Gag mRNA Research Articles

The Rev protein of human immunodeficiency virus type 1 promotes polysomal association and translation of gag/pol and vpu/env mRNAs.

Biochemical examination of the Rev-dependent expression of gag mRNAs produced from gag-Rev-responsive element (RRE) expression plasmids showed a large discrepancy between the level of cytoplasmic gag mRNA and the produced Gag protein. Significant levels of the mRNA produced in the absence of Rev were localized in the cytoplasm, while very low levels of Gag protein were produced. In the presence of Rev, the levels of mRNA increased by 4- to 16-fold, while the Gag protein production increased by 800-fold. These findings indicated that in addition to promoting nucleus-to-cytoplasm transport, Rev increased the utilization of cytoplasmic viral mRNA. Poly(A) selection and in vitro translation of cytoplasmic gag mRNA verified that the mRNA produced in the absence of Rev was functional. To analyze the translational defect in the absence of Rev, we examined the association of the cytoplasmic gag mRNA with ribosomes. gag mRNA produced in the absence of Rev was excluded from polysomes, while gag mRNA produced in the presence of Rev was associated with polysomes and produced Gag protein. These observations showed that the presence of Rev was required for efficient loading of gag mRNA onto polysomes. This effect required the presence of the RRE on the mRNA. Analysis of mRNAs produced from a rev-minus proviral clone confirmed that the presence of Rev promoted polysomal loading of both gag/pol and vpu/env mRNAs. The localization of gag mRNA was also examined by in situ hybridization. This analysis showed that in the presence of Rev, most of the gag mRNA was found in the cytoplasm, while in the absence of Rev, most of the gag mRNA was found in the nucleus and in the region surrounding the nucleus. These results suggest that a substantial fraction of the gag mRNA is retained in distinct cytoplasmic compartments in the absence and presence of Rev. These findings indicate that the presence of Rev is required along the entire mRNA transport and utilization pathway for the stabilization, correct localization, and efficient translation of RRE-containing mRNAs.

The Rev protein of human immunodeficiency virus type 1 promotes polysomal association and translation of gag/pol and vpu/env mRNAs.

Biochemical examination of the Rev-dependent expression of gag mRNAs produced from gag-Rev-responsive element (RRE) expression plasmids showed a large discrepancy between the level of cytoplasmic gag mRNA and the produced Gag protein. Significant levels of the mRNA produced in the absence of Rev were localized in the cytoplasm, while very low levels of Gag protein were produced. In the presence of Rev, the levels of mRNA increased by 4- to 16-fold, while the Gag protein production increased by 800-fold. These findings indicated that in addition to promoting nucleus-to-cytoplasm transport, Rev increased the utilization of cytoplasmic viral mRNA. Poly(A) selection and in vitro translation of cytoplasmic gag mRNA verified that the mRNA produced in the absence of Rev was functional. To analyze the translational defect in the absence of Rev, we examined the association of the cytoplasmic gag mRNA with ribosomes. gag mRNA produced in the absence of Rev was excluded from polysomes, while gag mRNA produced in the presence of Rev was associated with polysomes and produced Gag protein. These observations showed that the presence of Rev was required for efficient loading of gag mRNA onto polysomes. This effect required the presence of the RRE on the mRNA. Analysis of mRNAs produced from a rev-minus proviral clone confirmed that the presence of Rev promoted polysomal loading of both gag/pol and vpu/env mRNAs. The localization of gag mRNA was also examined by in situ hybridization. This analysis showed that in the presence of Rev, most of the gag mRNA was found in the cytoplasm, while in the absence of Rev, most of the gag mRNA was found in the nucleus and in the region surrounding the nucleus. These results suggest that a substantial fraction of the gag mRNA is retained in distinct cytoplasmic compartments in the absence and presence of Rev. These findings indicate that the presence of Rev is required along the entire mRNA transport and utilization pathway for the stabilization, correct localization, and efficient translation of RRE-containing mRNAs.

Distinct RNA sequences in the gag region of human immunodeficiency virus type 1 decrease RNA stability and inhibit expression in the absence of Rev protein

