Transcript Leader Research Articles

Mapping of the bovine herpesvirus 1 glycoprotein C promoter region and its specific transactivation by the viral BICP27 gene product.

We investigated the cis-acting sequences of the promoter regulating the gene encoding the glycoprotein C (gC) of bovine herpesvirus 1 (BoHV-1), a gene of the gamma1 class. S1 nuclease protection assays revealed that gC transcription initiated predominantly at position C18281 of the viral genome, corresponding to 21 and 56 bases downstream from putative TATA-like and CAAT boxes, respectively. To map the gC promoter (pgC), we measured the ability of a series of nested deletions of the region +71 to -1155 with respect to the major gC transcriptional start site, to drive expression of the firefly luciferase (Luc) reporter gene following transient transfection of a cell host, in the absence as well as in the presence of viral factors expressed in trans. We show that the minimal pgC sequences required to drive maximal BoHV-1 independent expression was within the region +71 to -76 which harbours the putative TATA and CAAT boxes, the mRNA start site, and the complete 5' transcript leader sequence. This small pgC fragment was highly trans-activated by the co-expression of the BoHV-1-encoded BICP27 protein, but not BICP0 nor BTIF. In contrast, the pgC fragment spanning the region +71 to -1155 was only minimally trans-activated by BICP27, but substantially stimulated by BICP0. These findings thus suggest that gC gene regulation may involve the combined action of several viral transactivators.

Abundant early expression of gpUL4 from a human cytomegalovirus mutant lacking a repressive upstream open reading frame.

The human cytomegalovirus UL4 gene encodes a 48-kDa glycoprotein, expression of which is repressed at the translational level by a short upstream open reading frame (uORF2) within the UL4 transcript leader. Mutation of the uORF2 initiation codon in the viral genome eliminates ribosomal stalling at the uORF2 termination site, resulting in early and abundant gpUL4 protein synthesis. This mutation does not appear to affect viral replication kinetics in human fibroblasts. These results reveal that the unusual uORF2 inhibitory mechanism is a principal determinant of the abundance and timing of gpUL4 expression but is nonessential for replication in cell culture.

Characterization and light-induced expression of theS-adenosylmethionine decarboxylase gene fromIpomoea nil

We screened a genomic library of Japanese morning glory(Ipomoea nil) and isolated theMGSDCgi clone (GenBank Accession Number U64927). The genomic clone comprised four exons and three introns.MCSDCgi contained a 660-bp 5′-untranslated region (UTR), a 1089-bp coding region of 362 amino acids, and a 209-bp 3′-UTR. A TATA box sequence (TATATAA) was found at position -29. The transcript leader of thisSAMDC gene contained a short, upstream open reading frame (uORF) of 51 amino acids, which was interrupted with a 107-bp intron. Two other introns were positioned upstream of the uORF in the 5’-UTR. Six-day-old seedlings were grown under a 12-h light/dark cycle, then treated with white light The amount ofSAMDC transcript dramatically increased from the first hour after light exposure, then returned to a basal level.SAMDC mRNA was accumulated more under high rather than low irradiance. These results suggest a relationship between photo-response and the physiological role of polyamines (PAs). The level ofSAMDC mRNA was increased only by brassinosteroids, not by any other plant hormone such as kinetin, IAA, ABA, and GA. This hormonal response may have replaced the effect of light onSAMDC gene expression in the dark. PAs decreased theSAMDC mRNA level, which could then be restored by their inhibitors. This indicates thatSAMDC gene expression is regulated by its cellular contents.

Transcript leader regions of two Saccharomyces cerevisiae mRNAs contain internal ribosome entry sites that function in living cells.

In higher eukaryotes, translation of some mRNAs occurs by internal initiation. It is not known, however, whether this mechanism is used to initiate the translation of any yeast mRNAs. In this report, we identify naturally occurring nucleotide sequences that function as internal ribosome entry sites (IRESes) within the 5' leader sequences of Saccharomyces cerevisiae YAP1 and p150 mRNAs. When tested in the 5' untranslated regions of monocistronic reporter genes, both leader sequences enhanced translation efficiency in vegetatively growing yeast cells. Moreover, when tested in the intercistronic region of dicistronic mRNAs, both sequences were shown to contain IRESes that functioned in living cells. The activity of the p150 leader was much greater than that of the YAP1 leader. The second cistron was not expressed in control dicistronic constructs that lacked these sequences or contained the 5' leader sequence of the CLN3 mRNA in the intercistronic region. Further analyses of the p150 IRES revealed that it contained several nonoverlapping segments that were able independently to mediate internal initiation. These results suggested a modular composition for the p150 IRES that resembled the composition of IRESes contained within some cellular mRNAs of higher eukaryotes. Both YAP1 and p150 leaders contain several complementary sequence matches to yeast 18S rRNA. The findings are discussed in terms of our understanding of internal initiation in higher eukaryotes.

