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.     

The simian immunodeficiency virus (SIV) rev gene regulates env expression

The rev gene product is a trans-acting nuclear regulatory protein that is essential for AIDS virus replication. To define the effect of the SIV rev gene on its gp160 expression, two SV40 constructs have been made: pBC17 is a rev+env+ construct, and pBD21 is a rev-env+ construct. After transfecting the constructs into COS-1 cells, RNA, protein, syncytium formation, and monoclonal antibody blocking assays were performed. The results indicated that the SIV rev gene positively influenced the level of full-length env mRNA and was required for expression of SIV gp160.     

Human immunodeficiency virus env expression becomes Rev-independent if the env region is not defined as an intron.

The human immunodeficiency virus (HIV) Rev protein functions to facilitate export of intron-containing HIV mRNA from the nucleus to the cytoplasm. We have previously shown that splice site recognition plays an important role in Rev regulation of HIV env expression. Here we have further analyzed the effects of splice sites on HIV env expression and Rev regulation, using a simian virus 40 late replacement vector system. env expression from the vector became completely Rev-independent when an excisable intron was positioned upstream of the env region, provided that env was not recognized as an intron. Complete Rev regulation was restored either by the insertion of a 5' splice site between the intron and the env open reading frame or by deletion of the 3' splice site of the upstream intron. These results show that 5' splice sites can function as cis-acting repressor sequence (CRS) elements to retain RNA in the nucleus in the absence of Rev. They also indicate that Rev regulation of HIV env expression is critically dependent on whether the env region is defined as an intron. This strengthens the hypothesis that Rev interacts with components of the splicing machinery to release splicing factors and enable export of the mRNA before splicing occurs.     

A splice hepadnavirus RNA that is essential for virus replication.

According to the current model of hepadnavirus gene expression, the viral envelope proteins are produced from unspliced subgenomic RNAs, in contrast to the retroviral mechanism, where the subgenomic env RNA is generated by RNA splicing. We now describe and characterize a novel duck hepatitis B virus RNA species which is derived from the RNA pregenome by loss of a 1.15 kb intron. This RNA (termed spliced L RNA) codes for the large surface protein (L protein), as does the previously described unspliced mRNA (the preS RNA); however, it differs in 5' leader sequence and promoter control. Mutational analysis indicates that the spliced L RNA is functionally important for virus replication in infected hepatocytes and ducks, but not for virus formation from transfected DNA genomes. This suggests that the newly discovered second pathway for L protein synthesis plays a distinct role in an early step in the viral life cycle.     

A tripartite HIV-1 tat-env-rev fusion protein.

A 26 kd protein reactive with antiserum to the transactivator tat of the Human Immunodeficiency Virus Type 1 (HIV-1) has been detected in virus producing cells. The 26 kd protein is shown to be a tripartite fusion protein including coding sequences of the tat, envelope (env) and regulator of virion expression (rev) genes. Fusion of these coding sequences occurs by use of a previously undescribed exon within env. This 26 kd protein, designated tnv, has tat but no rev activity detectable with the assay used. The existence of other less abundant tat and rev related proteins in HIV-1 producing cells is also noted.     

Messenger Activity of Virion RNA for Avian Leukosis Viral Envelope Glycoprotein

An intracellular assay for viral envelope glycoprotein (env) messenger was employed to analyze the RNA from virus particles of Rous-associated virus type 2. For this assay RNA was microinjected into cells infected by the env-deficient Bryan strain of Rous sarcoma virus [RSV(-) cells]. Only when the injected RNA could be translated by the recipient cells to produce viral envelope glycoprotein was the env deficiency of the RSV(-) cells complemented, enabling them to release focus-forming virus. RNA in a 21S size fraction from the Rous-associated virus particle promoted the release of numerous focus-forming virus from RSV(-) cells, whereas the major 35S virion RNA species was inactive. The env messenger activity sedimented as a sharp peak with high specific activity. RNase T1-generated fragments of virion 35S RNA were unable to promote the release of infectious virus from RSV(-) cells. Consequently, the active molecule was most likely to be env messenger which had been encapsulated by the virus particle from the cytoplasm of infected cells. Approximately 95% of the env messenger within the virion was associated with the virion high-molecular-weight RNA complex. The temperature required to dissociate env messenger from the high-molecular-weight complex was indistinguishable from the temperature required to disrupt the complex itself. Virion high-molecular-weight RNA that was associated with env messenger sedimented slightly more rapidly than the bulk virion RNA; this was the strongest evidence that the 21S messenger had been encapsulated directly from the infected cells. These data are considered along with a related observation [concerning the prolonged expression of env messenger after injection into RSV(-) cells] to raise the possibility that virus-encapsulated env messenger can become expressed within subsequently infected cells.     

