Viral mRNA Species Research Articles

Sars-cov-2leader-rna-primed transcription and rna-splicing prevention, control and treatment

Many viral and cellular mRNA species contain a leader sequence derived from a distant upstream site on the same gene by a process of RNA splicing. This process usually involves either nuclear functions or self-splicing of RNA molecules. Coronavirus, a cytoplasmic RNA virus, un- folds yet another mechanism of joining RNA, which involves the use of a free leader RNA molecule. This molecule is synthesized and dissociates from the template RNA, and subsequently re-associates with the template RNA at down- stream initiation sites of sub-genomic mRNAs to serve as the primer for transcription. This leader-primed transcriptional process thus generates viral mRNAs with a fused leader sequence. The purpose of the review to aggregate the anti-SARS drugs in the structural proteins from human SARS related coronavirus (SARS-CoV) while knowing little about the functional sites and possible mutations in these proteins. From a probabilistic viewpoint, we can theoretically select the amino acid pairs as potential candidates for anti-SARS drugs.

The nsp2 Hypervariable Region of Porcine Reproductive and Respiratory Syndrome Virus Strain JXwn06 Is Associated with Viral Cellular Tropism to Primary Porcine Alveolar Macrophages.

Porcine reproductive and respiratory syndrome virus (PRRSV) poses a major threat to global pork production and has been notorious for its rapid genetic evolution in the field. The nonstructural protein 2 (nsp2) replicase protein represents the fastest evolving region of PRRSV, but the underlying biological significance has remained poorly understood. By deletion mutagenesis, we discovered that the nsp2 hypervariable region plays an important role in controlling the balance of genomic mRNA and a subset of subgenomic mRNAs. More significantly, we revealed an unexpected link of the nsp2 hypervariable region to viral tropism. Specifically, a mutant of the Chinese highly pathogenic PRRSV strain JXwn06 carrying a deletion spanning nsp2 amino acids 323 to 521 (nsp2Δ323-521) in its hypervariable region was found to lose infectivity in primary porcine alveolar macrophages (PAMs), although it could replicate relatively efficiently in the supporting cell line MARC-145. Consequently, this mutant failed to establish an infection in piglets. Further dissection of the viral life cycle revealed that the mutant had a defect (or defects) lying in the steps between virus penetration and negative-stranded RNA synthesis. Taken together, our results reveal novel functions of nsp2 in the PRRSV life cycle and provide important insights into the mechanisms of PRRSV RNA synthesis and cellular tropism.IMPORTANCE The PRRSV nsp2 replicase protein undergoes rapid and broad genetic variations in its middle region in the field, but the underlying significance has remained enigmatic. Here, we demonstrate that the nsp2 hypervariable region not only plays an important regulatory role in maintaining the balance of different viral mRNA species but also regulates PRRSV tropism to primary PAMs. Our results reveal novel functions for PRRSV nsp2 and have important implications for understanding the mechanisms of PRRSV RNA synthesis and cellular tropism.

N4BP1 restricts HIV-1 and its inactivation by MALT1 promotes viral reactivation.

RNA-modulating factors not only regulate multiple steps of cellular RNA metabolism, but also emerge as key effectors of the immune response against invading viral pathogens including human immunodeficiency virus type-1 (HIV-1). However, the cellular RNA-binding proteins involved in the establishment and maintenance of latent HIV-1 reservoirs have not been extensively studied. Here, we screened a panel of 62 cellular RNA-binding proteins and identified NEDD4-binding protein 1 (N4BP1) as a potent interferon-inducible inhibitor of HIV-1 in primary T cells and macrophages. N4BP1 harbours a prototypical PilT N terminus-like RNase domain and inhibits HIV-1 replication by interacting with and degrading viral mRNA species. Following activation of CD4+ T cells, however, N4BP1 undergoes rapid cleavage at Arg 509 by the paracaspase named mucosa-associated lymphoid tissue lymphoma translocation 1 (MALT1). Mutational analyses and knockout studies revealed that MALT1-mediated inactivation of N4BP1 facilitates the reactivation of latent HIV-1 proviruses. Taken together, our findings demonstrate that the RNase N4BP1 is an efficient restriction factor of HIV-1 and suggest that inactivation of N4BP1 by induction of MALT1 activation might facilitate elimination of latent HIV-1 reservoirs.

