Translational suppression in retroviral gene expression.
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Amino Acid Sequence↗
Biomedical subjects
J G Levin
Publications and source records attributed to J G Levin.
Localization of DNA binding activity of HIV-1 integrase to the C-terminal half of the protein.
Human immunodeficiency virus type 1 (HIV-1) integrase (IN) is the viral protein required for integration of the HIV-1 genome into host cell DNA. A series of clones expressing portions of IN as lambda cII fusion proteins has been constructed in an Escherichia coli expression system; a Southwestern procedure was used to examine binding of the expressed proteins to DNA oligonucleotides. Proteins expressed by clone pHIP106, encoding the entire IN protein but no other pol sequence, and pKNA101, which expresses an IN fusion protein containing 23 amino acids of HIV-1 reverse transcriptase at its amino terminus, exhibited similar levels of oligonucleotide binding. Little DNA sequence specificity was associated with binding activity and there was a preference for Mn2+ over Mg2+ and Ca2+. Interestingly, the protein expressed by an N-terminal clone containing nucleotides coding for IN amino acids 1-141 (including a conserved His-Cys box) was unable to bind oligonucleotide, whereas the protein expressed by a C-terminal clone containing nucleotides coding for amino acids 142-288 exhibited binding equivalent to that of full-length IN. The C-terminal protein was unreactive with a MAb to the lambda cII leader peptide and with an antipeptide serum directed against amino acids 141-158. These results are consistent with the previously reported internal initiation of IN protein synthesis in E. coli at met 154, and indicate that the C-terminal clone does not express IN amino acids 142-153. These amino acids represent part of a conserved region termed D(35)E, containing amino acids 116-152, which has been implicated in IN DNA binding.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Bipartite signal for read-through suppression in murine leukemia virus mRNA: an eight-nucleotide purine-rich sequence immediately downstream of the gag termination codon followed by an RNA pseudoknot.
The pol gene of murine leukemia virus and other mammalian type C retroviruses is expressed by read-through suppression of an in-frame UAG codon which separates the gag and pol coding regions. In this study, we have analyzed the sequence requirements for read-through suppression by placing different portions of wild-type and mutant viral sequences from the gag-pol junction between reporter genes and testing transcripts of these constructs for suppression in reticulocyte lysates. We find that the read-through signal is contained within the first 57 nucleotides on the 3' side of the UAG codon. Our results indicate that the identities of six conserved bases in the eight-nucleotide, purine-rich sequence immediately downstream of the UAG codon are critical for suppression, as is the existence of a pseudoknot structure spanning the next 49 nucleotides. Thus, read-through suppression depends on a complex, bipartite signal in the mRNA.
Animals↗
Readthrough suppression in the mammalian type C retroviruses and what it has taught us.
Mammalian type C retroviruses use translational suppression to synthesize the enzymes which function in virus replication. The UAG termination codon at the end of the coding region for the viral core proteins is translated as glutamine at a frequency of approximately 5%, allowing synthesis of the enzymes as part of a large fusion protein. This unusual mechanism has several benefits for the virus: first, it modulates the relative levels of synthesis of the core proteins and the enzymes. This is essential for the proper assembly of the virus particle, since the fusion protein alone is apparently unable to assemble into particles. Second, the presence of the core protein moiety in the fusion protein probably provides a mechanism for targeting the enzymes to the virus particle. The mechanism of the suppression phenomenon is now under investigation. Recent studies have revealed that suppression in the viral context is dependent upon a complex cis-acting signal in the viral mRNA, including a pseudoknot beginning 9 nucleotides 3' of the termination codon. In addition, studies with viral mutants have shown that UAA and UGA, like UAG, are efficiently suppressed in the presence of this signal, and have identified the amino acids used in the suppression of these termination codons in reticulocyte lysates. In several cases, this analysis revealed the existence of previously unknown suppressor tRNAs. One important question which has not been answered is whether the suppression mechanism used by the virus has a parallel in the synthesis of host proteins.
