 to 0.51 on the unit genome) and the EcoRI-F, D region (0.70 to 0.83) of the genome were found to code for multiple transcripts. Three RNAs (28S, 22S, and 18S) are specified by SmaI-D, and four components, 28S, 22S, 18S, and 16S, are encoded by EcoRI-F,D. The RNA represented by each set of multiple transcripts exceeds the coding capacity of the respective region, and the species within each set of RNAs appear to contain common sequences. The relationship between the cytoplasmic RNA species synthesized at late times and early cytoplasmic RNAs was determined by hybridization-inhibition experiments. The multiple transcripts encoded by the EcoRI-D fragment were found to contain sequences that are present in early cytoplasmic RNA. These studies enabled preparation of a map which accounts for transcription of approximately 67% of the r strand of the adenovirus 2 genome.

A map of cytoplasmic RNA transcripts from lytic adenovirus type 2, determined by electron microscopy of RNA:DNA hybrids.

Abstract We have used electron microscopic RNA loop mapping to determine, to within 200 nucleotides, the chromosome coordinates of the 5′ and 3′ ends of adenovirus type 2 transcripts isolated from the cytoplasm of productively infected human KB cells. The major blocks of transcription and many of the individual transcripts have discrete lengths and well defined 5′ and 3′ termini. Early cytoplasmic RNA is transcribed from four separate regions of the adenovirus genome, in map intervals 1.3–11.1, 62.4–67.9, 78.6–86.1 and 91.5–96.8. The most frequent late RNA loop occurred between coordinates 51.9 and 62.2. A late transcript complementary to interval 86.3–91.5 formed a characteristic R loop with a long branch of displaced RNA from coordinate 89.8–91.5, possibly a result of secondary structure near coordinate 89.8. Other late cytoplasmic RNA loops were located between coordinates 11.1 and 14.9, and in the intervals from 30–52 and 68–83. Transcripts from several regions of the chromosome had variable lengths but common 5′ or 3′ termini. Few R loops were seen between map coordinates 15 and 30. Switches between template strands were observed at map positions 11.2, 62.4 and 91.5. Many of the R loops have been correlated with gene products mapped previously by translation of RNA complementary to different portions of the chromosome.

Adenovirus transcription. IV. Synthesis of viral-specific RNA in human cells infected with temperature-sensitive mutants of adenovirus 5.

Cytoplasmic RNA sequences produced in HeLa cells infected with the adeno-virus 5 temperature-sensitive mutants ts1, ts2, ts9, ts17, ts18, ts19, ts20, ts22, ts49, ts36, and ts125 were characterized by hybridization to DNA probes generated by strand separation of restriction endonuclease fragments of adenovirus 5 DNA. Two ""early'' mutants defective in DNA synthesis, ts125 and ts36, fail to make wild-type levels of all previously reported classes of late RNA at the nonpermissive temperature. At 40.5 degrees C, both ts125 and ts36 synthesize a wild-type complement of early cytoplasmic RNA 16 h after infection. Under these conditions, no ""late'' cytoplasmic RNA sequences were observed. Similarly, nuclear RNA present in these cells resembled early cytoplasmic RNA rather than late nuclear RNA. All the late adenovirus 5 temperature-sensitive mutants synthesized normal wild-type levels of late cytoplasmic RNA at the nonpermissive temperature, except ts2, which appears to overproduce certain cytoplasmic species.

Adenovirus type 12-specific RNA sequences during productive infection of KB cells.

The complementary strands of adenovirus type 12 DNA were separated, and virus-specific RNA was analyzed by saturation hybridization in solution. Late during infection whole cell RNA hybridized to 75% of the light (1) strand and 15% of the heavy (H) strand, whereas cytoplasmic RNA hybridized to 65% of the 1 strand and 15% of the h strand. Late nuclear RNA hybridized to about 90% of the 1 strand and at least 36% of the h strand. Double-stranded RNA was isolated from infected cells late after infection, which annealed to greater than 30% of each of the two complementary DNA strands. Early whole cell RNA hybridized to 45 to 50% of the 1 strand and 15% of the h strand, whereas early cytoplasmic RNA hybridized to about 15% of each of the complementary strands. All early cytoplasmic sequences were present in the cytoplasm at late times.