Toscana virus cap-snatching and initiation of transcription

Toscana virus (TOSV) is an arthropod-borne phlebovirus within the family Phenuiviridae in the order Bunyavirales. It seems to be an important agent of human meningoencephalitis in the warm season in the Mediterranean area. Because the polymerase of Bunyavirales lacks a capping activity, it cleaves short-capped RNA leaders derived from the host cell, and uses them to initiate viral mRNA synthesis. To determine the size and nucleotide composition of the host-derived RNA leaders, and to elucidate the first steps of TOSV transcription initiation, we performed a high-throughput sequencing of the 5' end of TOSV mRNAs in infected cells at different times post-infection. Our results indicated that the viral polymerase cleaved the host-capped RNA leaders within a window of 11-16 nucleotides. A single population of cellular mRNAs could be cleaved at different sites to prime the synthesis of several viral mRNA species. The majority of the mRNA resulted from direct priming, but we observed mRNAs resulting from several rounds of prime-and-realign events. Our data suggest that the different rounds of the prime-and-realign mechanism result from the blocking of the template strand in a static position in the active site, leading to the slippage of the nascent strand by two nucleotides when the growing duplex is sorted out from the active site. To minimize this rate-limiting step, TOSV polymerase cleaves preferentially capped RNA leaders after GC, so as to greatly reduce the number of cycles of priming and realignment, and facilitate the separation of the growing duplex.

A stably expressed llama single-domain intrabody targeting Rev displays broad-spectrum anti-HIV activity

The HIV Rev protein mediates the transport of partially and unspliced HIV mRNA from the nucleus to the cytoplasm. Rev multimerizes on a secondary stem-loop structure present in the viral intron-containing mRNA species and recruits the cellular karyopherin CRM1 to export viral mRNAs from the nucleus to the cytoplasm. Previously we have identified a single-domain intrabody (Nb190), derived from a llama heavy-chain antibody, which efficiently inhibits Rev multimerization and suppresses the production of infectious virus. We recently mapped the epitope of this nanobody and demonstrated that Rev residues K20 and Y23 are crucial for interaction while residues V16, H53 and L60 are important to a lesser extent.Here, we generated cell lines stably expressing Nb190 and assessed the capacity of these cell lines to suppress the replication of different HIV-1 subtypes. These cells stably expressing the single-domain antibody are protected from virus-induced cytopathogenic effect even in the context of high multiplicity of infection. In addition, the replication of different subtypes of group M and one strain of group O is significantly suppressed in these cell lines. Next, we analysed the natural variations of Rev amino acids in sequence samples from HIV-1 infected patients worldwide and assessed the effect of Nb190 on the most prevalent polymorphisms occurring at the key epitope positions (K20 and Y23) in Rev. We found that Nb190 was able to suppress the function of these Rev variants except for the K20N mutant, which was present in only 0.7% of HIV-1 sequence populations (n=4632).Cells stably expressing the single-domain intrabody Nb190 are protected against virus-induced cytopathogenic effect and display a selective survival advantage upon infection. In addition, Nb190 suppresses the replication of a wide range of different HIV-1 subtypes. Large-scale sequence analysis reveals that the Nb190 epitope positions in Rev are well conserved across major HIV-1 subtypes and groups. Altogether, our results indicate that Nb190 may have broad potential as a gene therapeutic agent against HIV-1.

Mapping of Betapapillomavirus Human Papillomavirus 5 Transcription and Characterization of Viral-Genome Replication Function

Betapapillomavirus replication and transcription have not been studied in detail because of a lack of suitable cellular systems supporting human papillomavirus (HPV) genome replication. We have recently shown that the human osteosarcoma cell line U2OS provides a useful environment for the genome replication of many different HPVs, including the betapapillomaviruses HPV5 and HPV8. Using mutational analysis and complementation assay, we demonstrated herein that the viral early proteins E1 and E2 are viral transfactors that are necessary and sufficient for HPV5 genome replication. We also identified four HPV5 early promoter regions with transcription start sites (TSSs) at nucleotides (nt) 184/191, 460, 840, and 1254, respectively, and the HPV late promoter with a TSS at nt 7640. In addition, we mapped the HPV5 early polyadenylation cleavage sites via 3' rapid amplification of cDNA ends (3'RACE) to nt 4457 and 4475. In total, 14 different viral mRNA species, originating from the HPV5 genome, were mapped in U2OS cells during transient and stable replication. The main splicing donor and acceptor sites identified herein are consistent with the data previously obtained in HPV5-positive skin lesions. In addition, we identified novel E8 open reading frame (ORF)-containing transcripts (E8^E1C and E8^E2C) expressed from the HPV5 genome. Similar to several other papillomaviruses, the product of the E8^E2C mRNA acts as a repressor of viral genome replication.