The expression of Gag, Pol, Vif, Vpr, Vpu, and Env proteins from unspliced and partially spliced human immunodeficiency virus type 1 (HIV-1) mRNAs depends on the viral protein Rev, while the production of Tat, Rev, and Nef from multiply spliced mRNAs does not require Rev. To investigate the difference between gag and tat mRNAs, we generated plasmids expressing tat-gag hybrid mRNAs. Insertion of the gag gene downstream of the tat open reading frame in the tat cDNA resulted in the inhibition of Tat production. This inhibition was caused, at least in part, by a decrease in the stability of the produced mRNA. Deletions in gag defined a 218-nucleotide inhibitory sequence named INS-1 and located at the 5' end of the gag gene. Further experiments indicated the presence of more than one inhibitory sequence in the gag-protease gene region of the viral genome. The inhibitory effect of INS-1 was counteracted by the positive effect mediated by the Rev-Rev-responsive element interaction, indicating that this sequence is important for Rev-regulated gag expression. The INS-1 sequence did not contain any known HIV-1 splice sites and acted independently of splicing. It was found to have an unusually high AU content (61.5% AU), a common feature among cellular mRNAs with short half-lives. These results suggest that HIV-1 and possibly other lentiviruses have evolved to express unstable mRNAs which require additional regulatory factors for their expression. This strategy may offer the virus several advantages, including the ability to enter a state of low or latent expression in the host.

Frequent Detection of HIV-1-Specific mRNAs in Infected Individuals Suggests Ongoing Active Viral Expression in All Stages of Disease

It has been shown that only a small fraction of CD4+ T cells are infected with human immunodeficiency virus type 1 (HIV-1) in vivo, particularly early in the course of infection. An even smaller proportion of cells have been shown to be expressing virus. Recent studies suggest that plasma viremia in asymptomatic HIV-infected individuals, representing active viral replication, is more common than was previously believed (range 23-100% of patients). To determine the in vivo state of HIV expression, samples of peripheral blood of 49 HIV-infected individuals at all stages of disease were examined. All subjects were positive for viral DNA by standard polymerase chain reaction (PCR), and a modified PCR was utilized to detect HIV-specific mRNAs (gag, major splice junction, env, and tat/rev). Patient's plasma was also assayed for p24 antigen and viremia. The results were as follows: (formula: see text) Overall, the findings suggest that active viral expression occurs at all stages of HIV infection. In particular, the presence of gag mRNA was determined in only 2 of 14 patients with T4% greater than 30% but in 20 of 35 patients with T4% less than or equal to 30% (p less than 0.05), demonstrating a direct association between the presence of message for a structural protein, and more advanced immunosuppression. Determination of the expression of certain HIV-specific messages from within a patient's cells adds a new dimension to understanding the pathogenesis of HIV infection. The presence of HIV-specific mRNAs, and in particular gag message, in many healthy seropositives may further argue for early initiation of antiviral therapy.

Altered mouse mammary tumor virus transcript synthesis in T-cell lymphoma cells

Proviral copies of mouse mammary tumor virus (MMTV) are known to be amplified in certain T-cell lymphomas. Transcription of the amplified MMTV proviruses was studied in detail in two T-cell lymphoma lines and showed the production of deletions and premature termination of env mRNAs and the premature termination of gag transcripts. EL-4 cells produce three env mRNAs, and sequence analysis of cDNAs of the two smaller transcripts revealed large deletions encompassing the 3' half of the env gene. The deletion in at least one of the altered transcripts appeared to be produced by a splicing mechanism. T-cell lymphoma line ML of DBA/2 mice also synthesizes two smaller env transcripts, both of which result from premature termination of transcription. Both lines transcribe high levels of gag mRNAs of about 0.8 kilobases in length, terminating at the end of the region encoding MMTV phosphoprotein pp21. Restriction enzyme BamHI analysis of the amplified proviruses of EL-4 and ML cells as well as of additional non-mammary tumor cell types containing amplified MMTV proviruses suggested that the amplified proviruses were derived from exogenous viruses, or activated endogenous provirus MTV-1 in the case of DBA/2 strain tumor cells.

Human immunodeficiency virus-like, nonreplicating, gag-env particles assemble in a recombinant vaccinia virus expression system

We report the assembly of human immunodeficiency virus (HIV)-like particles in African green monkey kidney cells coinfected with two recombinant vaccinia viruses, one carrying the HIV-1 gag and protease genes and the other the env gene. Biochemical analysis of particles sedimented from culture supernatants of doubly infected cells revealed that they were composed of gag proteins, primarily p24, as well as the env proteins gp120 and gp41. Thin-section immunoelectron microscopy showed that these particles were 100 to 120 nm in diameter, were characterized by the presence of cylindrical core structures, and displayed the mature gp120-gp41 complexes on their surfaces. Furthermore, thin-section immunoelectron microscopy analysis of infected cells showed that particle assembly and budding occurred at the plasma membrane. Nucleic acid hybridization suggested that the particles packaged only the gag mRNA but not the env mRNA. Therefore, the system we present is well suited for studies of HIV virion maturation. In addition, the HIV-like particles provide a novel and attractive approach for vaccine development.

Nucleolar targeting signal of human T-cell leukemia virus type I rex-encoded protein is essential for cytoplasmic accumulation of unspliced viral mRNA.