Base Sequence↗
Identification of amino acids inserted during suppression of UAA and UGA termination codons at the gag-pol junction of Moloney murine leukemia virus.
Expression of the murine leukemia virus pol gene occurs by translational readthrough of an in-frame UAG codon between the gag and pol coding regions. In a previous study, we mutated the UAG codon to UAA or UGA and demonstrated that both of these termination codons could be suppressed in reticulocyte lysates and in infected cells with the same efficiency as UAG. We now report the identity of the amino acids inserted in vitro in response to UAA and UGA in fusion products containing the gag-pol junction region. The results show that UAA, like UAG, directs the incorporation of glutamine, whereas UGA directs the incorporation of three amino acids, arginine, cysteine, and tryptophan. To our knowledge, this is the first report indicating misreading of UAA as glutamine and UGA as arginine and cysteine in higher eukaryotes. Interestingly, although our protein synthesis system presumably contains other known UAG and UGA suppressors, these tRNAs did not suppress the termination codons in our experiments. Thus, it seems possible that the sequence surrounding the gag-pol junction not only promotes suppression but also helps determine which tRNAs function in suppression.
Amino Acid Sequence↗
Expression of HIV-1 integrase in E. coli: immunological analysis of the recombinant protein.
Sequences encoding the human immunodeficiency virus type 1 (HIV-1) integrase gene have been cloned and expressed in Escherichia coli. The expressed protein is a lambda cII fusion protein of 37 kD containing the carboxyl-terminal 23 [corrected] amino acids of reverse transcriptase fused to the entire integrase sequence and is insoluble, a feature which allows partial purification away from soluble bacterial proteins. As judged by its reactivity with HIV positive sera in Western blot and in enzyme-linked immunosorbent assay (ELISA), the recombinant integrase retains antigenicity similar to native protein. Additionally, ELISA data obtained with the cloned protein indicate that patients infected with HIV-1 who are at different stages of progression to AIDS have antibodies reactive with the cloned integrase. HIV-2 positive human sera are also reactive with the cloned integrase. Rabbit antibodies produced against the recombinant protein react both by ELISA and Western blot with the homologous bacterially expressed protein, recognize both virion HIV-1 integrase and reverse transcriptase in Western blots, and immunoprecipitate an HIV-1 virion protein of 34 kD. Unlike human antisera from patients infected with HIV-1 or HIV-2 which are frequently reactive with both HIV-1 and HIV-2 integrase, the rabbit antibodies are type specific, reacting with HIV-1, but not with HIV-2 integrase by Western blot.
Acquired Immunodeficiency Syndrome↗
Chromatographic analysis of the aminoacyl-tRNAs which are required for translation of codons at and around the ribosomal frameshift sites of HIV, HTLV-1, and BLV.
An examination of the frameshift signals or proposed signals within published sequences of retroviruses and other genetic elements from higher animals shows that each site utilizes a tRNA which normally contains Wybutoxine (Wye) base or Queuine (Q) base in the anticodon loop. We find experimentally that most of the Phe-tRNA present in HIV-1 infected cells lacks the highly modified Wye base in its anticodon loop and most of the Asn-tRNA in HTLV-1 and BLV infected cells lacks the highly modified Q base in its anticodon loop. Interestingly, Phe-tRNA translates a UUU codon within the ribosomal frameshift signal in HIV and Asn-tRNA translates a AAC codon within the proposed frameshift signals in HTLV-1 and BLV. Thus, the lack of a highly modified base in the anticodon loop of tRNAs in retroviral infected cells is correlated with the participation of these undermodified tRNAs in the corresponding frameshift event. This suggests that the "shifty" tRNAs proposed by Jacks et al. (Cell 55, 447-458, 1988) to carry out frameshifting may be hypomodified isoacceptors.
Animals↗
Suppression of UAA and UGA termination codons in mutant murine leukemia viruses.