MicroRNA expression by an oncogenic retrovirus

Although >250 viral microRNAs (miRNAs) are expressed by a range of nuclear DNA viruses, efforts to identify miRNAs expressed by RNA viruses have so far been in vain (1, 2). In PNAS, Kincaid et al. (3) report the identification of five miRNAs encoded by the delta retrovirus bovine leukemia virus (BLV) that are expressed in BLV-transformed B cells. Interestingly, BLV uses RNA polymerase III (pol III) to directly transcribe the pre-miRNA hairpins that give rise to these viral miRNAs, thereby preventing the cleavage of viral genomic RNA and mRNA species as a result of pre-miRNA excision. Moreover, one of the BLV miRNAs, miR-B4, shares the same seed sequence as cellular miR-29 and therefore can down-regulate many of the same mRNA targets. As miR-29 overexpression can induce chronic lymphocytic leukemia (CLL) in transgenic mice (4), and BLV induces CLL-like B-cell neoplasms in infected cattle and sheep (5), it appears likely that these viral miRNAs play an important role in BLV pathogenesis.

An Intrabody Based on a Llama Single-domain Antibody Targeting the N-terminal α-Helical Multimerization Domain of HIV-1 Rev Prevents Viral Production

The human immunodeficiency virus, type 1 (HIV-1)-encoded Rev protein is essential for the expression of late viral mRNAs. Rev forms a large organized multimeric protein-protein complex on the Rev response element of these viral mRNA species and transports them from the nucleus to the cytoplasm, exploiting the CRM1-mediated cellular machinery. Here we report the selection of a nanobody, derived from a llama heavy-chain only antibody, that efficiently blocks the assembly of Rev multimers. The nanobody inhibits HIV-1 replication in cells and specifically suppresses the Rev-dependent expression of partially spliced and unspliced HIV-1 RNA. In HIV-susceptible cells, this nanobody thus has potential as an effective anti-HIV agent using genetic immunization strategies. Its binding site was mapped to Rev residues Lys-20 and Tyr-23 located in the N-terminal alpha-helical multimerization domain. In the presence of this nanobody, we observed an accumulation of dimeric Rev species, supporting a head-to-head/tail-to-tail molecular model for Rev assembly. The results indicate that the oligomeric assembly of Rev follows an ordered stepwise process and identify a new epitope within Rev that could guide strategies for the development of novel HIV inhibitors.

An Intrabody based on a Llama Single-domain Antibody Targeting the N-terminal α-Helical Multimerization Domain of HIV-1 Rev Inhibits Viral Replication

The human immunodeficiency virus, type 1 (HIV-1)-encoded Rev protein is essential for the expression of late viral mRNAs. Rev forms a large organized multimeric protein-protein complex on the Rev response element of these viral mRNA species and transports them from the nucleus to the cytoplasm, exploiting the CRM1-mediated cellular machinery. Here we report the selection of a nanobody, derived from a llama heavy-chain only antibody, that efficiently blocks the assembly of Rev multimers. The nanobody inhibits HIV-1 replication in cells and specifically suppresses the Rev-dependent expression of partially spliced and unsplicedHIV-1 RNA. InHIV-susceptible cells, this nanobody thushas potential as an effective anti-HIVagent using genetic immunization strategies. Its binding site wasmapped to Rev residues Lys-20 and Tyr-23 located in the N-terminal -helical multimerization domain. In the presence of this nanobody, weobserved an accumulationof dimericRev species, supporting a head-to-head/tail-to-tail molecular model for Rev assembly. The results indicate that the oligomeric assembly of Rev follows an ordered stepwise process and identify a new epitope within Rev that could guide strategies for thedevelopment of novelHIV inhibitors.