Upstream open reading frames as regulators of mRNA translation.

Continuing discoveries of new and surprising mechanisms of gene regulation suggest that our understanding of this complex and ubiquitous biological process remains incomplete. Emerging examples illustrate that many and perhaps all genes are regulated at multiple steps including transcription, posttranscriptional processing, nuclear export and localization, stability, and translation of mature mRNA molecules. Translation itself is regulated by a diverse collection of mechanisms that act not only at the initiation step but also during elongation and termination and even after termination. Among the various cis elements in mRNAs (43) that participate in regulating translation are AUG codons within transcript leaders (upstream AUGs [uAUGs]) and, in some cases, associated upstream open reading frames (uORFs). Based on a 1987 survey, less than 10% of eukaryotic mRNAs contain AUG codons within their transcript leader regions (often erroneously referred to as 5′ untranslated regions). However, uAUGs are conspicuously common in certain classes of genes, including two-thirds of oncogenes and many other genes involved in the control of cellular growth and differentiation (29, 31, 42). Despite the wealth of sequence data being generated by large-scale sequencing projects, extracting an up-to-date, comprehensive, and accurate estimate of the number of genes with uORFs is a formidable task. Only a minority of database entries are based on careful mRNA mapping data with annotations that identify the precise start of the transcript leader. Moreover, the use of alternative transcriptional start sites, alternative RNA processing, and alternative initiation codons complicates the determination of what exactly constitutes the transcript leader. Nonetheless, it is clear that uAUGs are not uncommon in genes with critical cellular roles, and identifying when and how they function is necessary if we are to achieve a comprehensive understanding of the interesting genes that contain these elements and of eukaryotic gene regulation in general. Some of the general principles by which uORFs participate in translational control are beginning to be understood. In this article, we first review these principles, which include the process of recognition of uORFs, regulation of reinitiation at downstream cistrons after translation of uORFs, and regulatory effects of peptides encoded by uORFs. We then illustrate how these principles are applied by reviewing several specific examples where the roles of uORFs in translational control have been well characterized.

Characterization and translational regulation of the arginine decarboxylase gene in carnation (Dianthus caryophyllus L.)

Arginine decarboxylase (ADC; EC 4.1.1.9) is a key enzyme in polyamine biosynthesis in plants. We characterized a carnation genomic clone, gDcADC8, in which the deduced polypeptide of ADC was 725 amino acids with a molecular mass of 77.7 kDa. The unusually long 5'-UTR that contained a short upstream open reading frame (uORF) of seven amino acids (MQKSLHI) was predicted to form an extensive secondary structure (free energy of approximately -117 kcal mol-1) using the Zuker m-fold algorithm. The result that an ADC antibody detected two bands of 45 and 33 kDa in a petal extract suggested the full length of the 78 kDa polypeptide precursor converted into two polypeptides in the processing reaction. To investigate the role of the transcript leader in translation, in vitro transcription/translation reactions with various constructs of deletion and mutation were performed using wheat germ extract. The ADC transcript leader affected positively downstream translation in both wheatgerm extract and primary transformant overexpressing ADC gene. It was demonstrated that heptapeptide (8.6 kDa) encoded by the ADC uORF was synthesized in vitro. Both uORF peptide, and the synthetic heptapeptide MQKSLHI of the uORF, repressed the translation of downstream ORF. Mutation of the uORF ATG codon alleviated the inhibitory effect. ORF translation was not affected by either a frame-shift mutation in uORF or a random peptide. To our knowledge, this is the first report to provide evidence that a uORF may inhibit the translation of a downstream ORF, not only in cis but also in trans, and that the leader sequence of the ADC gene is important for efficient translation.

Analysis of a translational enhancer present within the 5'-terminal sequence of the genomic RNA of potato virus S.