Genomes of mammalian type C retroviruses contain a UAG termination codon between the gag and pol coding regions. The pol region is expressed in the form of a gag-pol fusion protein following readthrough suppression of the UAG codon. We have used oligonucleotide-directed mutagenesis to change the UAG in Moloney murine leukemia virus to UAA or UGA. These alternate termination codons were also suppressed, both in infected cells and in reticulocyte lysates. Thus, the signal or context inducing suppression of UAG in wild-type Moloney murine leukemia virus is also effective with UAA and UGA. Further, mammalian cells and cell extracts contain tRNAs capable of translating UAA and UGA as amino acids. To our knowledge, this is the first example of natural suppression of UAA in higher eucaryotes.
Animals↗
Functional organization of the murine leukemia virus reverse transcriptase: characterization of a bacterially expressed AKR DNA polymerase deficient in RNase H activity.
The functional organization of the murine leukemia virus reverse transcriptase was investigated by expressing a molecular clone containing AKR MuLV reverse transcriptase-coding sequences in Escherichia coli. A purified preparation of the expressed enzyme (pRT250 reverse transcriptase) consisted primarily of a 69-kilodalton protein that has normal levels of murine leukemia virus polymerase activity but 10-fold-reduced levels of RNase H compared with the viral enzyme. The deficit in RNase H activity was correlated with the absence of 60 to 65 amino acids normally present at the carboxyl end of murine leukemia virus reverse transcriptase. The results provide additional experimental evidence for the localization of polymerase and RNase H domains to the N- and C-terminal regions of reverse transcriptase, respectively.
AKR murine leukemia virus↗
Murine leukemia virus pol gene products: analysis with antisera generated against reverse transcriptase and endonuclease fusion proteins expressed in Escherichia coli.
The organization of the murine leukemia virus (MuLV) pol gene was investigated by expressing molecular clones containing AKR MuLV reverse transcriptase or endonuclease or both gene segments in Escherichia coli and generating specific antisera against the expressed bacterial proteins. Reaction of these antisera with detergent-disrupted virus precipitated an 80-kilodalton (kDa) protein, the MuLV reverse transcriptase, and a 46-kDa protein which we believe is the viral endonuclease. A third (50-kDa) protein, related to reverse transcriptase, was also precipitated. Bacterial extracts of clones expressing reverse transcriptase and endonuclease sequences competed with the viral 80- and 46-kDa proteins, respectively. These results demonstrate that the antisera are specific for viral reverse transcriptase and endonuclease. Immunoprecipitation of AKR MuLV with antisera prepared against a bacterial protein containing only endonuclease sequences led to the observation that reverse transcriptase and endonuclease can be associated as a complex involving a disulfide bond(s).
AKR murine leukemia virus↗
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.
DNA, Viral↗
Selective packaging of host tRNA's by murine leukemia virus particles does not require genomic RNA.
The 4S RNA contained in RNA tumor virus particles consists of a selected population of host tRNA's. However, the mechanism by which virions select host tRNA's has not been elucidated. We have considered a model which specifies that 35S genomic RNA determines which tRNA's are to be encapsidated as well as the relative amounts of these tRNA's within the virion. The model was tested by comparing the free 4S RNA composition of normal murine leukemia virus (MuLV) particles and noninfectious virions from actinomycin D (ActD)-treated cells, which are deficient in genomic RNA (ActD virions). Viral 4S RNA was analyzed by two-dimensional polyacrylamide gel electrophoresis. Surprisingly, the patterns obtained for control and ActD 4S RNA were identical to each other and were clearly distinct from the cell 4S RNA pattern. The viral patterns had three prominent areas of radioactivity. One of the spots was identified on the basis of its oligonucleotide fingerprint as tRNA (Pro), the primer for MuLV RNA-directed DNA synthesis. These results were obtained with two different MuLV strains, AKR and Moloney, each grown in SC-1 cells. The demonstration that ActD virions contain primer tRNA and in general exhibit the characteristic MuLV tRNA pattern rather than the complete representation of cell 4S RNA leads to the conclusion that genomic RNA is not the major determinant in selective packaging of host tRNA's. A possible role for one or more viral proteins, including reverse transcriptase, is suggested.
