A structured retroviral RNA element that mediates nucleocytoplasmic export of intron-containing RNA.

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.

NXT1 (p15) is a crucial cellular cofactor in TAP-dependent export of intron-containing RNA in mammalian cells.

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 RevDelta78-79. 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 DeltaCAN, 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.

Vascular injury induces posttranscriptional regulation of the Id3 gene: cloning of a novel Id3 isoform expressed during vascular lesion formation in rat and human atherosclerosis.

The molecular mechanisms that regulate the proliferation of smooth muscle cells (SMCs) of the vasculature in response to injury are poorly understood. Members of the inhibitor of DNA binding (Id) class of helix-loop-helix transcription factors are known to regulate the growth of a variety of cell types; however, the expression of the various Id genes in SMCs and in vascular lesions has not been examined. In the present study, the yeast 2-hybrid system was used to clone Id genes from a cultured rat aortic SMC library. By use of ubiquitous E proteins as bait, Id3 and a novel isoform of Id3 (Id3a) were cloned. Id3a is the product of alternative splicing of the Id3 gene, resulting in inclusion of a 115-bp "coding intron," which encodes a unique 29-amino acid carboxyl terminus for the Id3a protein. Unlike Id3, Id3a mRNA was not detected in the normal rat carotid artery. However, after balloon injury, Id3a was abundantly expressed throughout the neointimal layer. In addition, mRNA of the human homologue of Id3a (Id3L) was detected in human carotid atherosclerotic plaques. Adenovirus-mediated overexpression of these Id3 isoforms in cultured rat aortic SMCs revealed that infection of SMCs with an adenovirus overexpressing Id3a (in contrast to Id3) resulted in a significant decrease in cell number versus AdLacZ-infected cells. DNA fragmentation analysis suggested that this decrease in SMC viability was due to increased apoptotic activity in cells infected with adenovirus overexpressing Id3a. These results provide evidence that alternative splicing of the Id3 gene may represent an important mechanism by which neointimal SMC growth is attenuated during vascular lesion formation.

High-level expression of the Epstein-Barr virus EBNA1 protein in CV1 cells and human lymphoid cells using a SV40 late replacement vector.

To construct a recombinant plasmid designed to yield large amounts of the Epstein-Barr virus (EBV) nuclear antigen, EBNA1, the EBV BamHI-K fragment (B95-8 strain) was inserted into an expression vector composed of SV40 and pBR322 DNA. The vector replicates in both Escherichia coli and eukaryotic cells. Introduction of such a BamHI-K-containing vector into CV1 monkey cells (using DEAE-dextran, glycerol and chloroquine diphosphate) gave high yields of the correct size EBNA1 protein in 40-50% of the transfected cells. Maximal amounts of EBNA1 could be extracted from the cells at 65-72 h post transfection. Using a quantitative ELISA assay, it was estimated that transfected cells express 500-1000 times more EBNA1 than lymphoid cells, latently infected with EBV. A monoclonal antibody directed against EBNA1 immunoprecipitated two proteins of 74 and 62 kDa from transfected cells. These same two proteins were detected in immunoprecipitation and immunoblot experiments using human EBV-positive polyclonal serum, although this serum also detected several other protein products in transfected cells. In vivo labelling of transfected cells with [32P]orthophosphate showed that the 74- and 62-kDa proteins are modified by phosphorylation. The same vector construction was also used to transfect an EBV-negative human lymphoblastoid cell line (Ramos). Expression of the EBNA1 protein was obtained in up to 20% of the cells.

Interaction of HIV-1 and human salivary mucins.

Previous studies have suggested that salivary secretions may act as inhibitors of HIV-1 replication in vitro. This inhibitory activity was determined to be associated mainly with secretions obtained from the human submandibular-sublingual glands, and subsequent electron micrographs revealed the association of viral particles with the salivary sediment. Fractionation of human submandibular-sublingual (HSMSL) saliva by size-exclusion chromatography was initiated, and resulting fractions were tested for their ability to modulate the replication of HIV-1 using a plaque assay on HeLa CD4+ cell monolayers. Results indicated that the filtration-sensitive inhibitory activity was primarily associated with the mucin-rich fractions, and the inhibitory activity was found to reduce the number of infectious units by 75%. To determine the identity of the salivary components involved, adsorption experiments involving the interaction of HIV particles with immobilized salivary components were performed. Immunological counter staining revealed an interaction of HIV particles as well as recombinant gp120 with the lower-molecular-weight mucin. Electron microscopic examination of the mucin-rich fractions-HIV incubates revealed the aggregation of virus particles by salivary components. These results suggest that human salivary mucins may have a role in modulating the infectivity of HIV-1.

