Jacob V. Maizel

Comparative sequence analysis of the 5' noncoding region of the enteroviruses and rhinoviruses.

A comparative sequence analysis of the 5 noncoding region of a subgroup of the picornaviruses, including the polioviruses, coxsackie B3, and the human rhinoviruses, reveals the conservation of certain features despite the divergence of sequence. In this subgroup, for which nine complete sequences are available, two long stretches of sequence, two pyrimidine-rich regions, and 22 hairpins are conserved. Based on these results, similar secondary structures encompassing the entire 5 noncoding regions of these viruses are predicted. The fact that sequence divergence occurred only in a manner that allowed conservation of these structures implicates a biologically functional role for this region. The possible roles it may have in the picornavirus life cycle are discussed.

A method for assessing the statistical significance of RNA folding.

We have developed a statistical method that is designed for analyzing potential RNA folded substructures. The statistical significance of RNA folding is assessed by the segment score. The segment score is defined as the difference between the lowest free energy calculated for the real biological sequence and the mean of the lowest free energies from random permutations of the real segment sequence, divided by the standard deviation of the random sample. This procedure was applied to the well-studied Escherichia coli 16S rRNA and potato spindle tuber viroid (PSTV) RNA. The results showed that the predictions of the locally significant secondary structures in these two molecules are in accord with the universally conserved local secondary structure elements (Gutell, Weiser & Noller, 1985, Prog. Nucl. Acid Res. molec. Biol. 32, 155-216; Riesner & Gross, 1985, A. Rev. Biochem. 54, 531-564). In addition, a statistical analysis indicated that the lowest free energies of a random sample set follow an approximately normal distribution. A reasonable size for the random sample set was determined statistically. Moreover, the statistical evaluation has been carried out using three different sets of energy rules--two sets (Salser, 1977, Cold Spring Harb. Symp. Quant Biol. 42, 985-1002; Freier, Kierzek, Jaeger, Sugimoto, Caruthers, Neilson & Turner, 1986, Proc. natn. Acad. Sci. U.S.A. 83, 9373-9377) take into account stacking energies and are based on experimental data and their computational extension (Salser, 1977)--the third set is a simplistic unitary matrix approach, where any base-pair is given a weight of minus one and an unpaired based is zero. The Freier energy rules usually yield the strongest indication of significant folding region. However, the results derived from paired comparisons test dont provide sufficient evidence for concluding that a different set of energy rules is effective in changing the segment score level for local stem-loop structures in the 16S rRNA.

Unusual folding regions and ribosome landing pad within hepatitis C virus and pestivirus RNAs

A statistically significant folding region is identified in the 5 untranslated region (5-UTR) of hepatitis C virus (HCV), bovine viral diarrhea virus and hog cholera virus. This unusual folding region (UFR) detected in HCV encompasses 199 nucleotides (nt) and coincides with the reported internal ribosome entry site or ribosome landing pad (RLP), as determined by the 5 and 3 deletions [Tsukiyama-Kohara et al., J. Virol. 66 (1992) 1476-1483]. The RNA structure predicted in the UFR of HCV consists of a large stem-loop and a pseudoknot. The proposed structural model is consistent with RNase sensitivity studies [Brown et al., Nucleic Acids Res. 20 (1992) 5041-5045]. Moreover, the structure is highly conserved among these divergent HCV and pestivirus RNAs. The covariation of paired bases in the helical regions offers support for the proposed structural models. The pseudoknot predicted in these UFR shares a similar structural feature to those proposed in the RLP of cardioviruses, aphthoviruses and hepatitis A virus. Based on the common structural motif, a putative base-pairing model between HCV RNA and 18S rRNA, as well as pestiviral RNAs and 18S rRNA are suggested. Intriguingly, the proposed base-pairing models in this study are comparable to those proposed in picornaviruses in terms of their folded shape and location of the predicted complementary sequences between viral RNAs and 18S rRNA. Taken together, we suggest that the common base-pairing model between the UFR detected in the 5-UTR of pestivirus and HCV and 18S rRNA have a general function in the internal initiation of cap-independent translation.

