A common feature of all replication-competent retroviruses is that the primary transcription product from the proviral DNA contains at least three open reading frames, gag, po0, and env, positioned 5' to 3' in the RNA. This product is always a genome-length RNA that is spliced to generate subgenomic mRNAs. In the case of the "simpler" retroviruses, a single 5' splice site is positioned near the 5' end ofthe primary transcript and splicing involves the use ofone or two 3' acceptor sites positioned downstream in the RNA. Thus the subgenomic molecules are always singly spliced and have had most or all of the gag-pol region removed. However, because splicing is inefficient, enough full-length RNA remains to function both as the mRNA for the gag andpol genes and as the molecule that is packaged into virus particles (1).The situation in human immunodeficiency virus type 1 (HIV-1) is more complex. In this case, the coding regions for several novel genes are positioned near the center of the primary transcript between gag-pol and env and at the 3' end of the genome (2). The central region of the genome also contains several 5' and 3' splice sites, which, in conjunction with the conventionally positioned 5' splice site near the 5' end of the RNA, are used for differential splicing of the primary transcript into over 20 different species of mRNA (3-5). These RNAs are either singly or multiply spliced.In most cases, cellular mRNAs contain introns that are removed by splicing before transport to the cytoplasm occurs. Intron-containing RNAs are usually prevented from exiting the nucleus due to the binding of splicing factors (6, 7), although there are examples of differentially spliced cellular transcripts that are transported with a retained intron (8). Little is known about how these mRNAs are transported.The HIV Rev protein functions to allow nuclear export of unspliced and singly spliced HIV RNA molecules (9-12). These RNAs contain complete introns and are retained in the nucleus in the absence of Rev. The details of how Rev functions are not known, although it is clear it binds to a specific element in the HIV RNA known as the Revresponsive element (RRE) (13,14).Another genus of more complex retroviruses, typified by human T-lymphotropic virus (HTLV) types I and II, seems to have evolved a mechanism similar to that of HIV to facilitate the transport of intron-containing RNA. These viruses utilize a protein called Rex, which, like Rev, must bind to a specific element present in the viral RNA (RxRE) (15). Rex has also been shown to substitute for Rev in promoting the transport of Rev-dependent mRNA (16,17).While the more complex retroviruses have developed Rev and Rex regulation to allow the cytoplasmic expression of their intron-containing RNA, the simpler retroviruses do not seem to have similar trans-acting proteins. Thus, it has been a puzzle how these viruses achieve nuclear export of their full-length RNA that contains the gag-pol intron.Here we report the identification of a 219-nt element from Mason-Pfizer monkey ...

TAP, the human homologue of the yeast protein Mex67p, has been proposed to serve a role in mRNA export in mammalian cells. We have examined the ability of TAP to mediate export of Rev response element (RRE)-containing human immunodeficiency virus (HIV) RNA, a well-characterized export substrate in mammalian cells. To do this, the TAP gene was fused in frame to either RevM10 or Rev⌬78-79. These proteins are nonfunctional Rev mutant proteins that can bind to HIV RNA containing the RRE in vivo but are unable to mediate the export of this RNA to the cytoplasm. However, the fusion of TAP to either of these mutant proteins gave rise to chimeric proteins that were able to complement Rev function. Significantly, cotransfection with a vector expressing NXT1 (p15), an NTF2-related cellular factor that binds to TAP, led to dramatic enhancement of the ability of the chimeric proteins to mediate RNA export. Mutant-protein analysis demonstrated that the domain necessary for nuclear export mapped to the C-terminal region of TAP and required the domain that interacts with NXT1, as well as the region that has been shown to interact with nucleoporins. RevM10-TAP function was leptomycin B insensitive. In contrast, the function of this protein was inhibited by ⌬CAN, a protein consisting of part of the FG repeat domain of CAN/Nup214. These results show that TAP can complement Rev nuclear export signal function and redirect the export of intron-containing RNA to a CRM1-independent pathway. These experiments support the role of TAP as an RNA export factor in mammalian cells. In addition, they indicate that NXT1 serves as a crucial cellular cofactor in this process.During recent years, it has become increasingly clear that regulation of molecular trafficking between the nucleus and cytoplasm of the eukaryotic cell plays an important role in the regulation of cellular gene expression (for reviews, see references 27, 33, and 39). Although the detailed mechanisms for nuclear export and import remain to be elucidated, numerous studies have shed light on these processes. It has been demonstrated that both protein and RNA substrates are recognized by specific import and export receptors. Several of these receptors have been identified, including receptors involved in export of RNA to the cytoplasm.Different classes of RNA are transported through separate pathways, and export of each of these classes is saturable, indicating the involvement of specific limiting factors (15,29,49,51). The details of the mRNA export pathway have not yet been elucidated. However, in higher eukaryotes, most primary mRNA transcripts contain introns, and as a general rule, these introns are all removed before export from the nucleus (21, 34). Nuclear retention until splicing is completed is believed to ensure that incompletely processed mRNAs do not reach the cytoplasm, where they could give rise to aberrant proteins.Retroviruses have, for several years, served as important model systems for the analysis of mRNA export (for reviews, see references 9 and 22). For al...

RNA interference (RNAi)-mediated viral inhibition has been used in a few organisms for eliciting viral resistance. In the present study, we report the use of RNAi in preventing baculovirus infection in a lepidopteran. We targeted the baculoviral immediate early-1 (ie-1) gene in both a transformed lepidopteran cell line and in the transgenic silkworm Bombyx mori L. Constitutive expression of double-stranded RNA was achieved by piggyBac-mediated transformation of Sf9 cell line with a transgene encoding double-stranded ie-1 RNA (dsie-1). Strong viral repression was seen at early stages of infection but subsequent recovery of viral proliferation was observed. In contrast, the same transgene inserted into the chromosomes of transgenic silkworms induced long-term inhibition of B. mori nucleopolyhedrovirus infection, with nearly 40% protection compared with nontransgenic animals. Protection was efficient at larval stages after oral infection with occlusion bodies or hemocoel injection of budded viruses. Virus injected pupae also displayed resistance. These results show that heritable RNAi can be used to protect silkworm strains from baculovirus infection.

We describe the generation of stable human immunodeficiency virus type 1 (HIV-1)-packaging lines that constitutively express high levels of HIV-1 structural proteins in either a Rev-dependent or a Rev-independent fashion. These cell lines were used to assess gene transfer by using an HIV-1 vector expressing the hygromycin B resistance gene and to study the effects of Rev, Tat, and Nef on the vector titer. The Rev-independent cell lines were created by using gag-pol and env expression vectors that contain the Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE). Vector titers approaching 10 4 CFU/ml were routinely obtained with these cell lines, as well as with the Rev-dependent cell lines, with HeLa-CD4 cells as targets. The presence of Nef and Tat in the producer cell each increased the vector titer 5-to 10-fold. Rev, on the other hand, was absolutely essential for gene transfer, unless the MPMV CTE was present in the vector. In that case, by using the Rev-independent cell lines for packaging, Rev could be completely eliminated from the system without a reduction in vector titer.

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 represssor 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.