When present as a transcript leader the 5' untranslated sequence from the potato virus S genomic RNA molecule enhances translation of a downstream open reading frame both in vitro and in vivo. Translational enhancement was 30-fold in rabbit reticulocyte lysate and 15 fold in wheat germ above translation from a transcript with a synthetic leader. Transient expression experiments using tobacco protoplasts and particle bombardment of leaf tissue resulted in enhancement of fourteen and five-fold, respectively, above translation with a synthetic leader. In stably transformed plants the PVS 5'UTR enhanced translation yield ca. 5-fold compared with a synthetic 5'UTR.

Cell type-dependent and -independent control of HER-2/ neu translation

Overexpression of the HER-2 oncogene occurs in a variety of human tumors, including 25–30% of breast carcinomas, and has been associated with an adverse prognosis. Amplification of the HER-2 gene is frequently detected in tumors, but by itself may not fully account for HER-2 overexpression since transcriptional and post-transcriptional mechanisms also regulate HER-2 protein synthesis. Our studies reveal that the efficiency of HER-2 translation differs between primary and transformed cells. In primary human fibroblasts and human mammary epithelial cells, the HER-2 mRNA is associated with monosome and small polysome fractions. In contrast, in BT474 and MCF-7 human breast cancer cell lines and in COS-7 cells the mRNA co-sedimented with larger polysomes, indicating that it is more efficiently translated in these transformed cells. Northern analysis revealed no detectable mRNA size difference, and nuclease S1 protection and sequence analyses showed no differences between the HER-2 transcript leader in primary cells compared to transformed human cells. The transcript leader in all cell types contains a short upstream open reading frame that is also conserved in other mammalian species. Transient transfection assays revealed that the HER-2 transcript leader repressed downstream translation approximately five-fold in both primary and transformed cells and mutation of the upstream initiation codon alleviated most of the inhibitory effect. These results indicate that HER2 expression is translationally controlled both by a short upstream open reading frame that represses HER-2 translation in a cell type-independent manner, and by a distinct cell type-dependent mechanism that increases translational efficiency of HER-2 in transformed cells.

The herpes simplex virus 2 kb latency associated transcript (LAT) leader sequence allows efficient expression of downstream proteins which is enhanced in neuronal cells: possible function of LAT ORFs.

Herpes simplex viruses (HSV)-1 and -2 enter latency in peripheral ganglia during which time a number of latency associated transcripts (LATs) are produced. The most abundant (2 kb) LAT contains ORFs of significant size which could play a role in latency. We hypothesized that the leader sequence of the 2 kb LAT might regulate expression of the LAT ORFs in neurons and might thus enhance some aspect of the latency process. We show that constructs with the HSV-1 2 kb LAT leader sequence in front of a CAT gene are efficiently translated, with enhanced activity in cells of neuronal origin, but that from within a larger LAT-derived RNA from which 2 kb LATs are efficiently spliced, expression levels are low. Thus some further regulation of expression must occur if LAT ORFs are expressed in vivo. Experiments to test any regulatory function of the LAT ORFs in suppressing or stimulating immediate early (IE) gene expression during latency did not, however, show effects on IE promoters in cotransfection assays, nor on an early gene promoter which has recently been shown to be expressed early in reactivation.

Signs of translational regulation within the transcript leader of a plant plasma membrane H(+)-ATPase gene.

Transcripts of most plant plasma membrane H(+)-ATPase genes possess a leader (5' untranslated region) that is unusually long and that contains a short upstream open reading frame (uORF), two features which suggest post-transcriptional regulation. To investigate the putative role of the transcript leader, we have placed the leader of pma3, one of the Nicotiana plumbaginifolia H(+)-ATPase genes, between the CaMV 35S promoter and the sequence coding for the beta-glucuronidase (GUS) reporter gene. Transient expression of this chimeric gene and of derived mutants was analysed in electroporated tobacco protoplasts. The whole leader had a positive effect on translation, since deletion of most of its sequence reduced GUS activity. Suppression of the uORF by point mutation of its initiating AUG increased GUS activity by about 55%. Analysis of various deletions and mutations suggested that the uORF is translated by at least two-thirds of scanning ribosomes, half of which subsequently reinitiate downstream translation under our experimental conditions. Reinitiation did not depend on the nucleotide sequence of the uORF, nor on that separating the uORF and the main open reading frame. We conclude that the pma3 transcript possesses features of translational regulation, whose mode of functioning has yet to be discovered.