AKR murine leukemia virus↗
Mutant of B-tropic murine leukemia virus synthesizing an altered polymerase molecule.
A nonconditional mutant of B-tropic murine leukemia virus (MuLV), defective in polymerase, has been isolated by cloning chronically infected cells. The cell clone containing the mutant produced virus particles which were noninfectious. However, superinfection of the cells by replication-competent XC-negative viruses resulted in the rescue of virus capable of forming plaques in a modified XC test, termed the "complementation plaque assay" (A. Rein and R. H. Bassin, J. Virol. 28:656-660, 1978). Analysis of the noninfectious virions produced without superinfection demonstrated that they contained only 2 to 5% of the wild-type level of reverse transcriptase activity. Purification of this activity indicated that it was associated with a smaller molecule than that produced by wild-type virus. Cells producing the mutant virions did not contain the gag-pol precursor, Pr180gag-pol; however the cells contained proteins of 147K and 114K daltons precipitable with anti-pol serum. All of the normal structural proteins as well as 70S genomic RNA could be detected in the mutant particles. An interference test indicated that a functional ecotropic glycoprotein was synthesized by the mutant. These results indicate that the mutant has a unique defect in the pol gene.
Animals↗
Interactions of murine leukemia virus core components: characterization of reverse transcriptase packaged in the absence of 70S genomic RNA.
Virions produced by cells in the presence of actinomycin D (Act D virions) contain reverse transcriptase but are deficient in 70S genomic RNA. To assess the role of genomic RNA in encapsidation of a functional reverse transcriptase and to study the interaction of the enzyme and its template in the cores of intact virions, the reverse transcriptase enzymes of normal and Act D virions were compared. The enzymes were indistinguishable by column chromatography, sedimentation velocity, or template/primer preferences. In addition, these enzymes showed equal sensitivity to inactivation by antibodies directed against Rauscher murine leukemia virus DNA polymerase. The enzymes from Act D and normal virions had similar thermal decay rates and were both protected against heat denaturation by natural and synthetic template/primers. By these criteria, the DNA polymerase molecules synthesized and assembled into virions in the absence of genomic RNA are identical to those packaged under normal conditions. Additional studies designed to measure protection of reverse transcriptase by genomic RNA were carried out by comparing the thermal lability of the enzyme in intact Act D and normal virions. The thermal decay rate of reverse transcriptase in Act D virions was identical to that in control virions. In contrast to the lability of the virion-associated enzyme, however, genomic RNA in control virions was stable to heat treatment.
AKR murine leukemia virus↗
Synthesis of murine leukemia virus proteins associated with virions assembled in actinomycin D-treated cells: evidence for persistence of viral messenger RNA.
Murine leukemia virus particles assembled in actinomycin D-treated cells were detected by determination of reverse transcriptase [RNA-dependent DNA polymerase (nucleotidyltransferase)] activity and by radioimmunoassay of the major virion protein, p30. The levels of enzyme activity and p30 protein were both 30-40% relative to the control over an 8 hr period, whereas after 3 or 4 hr infectivity was reduced by 95%. Thus, virions produced in the absence of RNA synthesis represent a fairly homogeneous population of defective particles. Although RNA synthesis is not necessary for virus assembly, protein synthesis is required. Treatment of cells with 10 mug/ml of cycloheximide reduced virus production by 80-85% within 2 hr, and by greater than 95% at later times. As might be expected from this finding, viral protein synthesis accompanies virus assembly in actinomycin D-treated cells. Newly synthesized proteins associated with the defective particles were identical with those found in standard virions and were present in the correct proportions. The results demonstrate that viral mRNA persists in cells in which RNA synthesis is blocked and continues to direct viral protein synthesis with a functional half-life of approximately 6-8 hr. Since viral mRNA is not packaged in virions even when viral RNA synthesis is shut off [Levin et al. (1974) J. Virol. 14, 152-161], we propose that murine leukemia virus-infected cells contain two nonequilibrating pools of intracellular viral RNA molecules, one associated with polyribosomes and one which is encapsidated into extracellular particles.
AKR murine leukemia virus↗