Arterivirus Nsp1 Modulates the Accumulation of Minus-Strand Templates to Control the Relative Abundance of Viral mRNAs

The gene expression of plus-strand RNA viruses with a polycistronic genome depends on translation and replication of the genomic mRNA, as well as synthesis of subgenomic (sg) mRNAs. Arteriviruses and coronaviruses, distantly related members of the nidovirus order, employ a unique mechanism of discontinuous minus-strand RNA synthesis to generate subgenome-length templates for the synthesis of a nested set of sg mRNAs. Non-structural protein 1 (nsp1) of the arterivirus equine arteritis virus (EAV), a multifunctional regulator of viral RNA synthesis and virion biogenesis, was previously implicated in controlling the balance between genome replication and sg mRNA synthesis. Here, we employed reverse and forward genetics to gain insight into the multiple regulatory roles of nsp1. Our analysis revealed that the relative abundance of viral mRNAs is tightly controlled by an intricate network of interactions involving all nsp1 subdomains. Distinct nsp1 mutations affected the quantitative balance among viral mRNA species, and our data implicate nsp1 in controlling the accumulation of full-length and subgenome-length minus-strand templates for viral mRNA synthesis. The moderate differential changes in viral mRNA abundance of nsp1 mutants resulted in similarly altered viral protein levels, but progeny virus yields were greatly reduced. Pseudorevertant analysis provided compelling genetic evidence that balanced EAV mRNA accumulation is critical for efficient virus production. This first report on protein-mediated, mRNA-specific control of nidovirus RNA synthesis reveals the existence of an integral control mechanism to fine-tune replication, sg mRNA synthesis, and virus production, and establishes a major role for nsp1 in coordinating the arterivirus replicative cycle.

An Exonic Splicing Silencer Downstream of the 3′ Splice Site A2 Is Required for Efficient Human Immunodeficiency Virus Type 1 Replication

Alternative splicing of the human immunodeficiency virus type 1 (HIV-1) genomic mRNA produces more than 40 unique viral mRNA species, of which more than half remain incompletely spliced within an HIV-1-infected cell. Regulation of splicing at HIV-1 3' splice sites (3'ss) requires suboptimal polypyrimidine tracts, and positive or negative regulation of splicing occurs through binding of cellular factors to cis-acting splicing regulatory elements. We have previously shown that splicing at HIV-1 3'ss A2, which produces vpr mRNA and promotes inclusion of HIV-1 exon 3, is repressed by the hnRNP A/B-dependent exonic splicing silencer ESSV. Here we show that ESSV activity downstream of 3'ss A2 is localized to a 16-nucleotide element within HIV-1 exon 3. HIV-1 replication was reduced by 95% when ESSV was inactivated by mutagenesis. Reduced replication was concomitant with increased inclusion of exon 3 within spliced viral mRNA and decreased accumulation of unspliced viral mRNA, resulting in decreased cell-associated p55 Gag. Prolonged culture of ESSV mutant viruses resulted in two independent second-site reversions disrupting the splice sites that define exon 3, 3'ss A2 and 5' splice site D3. Either of these changes restored both HIV-1 replication and regulated viral splicing. Therefore, inhibition of HIV-1 3'ss A2 splicing is necessary for HIV-1 replication.

A membrane-located glycosphingolipid of monocyte/granulocyte lineage cells induces growth arrest and triggers the lytic viral cycle in Epstein-Barr virus genome-positive Burkitt lymphoma lines

Gangliosides are known to influence cell growth and differentiation. The neolacto series ganglioside IV3NeuAc-nLc4 (2-->3-sialosylparagloboside) is present in members of the monocyte/granulocyte lineage, but is not found in cells that belong to the lymphocyte lineage. In this study we demonstrated that IV3NeuAc-nLc4 inhibits the proliferation of Epstein-Barr virus (EBV) genome-positive Burkitt lymphoma cells of the lines Raji and P3HR-1K. IV3NeuAc-nLc4-induced growth inhibition is associated with an increase in G0/G1 phase cells and a reduced expression of CD21 and HLA-DR antigens on Raji cells. These data suggest that IV3NeuAc-nLc4 may affect differentiation of lymphoma cells. Additionally, the increased expression of viral mRNA species which are characteristic for the lytic viral cycle in the non-producer line Raji and the enhanced release of virions from the producer line P3HR-1K demonstrate that IV3NeuAc-nLc4 activates the replication of EBV. Growth inhibition and termination of the viral latency suggest that IV3NeuAc-nLc4 present in monocyte/granulocyte lineage cells may be an effector of the natural defense against EBV persistency and transformation.