Secondary structure of poliovirus RNA: Correlation of computer-predicted with electron microscopically observed structure

A secondary structure map of poliovirus 1, strain Mahoney, RNA was determined by psoralen crosslinking the (+) strand and visualizing the structures in the electron microscope. Hairpins and looped hairpins were observed, and the size and distribution were measured. To orient map features the 3 end of the RNA was linked to polybromodeoxyuridine [poly(BUdR)]SV40 and histograms were constructed from these measurements. Secondary structure maps of the RNA were likewise constructed from the results of computer prediction programs for secondary structure. The programs used were those of M. Zuker (RNA2 and FOLD) which calculate a minimal global energy for a given sequence. Many single hairpins predicted by both RNA2 and FOLD showed a correlation with the histograms of hairpin structures of RNA crosslinked with psoralen. A secondary structure map was also constructed for the entire 7433 bases using the option in FOLD which allows multi-branch loops by folding uniformly stepped overlapping segments. Any structure that occurred at or greater than a given frequency was selected and mapped with respect to genome position. A correlation in structured regions was seen between psoralen-derived and computer-predicted maps of secondary structure. Furthermore, a region of large loops from base position 681 to 3899 was noted that corresponded to frequently observed large loop(s) in electron micrographs of psoralen preparations of RNA. Agreement between the two methods of determining secondary structure strengthens the credibility of the computer-aided methods used for predicting secondary structure and allows us to suggest an overall secondary structure map for poliovirus RNA.

The polypeptides of adenovirus: VII. Further studies of early polypeptides in vivo and localization of E2 and E2A to the cell plasma membrane

Abstract At early times, adenovirus-2-infected HeLa cells have been shown to contain four induced polypeptides with apparent molecular weights of 70–75,000 (E1), 45–48,000, 19–20,000 (E2), and 10–11,000 (E3). In this communication, a combination of high multiplicity infection, separation of infected cells into cytoplasmic and nuclear fractions, and high resolution SDS-polyacrylamide gel electrophoresis has allowed the characterization of three other early species: 42,000 (E1A), 17,000 (E2A) and 14,000 (E2B). In addition, further fractionation of cytoplasmic and nuclear fractions revealed an association of E2 and E2A with cell plasma membranes and E3 with nuclear particulate matter. Pulsechase data, behavior of early proteins on SDS-hydroxylapatite columns, and fractionation with ammonium sulfate also have served to further characterize the early proteins. The 45–48K protein was not seen in our system.

Distance analysis helps to establish characteristic motifs in intron sequences

Computer-assisted sequence analysis was applied to detect the most apparent nonrandom sequence motifs in eukaryotic introns. We describe in detail a method, which we call distance analysis, that we applied to the extensive study of 405 eukaryotic intron sequences. We observed very strong two-base periodicities for almost all tetranucleotides that are tandem repeats of nonhomopolymeric dinucleotides (the exception was GCGC and CGCG). We also observed, by using a fixed-point alignment method, that these periodic sequence motifs belong to large clusters of dinucleotides repeated tandemly as many as 15-35 times, which corresponds to the cluster lengths of 30-70 bases. We did not observe two-base periodicity of tetranucleotides in the collections of either 262 spliced eukaryotic exons or 107 bacterial genes. Instead, these sequences displayed strong three-base periodicity of some other tetranucleotides. These findings suggest that introns and exons display distinct sequence properties that can be used for mapping purposes.

Sequence signals in eukaryotic upstream regions

Two DNA sequence elements are known to recur frequently upstream of eukaryotic polymerase II-transcribed genes. The TATAAA, at position -40, specifies the transcription initiation site. The GGCCAATCT is less frequent around -80. Sequence analysis of upstream regions reveals that the underlined yeast UAS2 consensus sequence, TGATTGGT, is also very frequent at -80 in higher polymerase II-transcribed animal sequences. The underlined CCAAT box and yeast UAS sequences are complementary. Structural analysis suggests some symmetry in their DNA structures. Upstream of the TATAAT-rich region there is an abundance of GC sequences. Analysis of nucleotide tracts indicates that these are preferentially flanked by their complementary nucleotides with a pyrimidine-purine junction, i.e., TTAN, CCGn, CnGG, TnAA. Here, I discuss DNA structural consideration in upstream regions along with protein readout of the major and minor groove information content. These sequence-structure aspects are put in the general context of protein (factors)-DNA (elements) recognition and regulation.