Epstein-barr virus latent membrane protein (LMP1) induces specific NFkappaB complexes in human T-lymphoid cells.

The Epstein-Barr virus (EBV) latent membrane protein (LMP1) is believed to play a crucial role in oncogenesis mediated by this virus. We and others previously showed that LMP1 can induce NFkappaB activity in several non-lymphoid cells and B-lymphoid cell lines. Here we show that LMP1 is also able to efficiently induce NFkappaB in human T-lymphoid and monocytic cells. Specific NFkappaB complexes were detected in the nuclei of transfected Jurkat cells using gel mobility shift assays and Western blot analyses. Using antibodies, we demonstrated that these complexes contain NFkappaB subunits NFkB1, NFkB2, RelA and c-Rel. Our results also showed that the NFkappaB complexes induced by LMP1 are able to bind to the NFkappaB consensus sequence in the promoter of the interleukin-2alpha receptor gene and induce expression from a minimal promoter linked to four tandem copies of this sequence. This suggests a possible mechanism by which LMP1 could induce T-cell activation and proliferation.

Antibodies in human sera against the Epstein-Barr virus encoded latent membrane protein (LMP).

Antibodies reactive with the Epstein-Barr (EBV)-encoded latent membrane protein, LMP, were detected in human sera. Membrane fractions of the EBV-carrying Raji cells and a fusion protein (LMFP), that represents the carboxy part of LMP, were used as antigens. These were assayed by the reduction of the leukocyte migration inhibition (LMI) reaction. Reactivity with LMFP was detected in 10 of 14 sera from patients with Burkitt's lymphoma (BL) and in 3 of 23 sera from healthy EBV-seropositive individuals. The antibody levels were higher in the BL sera. Since the tumor cells do not express LMP, this may be due to the high virus load in the patients.

Expression and purification of the HIV type 1 Rev protein produced in Escherichia coli and its use in the generation of monoclonal antibodies.

We have developed a simple and rapid procedure for the purification of large amounts of Rev protein overexpressed in E. coli. The purification method, which does not require denaturation of the protein, takes advantage of the positively charged nature of Rev and the ability of Rev to interact with nucleic acids. The purified protein was used to develop three novel murine monoclonal antibodies against Rev. Using fusion proteins between glutathione S-transferase (GST) and various fragments of the Rev protein, we mapped the specificity of these antibodies to different regions of the Rev protein. One antibody, 3H6, is directed against the nucleolar localization/RRE-binding domain of Rev between amino acids 38 and 44. Another antibody, 3G4, recognizes an epitope between amino acids 90 and 116 of Rev. A third antibody, 2G2, does not recognize any of the fusion proteins, and may be directed against a conformational epitope. All three antibodies are able to detect Rev on Western blots and to immunoprecipitate Rev under native conditions. However, only 3H6 and 3G4 immunoprecipitate Rev under denaturing conditions and are able to detect Rev expressed in transfected cells by indirect immunofluorescence. These antibodies should prove useful in further studies of Rev function.

Immunoprecipitation of Epstein-Barr virus EBNA1 protein using human polyclonal serum.

We have developed a method that permits the use of human polyclonal serum to immunoprecipitate BamH1-K EBNA(EBNA1) from EBV transformed cell lines and from cells transfected with an expression vector containing the Bam K region of EBV. Serum from healthy seropositive donors is preabsorbed once with lysate of EBV-negative Burkitt lymphoma cells, then fractionated by gel filtration. The main IgG fraction is then used for the immunoprecipitations. Immunoprecipitated material is visualized by immunoblotting using the same serum. Two proteins with apparent molecular weights of 74 and 62 kD are specifically precipitated from extracts of B95-8 cells. Several proteins are immunoprecipitated from cells transfected with the Bam K containing vector, the apparent molecular weights of the 4 major bands are 74, 68, 62 and 57 kD. Labelling of transfected cells with [3H]glycine and [32P]orthophosphate shows that the 74 and 62 kD proteins can be labelled with both isotopes.

Human T-cell lymphotrophic virus type-I (HTLV-I) retrovirus and human disease.

Human T-cell lymphotrophic virus type-I (HTLV-I) was the first pathogenic retrovirus identified in humans. HTLV-I is now linked to a number of clinical diseases, most notably adult T-cell leukemia/lymphoma and the syndrome known as HTLV-associated myelopathy or tropical spastic paraparesis (HAM/TSP). For the emergency physician practicing among patients from high-risk groups, HTLV-I infection and its associated diseases are presenting an increasing challenge. This report describes its transmission, seroprevalence, associated diseases, and methods to control the spread of this retrovirus.

Regulation of retroviral RNA export.