Strand-Specific Transcription of Polyoma Virus DNA Early in Productive Infection and in Transformed Cells

The DNA strand origin of nuclear and cytoplasmic polyoma-specific RNA in productively infected mouse cells and in a line of polyoma-transformed hamster cells was determined by hybridization of unlabeled RNA with radioactively labeled separated strands of polyoma DNA. Early in the productive cycle (10 h postinfection) nuclear viral RNA is complementary to only about 40% of the E strand of viral CNA. No RNA complementary to the L strand was detected even when the RNA was first self-annealed to enrich for possible minor species. Early cytoplasmic RNA is complementary to the same 40% of the E strand. Thus, only that part of the poloma genome which codes for early virual messenger RNA appears to be transcribed. Late in infection, nuclear viral RNA is complementary to most or all of the L strand and to at least 60% of the E strand. Late cytoplasmic viral RNA hybridizes to 40 to 45% of the E strand and 50 to 55% of the L strand. The transformed cell nuclear viral RNA is complementary to 60% of the E strand, whereas cytoplasmic RNA is complementary to 40% of the E strand and comprises the same polyoma-specific sequences as are found in RNA early in productive infection. No L strand transcripts could be detected. Thus, in the transformed cells and late in productive infection, viral RNA sequences in the cytoplasm are a specific subset of those in the nucleus.

Identification of early adenovirus type 2 RNA species transcribed from the left-hand end of the genome.

Unique fragments of adenovirus type 2 DNA generated by cleavage with endonuclease R-Eco RI or endonuclease R-Hsu I (Hin dIII) were used to map cytoplasmic viral RNAs transcribed early in productive infection. Radioactive early viral RNA was first fractionated by polyacrylamide gel electrophoresis. Eluted viral RNAs were then tested for hybrid formation with DNA fragments. The Eco RI DNA fragment (Eco RI-A) which contains the left-hand 58% of the genome hybridized 13S and 11S RNAs. More detailed mapping of these RNAs was achieved by hybridization to the seven Hsu I fragments of Eco RI-A. The early RNA annealed only to Hsu I-G and C, two fragments which comprise the extreme left-hand 17% of the genome. Viral RNA migrating as 13S and 11S annealed to Hsu I-G, and 13S RNA annealed to Hsu I-C. A 13S RNA is transcribed from Eco RI-A late in infection (18 h). Hybridization-inhibition studies with Eco RI-A DNA, early cytoplasmic RNA, and 3H-labeled 13S late RNA demonstrated that this RNA synthesized at late times is an early RNA species which continues to be synthesized in large amounts at 18 h. This 13S RNA synthesized at 18 h hybridized to Hsu I-C but not to Hsu I-G DNA. These results establish that the 13S RNAs transcribed from Hsu I-G and C at early times must be different species.