Characterization and expression of two members of the S-adenosylmethionine decarboxylase gene family in carnation flower.

S-adenosylmethionine decarboxylase (SAMDC; EC 4.1.4.50) is one of the key enzymes in polyamine biosynthesis, and the product of its catalytic reaction, decarboxylated S-adenosylmethionine (dcSAM), serves as an aminopropyl donor in the biosynthesis of spermidine and spermine. In order to provide information on the structure and regulation of SAMDC, we have isolated and sequenced two different SAMDC cDNA clones from carnation petals. The nucleotide sequences of CSDC9 and CSDC16 show 78.3% identity, and the deduced amino acid sequences show 81.7% identity and 86.5% similarity [12]. There are several regions with highly conserved sequences among SAMDC cDNAs of potato, spinach, periwinkle, man and yeast. These conserved regions include a cleavage site for the processing of SAMDC proenzyme and a putative PEST sequence that may be relevant to the rapid degradation of SAMDC protein. Carnation SAMDC cDNAs have long transcript leaders of 472 bp and 502 bp for CSDC9 and CSDC16, respectively. Both sequences contain short upstream open reading frames (uORFs) in their 5'-untranslated regions. The CSDC9 uORF is 54 amino acids from 152 to 317 while the corresponding sequence in CSDC16 is 52 amino acids located from 156 to 314 in each 5'-untranslated region. The nucleotide sequences of uORFs in CSDC9 and CSDC16 were 89.9% identical. In vitro transcription/translation experiments showed: (1) each proenzyme of both cDNAs of SAMDC was converted to two polypeptides consisting of a large subunit (calculated as 31,544 Da and 32,537 Da, respectively) and a small subunit (calculated as 9704 and 9041 Da, respectively) after 20 min of translation; (2) the processing occurs rapidly during the translation of protein. But once the translation process is stopped accumulation of the subunits slows and never reaches completion even after 300 min. The processing of carnation SAMDC enzyme is not stimulated by putrescine in in vitro transcription/translation reaction.

Inhibition of nascent-peptide release at translation termination.

The transcript leader of the human cytomegalovirus (CMV) gpUL4 (gp48) gene contains a 22-codon upstream open reading frame (uORF2) that represses translation of the downstream cistron. Previous work demonstrated that ribosomes stall at the termination codon of uORF2 and, remarkably, that the coding information of uORF2 is required for both the translational repression and ribosomal stalling. We now provide evidence that the peptide product of uORF2 is synthesized and is retained in the ribosome in the form of a peptidyl-tRNA. Translation of the gp48 transcript leader in cell extracts produces the 2.4-kDa uORF2 peptide and a second product migrating with an apparent molecular mass of 20 kDa that represents the uORF2 peptide covalently linked to tRNA(Pro), the tRNA predicted to decode the carboxy-terminal codon of uORF2. The uORF2 peptidyl-tRNA is only detected after translation of RNAs containing uORF2 sequences that also inhibit downstream translation and cause ribosomal stalling. These data support a model in which the nascent uORF2 peptide blocks translation termination prior to hydrolysis of the peptidyl-tRNA bond. This blockade results in ribosomal stalling on the transcript leader which in turn impedes the access of ribosomes to the downstream cistron. This system illustrates that translation termination may be a critical step controlling expression of some eukaryotic genes.

Coding sequence-dependent ribosomal arrest at termination of translation.

A remarkably high percentage of proto-oncogene, growth factor, cellular receptor, and viral transcript leaders contain short upstream open reading frames (uORFs), yet the significance and regulatory effects of these uORFs have not been well characterized. In the case of the human cytomegalovirus gpUL4 (gp48) transcript, the second of three uORFs (uORF2) inhibits translation of the downstream cistron by a process that depends on the uORF2 amino acid coding information. To investigate the mechanism underlying this unusual regulatory element, we adapted the toeprinting (or reverse transcriptase extension inhibition) assay for use in detecting positions of ribosomal stalling on gp48 transcripts. Using a cell-free translation system, we demonstrate that ribosomes arrest at the termination codon of uORF2 by a uORF2 coding sequence-dependent mechanism. Further, the sequence requirements for ribosomal stalling are the same as for inhibition of downstream translation. We also provide evidence for ribosomal stalling in vivo, on the natural viral mRNA. These data support the hypothesis that the inhibition of downstream translation results from uORF2 peptide-dependent ribosomal arrest at termination and suggest that translation termination may be a regulatory step in expression of some eukaryotic genes.