Viruses are obligate intracellular parasites and have to use the host cell machinery for their replication. Many viruses are able to divert different parts of this machinery to preferentially enhance virus replication at the expense of the cell. The mechanisms by which different viruses do this have, over the years, given us great insight into many cellular processes. Although we still know relatively little about how RNA is exported from the nucleus to the cytoplasm and how this process is regulated, retroviruses have already emerged as one of the most important model systems for these studies. This review will attempt to summarize what we have learnt from these viruses to date and what we hope to achieve in the near future.

Epstein-Barr virus latent membrane protein transactivates the human immunodeficiency virus type 1 long terminal repeat through induction of NF-kappa B activity.

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-kappa B elements in the LTR abolished the transactivation, indicating that the LMP effect on HIV expression was due to induction of NF-kappa B 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.

Isolation of DNA from agarose gels using DEAE-paper. Application to restriction site mapping of adenovirus type 16 DNA.

A new method for isolating DNA from agarose gels is described. The method involves the simultaneous transfer of all DNA-fragments from an agarose slab gel onto DEAE-cellulose paper and the elution of the individual fragments from the paper with 1 M NaCl. DNA isolated from agarose gels in this way is susceptible to cleavage with several restriction endonucleases, and can be labeled in vitro with E coli DNA-polymerase I, T4 DNA-polymerase and T4 polynucleotide kinase. We have used the method to construct restriction endonuclease maps of adenovirus type 16 DNA.

Characterization of deletion mutations in the capsid region of human immunodeficiency virus type 1 that affect particle formation and Gag-Pol precursor incorporation.

The core of human immunodeficiency virus type 1 is derived from two precursor polyproteins, Pr55gag and Pr160gag-pol. The Gag precursor can assemble into immature virus-like particles when expressed by itself, while the Gag-Pol precursor lacks particle-forming ability. We have shown previously that the Gag precursor is able to "rescue" the Gag-Pol precursor into virus-like particles when the two polyproteins are expressed in the same cell by using separate simian virus 40-based plasmid expression vectors. To understand this interaction in greater detail, we have made deletion mutations in the capsid-coding regions of Gag- and Gag-Pol-expressing plasmids and assayed for the abilities of these precursors to assemble into virus-like particles. When we tested the abilities of Gag-Pol precursors to be incorporated into particles of Gag by coexpressing the precursors, we found that mutant Gag-Pol precursors lacking a conserved region in retroviral capsid proteins, the major homology region (MHR), were excluded from wild-type Gag particles. Mutant precursors lacking MHR were also less efficient in processing the Gag precursor in trans. These results suggest that the MHR is critical for interactions between Gag and Gag-Pol molecules. In contrast to these results, expression of mutated Gag precursors alone showed that deletions in the capsid region, including those which removed the MHR, reduced the efficiency of particle formation by only 40 to 50%. The mutant particles, however, were clearly lighter than the wild type in sucrose density gradients. These results indicate that the requirements for Gag particle formation differ from the ones essential for efficient incorporation of the Gag-Pol precursor into these particles.

An intact TAR element and cytoplasmic localization are necessary for efficient packaging of human immunodeficiency virus type 1 genomic RNA.

Although most reports defining the human immunodeficiency virus type 1 (HIV-1) genomic RNA packaging signal have focused on the region downstream of the major 5' splice site, others have suggested that sequences upstream of the splice site may also play an important role. In this study we have directly examined the role played by the HIV-1 TAR region in RNA packaging. For these experiments we used a proviral expression system that is largely independent of Tat for transcriptional activation. This allowed us to create constructs that efficiently expressed RNAs carrying mutations in TAR and to determine the ability of these RNAs to be packaged. Our results indicate that loss of sequences in TAR significantly reduce the ability of a viral RNA to be packaged. The requirement for TAR sequences in RNA packaging was further examined by using a series of missense mutations positioned throughout the entire TAR structure. TAR mutations previously shown to influence Tat transactivation, such as G31U in the upper loop region or UCU to AAG in the bulge (nucleotides [nt] 22 to 24), failed to have any effect on RNA packaging. Mutations which disrupted the portion of the TAR stem immediately below the bulge also had little effect. In contrast, dramatic effects on RNA packaging were observed with constructs containing mutations in the lower portion of the TAR stem. Point mutations which altered nt 5 to 9, 10 to 15, 44 to 49, or 50 to 54 all reduced RNA packaging 11- to 25-fold. However, compensatory double mutations which restored the stem structure were able to restore packaging. These results indicate that an intact lower stem structure, rather than a specific sequence, is required for RNA packaging. Our results also showed that RNA molecules retained within the nucleus cannot be packaged, unless they are transported to the cytoplasm by either Rev/Rev response element or the Mason-Pfizer monkey virus constitutive transport element.