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 ...

A common feature of gene expression in all retroviruses is that unspliced, intron-containing RNA is exported to the cytoplasm despite the fact that cellular RNAs which contain introns are usually restricted to the nucleus. In complex retroviruses, the export of intron-containing RNA is mediated by specific viral regulatory proteins (e.g., human immunodeficiency virus type 1 [HIV-1] Rev) that bind to elements in the viral RNA. However, simpler retroviruses do not encode such regulatory proteins. Here we show that the genome of the simpler retrovirus Mason-Pfizer monkey virus (MPMV) contains an element that serves as an autonomous nuclear export signal for intron-containing RNA. This element is essential for MPMV replication; however, its function can be complemented by HIV-1 Rev and the Rev-responsive element. The element can also facilitate the export of cellular intron-containing RNA. These results suggest that the MPMV element mimics cellular RNA transport signals and mediates RNA export through interaction with endogenous cellular factors.The mechanisms that govern the export of RNA from the nucleus to the cytoplasm in eukaryotic cells are to a large extent still unknown. Although some proteins have been shown to be involved in the export process, it is not at all clear how they interact or how export is regulated. Experiments performed by microinjection of oocytes do suggest, however, that RNA export is an energy-dependent, saturable process and that different pathways are utilized by different RNA species (17,28,29,32). It is believed that the export pathways, in all cases, involve passage of the RNA through the nuclear pore.Some specific export signals have been identified in RNA. In the case of spliceosomal small nuclear RNAs (snRNAs), the m 7 G cap structure present in these RNAs is known to constitute an important signal (20,27,29,61). This kind of cap may also play a role in mRNA export since it is also present on mRNAs. For mRNA, it has also been shown that export, in most cases, requires removal of all complete introns by splicing (6, 38). If splice sites are mutated so that intron removal is prevented or slowed, the resulting incompletely spliced mRNAs are retained in the nucleus. These types of experiments have led to the hypothesis that splicing factors serve to retain mRNA in the nucleus and that the RNA's association with the export machinery requires prior removal of all complete introns.In accordance with this hypothesis, export of intron-containing cellular mRNAs from the nucleus to the cytoplasm seems to be a rare occurrence, although there are a few examples in the literature (41). In contrast, the export of unspliced introncontaining RNA is an imperative for the replication of all retroviruses. This is due to the fact that individual viral mRNAs are generated by alternative splicing of a single primary transcript that serves as the viral genome and as the mRNA for the gag and gag-pol gene products (8,33,63). This transcript is capped and polyadenylated by the normal cellular machinery.In t...

e HIV-1 Rev and the Rev response element (RRE) enable a critical step in the viral replication cycle by facilitating the nuclear export of intron-containing mRNAs, yet their activities have rarely been analyzed in natural infections. This study characterized their genetic and functional variation in a small cohort of HIV-infected individuals. Multiple Rev and RRE sequences were obtained using single-genome sequencing (SGS) of plasma samples collected within 6 months after seroconversion and at a later time. This allowed the identification of cognate sequences that were linked in vivo in the same viral genome and acted together as a functional unit. Phylogenetic analyses of these sequences indicated that 4/5 infections were founded by a single transmission event. Rev and RRE variants from each time point were subjected to functional analysis as both cognate pairs and as individual components. While a range of Rev-RRE activities were seen, the activity of cognate pairs from a single time point clustered to a discrete level, which was termed the set point. In 3/5 patients, this set point changed significantly over the time period studied. In all patients, RRE activity was more sensitive to sequence variation than Rev activity and acted as the primary driver of the cognate set point. Selected patient RREs were also shown to have differences in Rev multimerization using gel shift binding assays. Thus, rather than acting as a simple on-off switch or maintaining a constant level of activity throughout infection, the Rev-RRE system can fluctuate, presumably to control replication. Infection with HIV most often results from the transmission of a single viral particle, as evident from analysis of env, gag, and pol gene sequences in acutely infected individuals (1-6). Populationlevel analysis has shown that single-variant HIV infections have low sequence diversity at early time points after seroconversion (7-10), but that multiple sequence variants arise over time to form a quasispecies. Various selective pressures, including antibody and cytotoxic-T-lymphocyte (CTL) immune-mediated responses (11-16) and other less well-defined viral and host characteristics, appear to drive the expansion and contraction of HIV subpopulations throughout infection. Single-genome sequencing (SGS) techniques have been used to determine the evolution of env and gag genes (both of which encode structural components of the virus) during infection (3, 4, 6, 16). However, few reports have examined how other HIV genes, such as the essential regulatory gene rev, evolve.The rev gene product (Rev) acts at the posttranscriptional level to mediate the expression of viral genomic RNA and singly spliced mRNAs that encode many of the viral proteins (for reviews, see references 17 and 18). These mRNAs all retain introns and would be expected to be restricted in their nucleocytoplasmic export. However, the Rev protein functions as a bridge between the cellular export machinery and the viral RNA by binding and multimerizing onto the viral Rev response element...

The Epstein-Barr virus latent membrane protein (LMP) is an integral membrane protein that is expressed in cells latently infected with the virus. LMP is believed to play an important role in Epstein-Barr virus transformation and has been shown to induce expression of several cellular proteins. We performed a series of experiments that demonstrated that LMP is an efficient transactivator of expression from the human immunodeficiency virus type 1 long terminal repeat (HIV-1 LTR). Mutation or deletion of the NF-cB elements in the LTR abolished the transactivation, indicating that the LMP effect on HIV expression was due to induction of NF-KB activity. Experiments in which the HIV-1 Tat protein was coexpressed in cells together with LMP showed that Tat was able to potentiate the transactivation. Surprisingly, a synergistic effect of the two proteins was observed even in the absence of the recognized target region for Tat (TAR) in the HIV-1 LTR.

Baseline HIV-1 resistance data are important for resistance monitoring purposes especially in regions initiating large-scale antiretroviral treatment programs. We examined 40 protease and 35 reverse transcriptase amino acid sequences of HIV-1 subtype C from drug inexperienced patients from rural settings in South Africa for resistance mutations. Samples were collected between 2001 and 2004 prior to the availability of antiretrovirals through public health institutions. Ninety-five percent of patients had no major mutations in the protease gene, although substitutions M46L (2.5%) and G73S (2.5%), which according to the Stanford Genotypic Resistance Interpretation Algorithm are considered major mutations, were detected. In addition, a high prevalence of minor mutations was observed in the protease, with at least three minor resistance-associated mutations in 37% of the isolates. An isoleucine insertion at codon 37 was detected in one sequence. Most of the RT sequences were wild-type, although V118I (8.5%) and Y318F (5.7%) associated with resistance to lamivudine and nevirapine, respectively, were observed. Our data suggest that major resistance mutations among the drug-inexperienced population in South Africa may be rare, and routine resistance testing before the initiation of therapy in this initial stage of the treatment program may not be necessary.