The posttranscriptional regulator (rex) of human T-cell leukemia virus type I is known to be located predominantly in the cell nucleolus and to induce the accumulation of gag and env viral mRNAs. The N-terminal 19 amino acids of rex-encoded protein (Rex) has been shown to be sufficient to direct hybrid proteins to the cell nucleolus. We have studied the function of the nucleolar targeting signal (NOS) of rex by using full-length proviral DNA and mutant rex expression plasmids. Partial deletions of the NOS sequence abolished the accumulation of unspliced cytoplasmic mRNA, although the gene products of rex mutants were found in the nucleoplasm. These results indicate that NOS sequence, or nucleolar localization of Rex, is essential for Rex function.

The rev (trs/art) protein of human immunodeficiency virus type 1 affects viral mRNA and protein expression via a cis-acting sequence in the env region.

The study of expression of several human immunodeficiency virus type 1 proviral mutants in human cells in the presence or absence of rev (trs/art) protein reveals that rev increases the levels of unspliced and env mRNA and the accumulated structural viral proteins. rev protein produced from appropriate expression vectors fully complements the rev-defective mutants. rev requires the presence of a specific cis-acting sequence for its function. This rev-responsive element sequence has been localized within a 520 base-pair fragment in the env region of human immunodeficiency virus type 1. gag and env expression is coordinately regulated by rev. Two independent cis-acting elements localized in the gag and env regions are responsible for the low levels of gag and env mRNA in the absence of rev. These elements are different than the rev-responsive element and act independent of each other.

Molecular mechanisms of regulation of HTLV-1 gene expression and its association with leukemogenesis.

Human T-cell leukemia virus type 1 (HTLV-1) has two trans-acting regulator genes, tax and rex, in the pX region. The tax gene is a trans-acting transcriptional activator of the long terminal repeat (LTR) and also of the cellular gene for IL-2R alpha. The latter seems to explain initiation of abnormal growth of HTLV-1 infected cells. The rex gene is a posttranscriptional regulator accumulating gag and env mRNA and also indirectly suppressing the transcription. The regulation requires two cis-acting elements, the LTR sequence at the 3' terminus and 5' splice signal, suggesting a novel mechanism of RNA processing in the nucleus. These two trans-activator genes are essential for efficient replication of HTLV-1 and also explain its poor replication competence and tendency to be latent in vivo.

Two cis-acting elements responsible for posttranscriptional trans-regulation of gene expression of human T-cell leukemia virus type I.

The pX sequence of human T-cell leukemia virus type I codes for two nuclear proteins, p40tax and p27rex, and a cytoplasmic protein, p21x-III.p40tax activates transcription from the long terminal repeat (LTR), whereas p27rex modulates posttranscriptional processing to accumulate gag and env mRNAs that retain intron sequences. In this paper, we identify two cis-acting sequence elements needed for regulation by p27rex: a 5' splice signal and a specific sequence in the 3' LTR. These two sequence elements are sufficient for regulation by p27rex; expression of a cellular gene (metallothionein I) became sensitive to rex regulation when the LTR was inserted at the 3' end of this gene. The requirement for these two elements suggests an unusual regulatory mechanism of RNA processing in the nucleus.

Transcriptional (p40x) and post-transcriptional (p27x-III) regulators are required for the expression and replication of human T-cell leukemia virus type I genes.

The pX sequence of human T-cell leukemia virus type I codes for three products: p40x, p27x-III, and p21x-III. p40x is a transcriptional trans-activator that activates not only the viral long terminal repeat but also cellular genes for interleukin 2 and its receptor. p27x-III and p21x-III are not required for transcriptional activation, and their functions were unknown. Cotransfection experiments with defective human T-cell leukemia virus type I proviruses and various pX expression plasmids revealed that p27x-III, in addition to p40x, was required for gag gene expression. Furthermore, it was shown that p27x-III induced accumulation of a high level of unspliced viral gag mRNA. These results indicate that p27x-III is a post-transcriptional modulator of viral RNA whose transcription has been fully activated by p40x.

The second pX product p27 x-III of HTLV-1 is required for gag gene expression

The human T-cell leukemia virus type-1 (HTLV-1) contains a unique pX region, which encodes the gene products p40 x , p27 x-III and p21 x-III . p40 x is required for transcriptional trans-activation, whereas p27 x-III and p21 x-III have no such function. Transfection of pX expression plasmids containing different combinations for the three gene products into cells integrated with HTLV-1 proviruses defective in pX expression revealed that both p40 x and p27 x-III are required for expression of the gag protein and accumulation of gag mRNA. These observations suggest that the pX product p40 x activates transcription and p27 x-III controls the level of gag mRNA by post-transcriptional modulation.