Translational inhibition by a human cytomegalovirus upstream open reading frame despite inefficient utilization of its AUG codon.

The second of three short upstream open reading frames (uORF2) in the transcript leader of the human cytomegalovirus gp48 (gpUL4) virion glycoprotein gene inhibits downstream translation approximately 10-fold. Remarkably, this inhibition depends on the amino acid coding information of uORF2. In the current studies we demonstrate that expression of the cistron downstream from uORF2 depends on ribosomes bypassing the uORF2 AUG codon (AUG2) by a leaky scanning mechanism. Replacing the nucleotides surrounding the wild-type AUG2 codon with those optimal for translation initiation reduces downstream translation approximately 10-fold. Analyses of mutants in which uORF2 either overlaps or is in frame with the downstream reading frame reveal that the initiation frequency at the wild-type AUG2 codon is surprisingly low; rather, the majority of ribosomal subunits bypass the wild-type AUG2 codon because of its suboptimal context. We propose a model to explain this unprecedented example of a paradoxically strong inhibitory effect of an upstream ORF despite inefficient utilization of its initiation codon.

Mutational Analysis of the Translational Signal in the Human Cytomegalovirus gpUL4 (gp48) Transcript Leader by Retroviral Infection

A short upstream open reading frame (uORF2) in the human cytomegalovirus (CMV) gpUL4 (gp48) transcript leader is conserved among CMV strains and inhibits translation of a downstream cistron. Remarkably, this inhibitory effect depends on the amino acid coding information of uORF2, at least in transient transfection assays in diploid human fibroblasts. Using retroviral vectors, we now report that the gp48 leader inhibits downstream translation in multiple additional cell types, even when expressed from a stably integrated gene, and on a transcript containing an additional kilobase of complex leader sequences. The magnitude of inhibition can be augmented approximately 3- to 10-fold by replacing the context of nucleotides flanking the wild-type initiation codon of uORF2 with an optimal context, suggesting that leaky scanning past the wild-type AUG codon accounts for translation of the downstream cistron. Using an in vivo mutagenesis protocol that relies on reverse transcriptase infidelity, we isolated mutants in which the inhibitory effect of the gp48 leader was inactivated as a result of alterations in the coding information of uORF2. These studies demonstrate that, independent of the cell type or expression system used, CMV gp48 uORF2 is a potent translational inhibitory element.

Cell-specific translational regulation of S-adenosylmethionine decarboxylase mRNA. Influence of the structure of the 5' transcript leader on regulation by the upstream open reading frame.

A small upstream open reading frame (uORF), located 14 nucleotides from the cap in the 5' transcript leader (5' TL) of the mRNA encoding S-adenosylmethionine decarboxylase (AdoMetDC), suppresses translation of the downstream cistron in normal T cells and T cell lines. In the present study, we examined the structural features of the 5' TL that overcome this suppressive influence in cells of nonlymphoid origin. Initiation at the downstream cistron in nonlymphoid cells is by a cap-dependent mechanism that requires ribosome scanning along the 5' TL and does not involve an internal ribosome entry site. Extending the uORF so that it overlapped the major cistron by 101 nucleotides had no effect on translation of the downstream cistron in either HeLa or Jurkat cells. When the distance between the uORF and the cap was extended to 47 nucleotides, using sequence previously found to be neutral, translation of the major cistron was inhibited 5-fold in HeLa cells and the mRNA was moved from polysomes to monosomes, a location identical to that of the wild type mRNA in Jurkat cells. Therefore, in contrast to T cells, initiation at the uORF seems to be relatively infrequent in non-lymphoid cells due to its proximity to the cap, allowing efficient translation of the downstream cistron.

An upstream open reading frame represses expression of Lc, a member of the R/B family of maize transcriptional activators.

The R/B genes of maize encode a family of basic helix-loop-helix proteins that determine where and when the anthocyanin-pigment pathway will be expressed in the plant. Previous studies showed that allelic diversity among family members reflects differences in gene expression, specifically in transcription initiation. We present evidence that the R gene Lc is under translational control. We demonstrate that the 235-nt transcript leader of Lc represses expression 25- to 30-fold in an in vivo assay. Repression is mediated by the presence in cis of a 38-codon upstream open reading frame. Furthermore, the coding capacity of the upstream open reading frame influences the magnitude of repression. It is proposed that translational control does not contribute to tissue specificity but prevents overexpression of the Lc protein. The diversity of promoter and 5' untranslated leader sequences among the R/B genes provides an opportunity to study the coevolution of transcriptional and translational mechanisms of gene regulation.