The polypeptides of adenovirus VIII. The enrichment of E3 (11,000) in the nuclear matrix fraction

Abstract Subnuclear fractions have been studied in order to localize E3 (11,000 daltons) which is an intranuclear adenovirus type 2 (Ad 2 ) -induced early protein. Of the various subnuclear fractions that were generated from [ 35 S]methionine-labeled Ad 2 -infected HeLa nuclei and whose polypeptides were analyzed by SDS-disc-PAGE, it was the nuclear matrix fraction (NM) that was enriched for E3. The nuclear matrix fraction corresponds to the pelleted material obtained after high salt and DNase treatment of disrupted detergent-washed nuclei and contains a polypeptide pattern that is qualitatively different from that of the whole nucleus. Although NM contained only 10–12% of the total nuclear-incorporated [ 35 S]methionine, it contained about 30–45% of the total E3. Thus, there is a 3.0- to 4.5-fold increase of E3 in the NM fraction. Moreover, the association of E3 with the NM is a strong one. Only denaturant combinations, such as 1% Triton X-100 and 5 M urea, or 2 M guanidine HCl and 2 M NaCl, were able to extract most of the associated E3, while 1% Triton X-100 or 2 M NaCl alone could not. Reconstruction experiments confirmed that the association of E3 to the NM fraction is not merely an artifact of the fractionation procedure. In addition, E2 (19,000) and a new early protein (39,000–40,000) were the only other early polypeptides to be resolved in the infected NM; in particular, neither El (72,000 single-strand DNA-binding protein) nor its known breakdown products were observed in this fraction.

Cell architecture during adenovirus infection.

Abstract These experiments are designed to probe the morphological consequences of adenovirus early gene expression. At later times after infection (18 hr) changes in the nucleus are conspicuous. In particular, the nucleoplasm appears compartmentalized, and the nuclear envelope becomes characteristically convoluted and vesiculated. At least some of these changes are the result of early events in infection, since by 6 hr perceptible alterations in the cell architecture can be seen, especially in the nuclear envelope. These morphological changes presumably reflect one aspect of the activities of the newly synthesized viral products. PAGE of the viral proteins demonstrates that nearly the entire spectrum of early polypeptides is found associated with the nucleus and its appurtenant cytoplasmic matrix. These include proteins of approximate molecular weights 58,000 and 40,000 which have been assigned to the left-end fragment of the adenovirus genome capable of transforming cells in vitro . The interaction of the early adenovirus proteins with the cytoskeletal lattice shown here can be considered as demonstrating one aspect of these proteins function and may be a basis for the physiological changes they effect.

Functional analysis of reverse transcription by a frameshift pol mutant of murine leukemia virus

Endogenous reverse transcription by wild-type murine leukemia virus (MuLV) was compared to that catalyzed by clone 23, a pol mutant containing a reverse transcriptase protein which lacks the carboxyl-terminal third of the molecule (J. G. Levin, S. C. Hu, A. Rein, L. I. Messer, and B. I. Gerwin (1984), J. Virol. 51, 470-478). Competition immunoassays revealed that mutant virions contain normal amounts of polymerase protein, indicating that the lack of carboxyl-terminal sequences does not alter normal processing of enzyme precursors. Although the mutant enzyme was previously shown to have the ability to copy and degrade RNA:DNA hybrids, the present study demonstrates that it is defective in functions required to generate full-length copies of viral DNA. Analysis of products of endogenous reverse transcription showed that minus-strand strong-stop DNA is formed and that mutant virions synthesize a series of minus-strand DNA intermediates up to 2.2 kb in length. Comparison of mutant and wild-type MuLV reaction products indicated that the 2.2-kb termination site of the mutant corresponds to a normal pausing region for the wild-type enzyme. Computer analysis of sequences and structure within pausing regions suggested the involvement of C-rich consensus sequences plus multibranch loop structures in the general phenomenon of enzyme-pausing during reverse transcription.