Evidence for a direct interaction of Rev protein with nuclear envelop mRNA-translocation system.

The interaction of the Rev protein from human immunodeficiency virus type 1 (HIV-1) with the nucleocytoplasmic mRNA-transport system was investigated. In gel-shift assay, the recombinant Rev protein used in this study selectively bound to the Rev-responsive element (RRE) region of HIV-1 env-specific RNA. Nitrocellulose-filter-binding studies and Northern/Western-blotting experiments revealed an association constant of approximately 1 x 10(10) M-1. The Rev protein also strongly bound to isolated nuclear envelopes from H9 cells, containing the poly(A)-binding site (= mRNA carrier) and the nucleoside triphosphatase (= NTPase), which are thought to be involved in nuclear export of poly(A)-rich mRNA. Binding of 125I-Rev to a 110-kDa nuclear-envelope protein, the putative mRNA carrier, could be demonstrated in in vitro experiments. Both efflux of cellular poly(A)-rich RNA, such as actin RNA [but not efflux of poly(A)-free RNA] from isolated nuclei and the nuclear-envelope NTPase activity were strongly inhibited by Rev protein. On the other hand, transport of viral env RNA, containing the Rev-responsive element, was increased in the presence of Rev. Studying the release of RNA from closed nuclear-envelope vesicles containing entrapped RNA, the action of Rev was found to occur at the level of translocation of RNA through the nuclear pore. Evidence is presented that Rev down-regulates the NTPase-driven transport of mRNA lacking the RRE, most likely via binding to the mRNA carrier within the envelope. In contrast to the efflux of RRE-free RNA, ATP-dependent efflux of RRE-containing RNA from resealed nuclear-envelope vesicles was found to be increased, if the RNA was entrapped in the vesicles together with Rev protein. In addition, it was found that phosphorylated Rev, which is transported together with RRE-containing RNA out of the vesicles, becomes dephosphorylated during transport. In the vesicle experiments it is demonstrated for the first time that a protein selectively channels a specific mRNA across the nuclear-envelope pore complex.     

A novel human immunodeficiency virus type 1 protein, tev, shares sequences with tat, env, and rev proteins

We have characterized a novel 28-kilodalton protein, p28tev, detected in human immunodeficiency virus type 1-infected cells. tev is recognized by both tat and rev monospecific antibodies. tev is initiated at the tat AUG and contains the first exon of tat at its amino terminus, a small portion of env in the middle, and the second exon of rev at its carboxy terminus. A cDNA clone producing tev was cloned and expressed in human cells. Sequence analysis revealed that the tev mRNA is generated by splicing to a novel exon located in the env region. This identifies a fourth class of multiply spliced human immunodeficiency virus mRNAs, produced in infected and transfected cells. tev is regulated during the virus life cycle similarly to the other regulatory proteins, tat, rev, and nef, and displays both tat and rev activities in functional assays. Since tev contains important functional domains of tat and rev and is produced very early after transfection, it may be an important regulator in the initial phase of virus expression. Another rev-related protein, p18(6)Drev, containing env and rev sequences, was characterized and was found not to have detectable rev activity.     

Expression of a subgenomic retroviral messenger RNA

When mRNA for avian retroviral envelope glycoprotein (env) was injected into cells transformed by env-deficient Bryan Rous sarcoma virus, the env deficiency of the injected cells was complemented to allow the release of transforming virus for up to 40 hr. When virus spread within the injected culture was allowed to occur, a second phase of transforming virus production by the injected culture began approximately 2 days following injection, continued for many days and often increased to titers well above those seen soon after injection. The requirement for virus spread, along with the genetic properties of virus released long after injection, supported the hypothesis that the second phase of virus production resulted when injected env mRNA was packaged into virus released by injected cells. When this virus infected other cells within the culture the env mRNA was reverse-transcribed to form a subgenomic, proviral-like molecule able to direct the synthesis of env mRNA. Accordingly, it was shown that neither DNA nor full genomic viral RNA contaminating injected mRNA preparations could account for the results. Evidence that an mRNA can be reverse-transcribed into an active, proviral-like molecule may be of importance in the relationship between retroviruses and their hosts.     

Comparison of Rous sarcoma virus RNA processing in chicken and mouse fibroblasts: evidence for double-spliced RNA in nonpermissive mouse cells.