Analysis of early adenovirus 2 RNA using Eco R-R1 viral DNA fragments

Adenovirus 2 RNA synthesized early in productive infection was analyzed by RNA-DNA hybridization. Hybridization experiments were performed with adenovirus 2 DNA and wit, the six adenovirus 2 DNA fragments generated 0y digestion with the restriction endonuclease Eco R.R1. Duplex formation between RNA and -32P-labeled viral DNA was assayed by S(1) nuclease digestion. RNA from the cytoplasm annealed 12 percent of the total viral DNA and the following percentage of each of the R.R1 fragments: 6 percent of R1-A, 24 percent of R1-B, 0 percent of R1-F, 40 percent of R1-D, 13 percent of R1-E, and 22 percent of R1-C. The early cytoplasmic RNA is composed of two sequence classes: class I, present in greatly reduced quantities at late times in infection (18 h), and class II, which remains at high concentrations at 18 h. In hybridization-inhibition experiments, hybridization of class II RNA is inhibited by late cytoplasmic RNA, whereas hybridization of class I RNA is not blocked by late cytoplasmic RNA (J. J. Lucas and H. S. Ginsberg, 1971; E.A. Craig and H. J. Raskas, 1971). To determine the location of class I and II sequences on the genome, membrane bound DNA fragments were used in hybridization-inhibition experiments. These studies demonstrated that the early cytoplasmic transcripts of R1-D belong to class II, whereas R1-C transcripts are class I sequences. The cytoplasmic RNAs transcribed from fragments A and B contain both class I and class II sequences. Analysis of cytoplasmic RNA fractionated by size demonstrated that the class I sequences include a 19 S RNA transcribed from R1-B and class II sequences include a 20S RNA derived from R1-D. Nuclear RNA purified from cultures early in infection was annealed with -32P-labeled R1 fragments. With all six fragments the nuclear RNA annealed as much or more of the DNA than did cytoplasmic RNA. Eco R1-F annealed at least 25 percent with early nuclear RNA, whereas no sequences homologous to R1-F were detected in early cytoplasmic RNA. When cultures were labeled from 2 to 6 h after infection, at least 5 percent of the -3H-labeled early nuclear viral RNA annealed to Eco R1-F. Some of these nuclear transcripts from R1-F appear to be covalently linked to sequences transcribed from a contiguous region of the genome (Eco R1-B). 8.4 percent of the RNA selected by hybridization of R1-F reannealed to R1-B, whereas no more than 1.5 percent reannealed to R1 fragments A, D, E, or C.

Mapping of adenovirus 2 RNA sequences in lytically infected cells and transformed cell lines.

The strands of the six EcoRI fragments and the HpaI fragments E and C of Ad2 DNA were separated by electrophoresis in agarose gels. Using 32P-labeled fragment strands in solution hybridization experiments, the fraction of each strand complementary to RNA extracted from infected or transformed cells was assayed by chromatography on hydroxylapatite. In this manner, a tentative map of the cytoplasmic RNA sequences has been constructed for viral RNA extracted from cells both early and late during infection (see Fig. 16; in the map shown, the two strands of Ad2 are named the r and l strands following the bacteriophage convention). Since early cytoplasmic RNA anneals to four distinct regions of the genome, Ad2 probably codes for at least four early gene functions. Summation experiments have shown that all RNA sequences found in the cytoplasm of cells early during infection are also present in the cells' cytoplasm at late times. Viral RNA sequences in five independently isolated and cloned transformed rat cell lines were also mapped on the Ad2 genome. One class of Ad2-transformed rat cells contains RNA sequences complementary to only the segment of Ad2 DNA from 0.03-0.10 on the physical map, and this corresponds to one of the four regions of the genome expressed early during infection. If a viral gene product is necessary to maintain the transformed phenotype of the cell or codes for the virus-specific tumor (T) antigen, this genetic information must be at the left end of the genome (see Fig. 16). The two other classes of Ad2-transformed rat cells contain viral RNA sequences complementary to two or three of the regions of the genome transcribed into early cytoplasmic RNA. At both early and late times during the lytic cycle, the nucleus of the infected cell contains viral RNA sequences that are not transported to the cell's cytoplasm, suggesting that RNA processing and selection may play a role in the regulation of viral mRNA production.

Biochemical studies on adenovirus multiplication: XV. Transcription of the adenovirus type 2 genome during productive infection

The viral genome is transcribed sequentially in adenovirus type 2-infected KB cells. About one-fifth of the viral genome is transcribed prior to DNA synthesis by 6 hours after infection (“early” virus-specific RNA). All early virus-specific RNA sequences are present “late” after infection (18 hours). Early cytoplasmic RNA, labeled during a 30-min pulse with 32P-labeled orthophosphate, has a lower G + C content (52–54% G + C) than early nuclear virus-specific RNA (58–59% G + C). Moreover, the cytoplasm contains only a portion of the virus-specific nucleotide sequences synthesized in the nucleus at 6 hours after infection. Late after infection, pulse-labeled nuclear and cytoplasmic virus-specific RNA possess the same base composition and the same nucleotide sequences. These findings suggest that the regulation of viral gene expression early after infection involves both transcription and translation controls.