Cell-specific translational regulation of S-adenosylmethionine decarboxylase mRNA. Dependence on translation and coding capacity of the cis-acting upstream open reading frame.

The mRNA encoding S-adenosylmethionine decarboxylase (AdoMetDC) has a 330-nucleotide 5'-transcript leader containing an open reading frame (uORF) that codes for the hexapeptide MAGDIS. The uORF restricts the intracellular distribution of AdoMetDC mRNA primarily to monosomes in normal T-lymphocytes and in T-cell lines. In contrast, non-lymphoid cells normally carry an average of seven to nine ribosomes per AdoMetDC mRNA molecule (Hill, J.R., and Morris, D. R. (1992) J. Biol. Chem. 267, 21886-21893). Several alterations abolish the negative regulatory effect of the uORF in T-cells. These include removing the site of translational initiation; weakening the context of the translational initiation site; changing the coding capacity of the fourth, fifth, and sixth codons; increasing the length of the uORF at either the 5' or 3' end; or changing the primary order of the codons. In contrast, altering the nucleic acid sequence of the uORF at degenerative positions without changing the amino acid coding capacity did not cause deregulation. The uORF does not regulate translation in the trans-configuration. Our results support a model in which translation of the uORF generates a nascent hexapeptide that interacts with its translating ribosome to suppress translation of AdoMetDC mRNA in a cell-specific manner. Structural features of the carboxyl-terminal 3 amino acids of the putative hexapeptide govern the interaction of the peptide with a component of the translation machinery.

Cell-specific translation of S-adenosylmethionine decarboxylase mRNA. Regulation by the 5' transcript leader.

The mRNA encoding S-adenosylmethionine decarboxylase (AdoMetDC) has an unusual distribution in polysomes from cells of T lymphocyte origin. It associates predominantly with monosomes and small polysomes with none located in the preribosomal or ribonucleoprotein pool. In sharp contrast, it associates broadly with larger polysomes in several nonlymphoid cell lines, including fibroblasts and the adrenal carcinoma line, Y1. The AdoMetDC 5'-transcript leader (5'-TL) is highly conserved between human and bovine mRNAs. It has a length of about 330 nucleotides and contains a 21-nucleotide upstream open reading frame (uORF) approximately 14 nucleotides downstream of the cap site. The AdoMetDC 5'-TL, when used to replace the 5'-TL of the human growth hormone gene in a chimeric expression construct, causes a suppressed polysomal distribution of the chimeric mRNA identical to that of the endogenous AdoMetDC mRNA in the T cell line, Jurkat. In contrast, mRNA encoded by the same chimeric construct, when expressed in Y1 cells, mimics the broad polysomal distribution of the endogenous AdoMetDC mRNA. Mutations that remove the uORF, or prevent its initiation, abolish the translational suppression in T cells, establishing that the uORF is a negative element that modulates the cell-specific polysomal distribution of AdoMetDC mRNA.

Variable inhibition of cell-free translation by HIV-1 transcript leader sequences.

The 5' ends of all human immunodeficiency virus type I (HIV-1) transcripts have the potential to coordinately regulate translation of HIV-1 mRNAs. Conflicting observations of the translational impact of these sequences in various systems stimulated these analyses of translation in reticulocyte lysates. We report a sensitive, rapid, quantitative, and inexpensive cell-free translation assay in which translational efficiency is monitored by enzymatic assay of the translation products. Using this assay and conventional radiolabeling assays, we demonstrate that the HIV-1 transcript leader inhibits downstream translation and that the stem-loop structure is required. Under our assay conditions, this inhibition occurs predominantly in cis and is not mediated by the 68 kD, interferon-induced, double-stranded RNA-activated kinase (p68). However, under other assay conditions the HIV-1 leader may activate p68 and inhibit translation in trans. We show that variation between individual preparations of cell-free extracts can dramatically alter the magnitude of the translational inhibition by the HIV-1 leader. Further, we provide evidence that a heat-labile factor is required for efficient translation of transcripts containing the HIV-1 leader. These observations provide a foundation for identifying factors required for translation of HIV-1 transcripts.