Rous sarcoma virus, an avian retrovirus, transforms but does not replicate in mammalian cells. To determine to what extent differences in RNA splicing might contribute to this lack of productive infection, cloned proviral DNA derived from the Prague A strain of Rous sarcoma virus was transfected into mouse NIH 3T3 cells, and the viral RNA was compared by RNase protection with viral RNA from transfected chicken embryo fibroblasts by using a tandem antisense riboprobe spanning the three major splice sites. The levels of viral RNA in NIH 3T3 cells compared with those in chicken embryo fibroblasts were lower, but the RNA was spliced at increased efficiency. The difference in the ratio of unspliced to spliced RNA levels was not due to the increased lability of unspliced RNA in NIH 3T3 cells. Although chicken embryo fibroblasts contained equal levels of src and env mRNAs, spliced viral mRNAs in NIH 3T3 cells were almost exclusively src. In NIH 3T3 cells the env mRNA was further processed by using a cryptic 5' splice site located within the env coding sequences and the normal src 3' splice site to form a double-spliced mRNA. This mRNA was identical to the src mRNA, except that a 159-nucleotide sequence from the 5' end of the env gene was inserted at the src splice junction. Smaller amounts of single-spliced RNA were also present in which only the region between the cryptic 5' and src 3' splice sites was spliced out. The aberrant processing of the viral env mRNA in NIH 3T3 cells may in part explain the nonpermissiveness of these cells to productive Rous sarcoma virus infection.     

Characterization of multiple mRNA species of simian immunodeficiency virus from macaques in a CD4+ lymphoid cell line.

Cytoplasmic poly(A)+ RNA was isolated from CEMX721.174 cells 5 to 10 days after infection with molecularly cloned simian immunodeficiency virus SIVmac239. Expression of SIV RNA was analyzed by Northern (RNA) blot hybridization and by sequencing of cDNA clones. As expected, a splice donor site was demonstrated in the untranslated leader sequence outside the left long terminal repeat. The region between pol and env was found to contain at least two splice donor and six splice acceptor sites. Splice acceptor and donor sites in the intergenic region were suitably positioned for expression of vpx, vpr, tat, and rev. Splice acceptor sites at nucleotides 8802 and 8805 were demonstrated in singly and doubly spliced RNAs with the potential of expressing nef and the second exons of tat and rev. Our results demonstrate a complex pattern of alternative splicing of SIV mRNAs. The results are very similar to what has been observed in human immunodeficiency virus type 1-infected cells, suggesting that both human and simian immunodeficiency viruses are subject to multiple levels of regulation.     

Role of intron-contained sequences in formation of moloney murine leukemia virus env mRNA.

Formation of the Moloney murine leukemia virus envelope mRNA involves the removal of a 5,185-base pair-long intron. Deletion analysis of two Moloney murine leukemia virus-derived expression vectors revealed the existence of two short regions within the viral intron which are required for the efficient formation of the spliced RNA species. One region was present upstream from the 3' splice junction, extended at least 85 nucleotides beyond the splice site, and was not more than 165 nucleotides long. As yeast polymerase II introns, the Moloney murine leukemia virus intron contains the sequence 5'-TACTAAC-3' 15 nucleotides upstream from the 3' splice site. A second region located in the middle of the intron, within a 560-nucleotide-long sequence, was also essential for formation of the spliced RNA species. The efficient splicing of the env mRNA in the absence of expression of viral genes raises the possibility that similar mechanisms are used to remove introns of (some) cellular genes.     

Role of the nuclear envelope in synthesis, processing, and transport of membrane glycoproteins

The outer nuclear membrane is morphologically similar to rough endoplasmic reticulum. The presence of ribosomes bound to its cytoplasmic surface suggests that it could be a site of synthesis of membrane glycoproteins. We have examined the biogenesis of the vesicular stomatitis virus G protein in the nuclear envelope as a model for the biogenesis of membrane glycoproteins. G protein was present in nuclear membranes of infected Friend erythroleukemia cells immediately following synthesis and was transported out of nuclear membranes to cytoplasmic membranes with a time course similar to transport from rough endoplasmic reticulum (t 1/2 = 5-7 min). Temperature-sensitive mutations in viral membrane proteins which block transport of G protein from endoplasmic reticulum also blocked transport of G protein from the nuclear envelope. Friend erythroleukemia cells and NIH 3T3 cells differed in the fraction of newly synthesized G protein found in nuclear membranes, apparently reflecting the relative amount of nuclear membrane compared to endoplasmic reticulum available for glycoprotein synthesis. Nuclear membranes from erythroleukemia cells appeared to have the enzymatic activities necessary for cleavage of the signal sequence and core glycosylation of newly synthesized G protein. Signal peptidase activity was detected by the ability of detergent-solubilized membranes of isolated nuclei to correctly remove the signal sequence of human preplacental lactogen. RNA isolated from the nuclear envelope was highly enriched for G protein mRNA, suggesting that G protein was synthesized on the outer nuclear membrane rather than redistributing to nuclear membranes from endoplasmic reticulum before or during cell fractionation. These results suggest a mechanism for incorporation of membrane glycoproteins into the nuclear envelope and suggest that in some cell types the nuclear envelope is a major source of newly synthesized membrane glycoproteins.