A herpesvirus ribosome-associated, RNA-binding protein confers a growth advantage upon mutants deficient in a GADD34-related function.

The herpes simplex virus type 1 gamma34.5 gene product and the cellular GADD34 protein both contain similar domains that can regulate the activity of eukaryotic initiation factor 2 (eIF2), a critical translation initiation factor. Viral mutants that lack the GADD34-related function grow poorly on a variety of malignant human cells, as activation of the cellular PKR kinase leads to the accumulation of inactive, phosphorylated eIF2 at late times postinfection. Termination of translation prior to the completion of the viral reproductive cycle leads to impaired growth. Extragenic suppressors that regain the ability to synthesize proteins efficiently in the absence of the viral GADD34-related function have been isolated. These suppressor alleles are dominant in trans and affect the steady-state accumulation of several viral mRNA species. We demonstrate that deregulated expression of Us11, a virus-encoded RNA-binding, ribosome-associated protein is necessary and sufficient to confer a growth advantage upon viral mutants that lack a GADD34-related function. Ectopic expression of Us11 reduces the accumulation of the activated cellular PKR kinase and allows for sustained protein synthesis. Thus, an RNA-binding, ribosome-associated protein (Us11) and a GADD34-related protein (gamma34.5) both function in a signal pathway that regulates translation by modulating eIF2 phosphorylation.

Signaling via the CD2 Receptor Enhances HTLV-1 Replication in T Lymphocytes

Human T lymphotropic virus type 1 (HTLV-1) is considered the etiologic agent of adult T cell leukemia/lymphoma and several chronic progressive immune-mediated diseases. Approximately 1–4% of infected individuals develop disease, generally decades following infection. Increased proviral transcription, mediated by the viral 40-kDatrans-activating protein, Tax, has been implicated in the pathogenesis of HTLV-1-associated diseases. Since the HTLV-1 promoter contains sequences responsive to cyclic AMP and protein kinase C, we hypothesized that lymphocyte activation signals initiated through the TCR/CD3 complex or CD2 receptor promote viral replication in HTLV-1-infected lymphocytes. We demonstrate that mAbs directed against the CD2, but not the CD3 receptor increase viral p24 capsid protein 1.5- to 5.7-fold in CD2/CD3+ HTLV-1-infected cell culture supernatants. Northern blot analysis demonstrated a 2.5- to 4-fold increase in all species of viral mRNA following CD2 cross-linking of OSP2/4 cells, an immortalized HTLV-1 cell line. Consistent with transcriptional regulation, reporter gene activity increased approximately 11-fold in CD2-stimulated Jurkat T cells cotransfected with a Tax-expressing plasmid and a CAT reporter gene construct under control of the HTLV-1 promoter. These data suggest a possible physiologic mechanism, whereby CD2-mediated cell adhesion and lymphocyte activation may promote viral transcription in infected lymphocytes.

Transdominant mutants of I kappa B alpha block Tat-tumor necrosis factor synergistic activation of human immunodeficiency virus type 1 gene expression and virus multiplication.

The human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) contains two binding sites for the NF-kappa B/Rel family of transcription factors which are required for the transcriptional activation of viral genes by inflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha) and interleukin-1. In the present study, we examined the effect of transdominant mutants of I kappa B alpha on the synergistic activation of the HIV-1 LTR by TNF-alpha and the HIV-1 transactivator, Tat, in Jurkat T cells. The synergistic induction of HIV-1 LTR-driven gene expression represented a 50- to 70-fold stimulation and required both an intact HIV-1 enhancer and Tat-TAR element interaction, since mutations in Tat protein (R52Q, R53Q) or in the bulge region of the TAR element that eliminated Tat binding to TAR were unable to stimulate LTR expression. Coexpression of I kappa B alpha inhibited Tat-TNF-alpha activation of HIV LTR in a dose-dependent manner. Transdominant forms of I kappa B alpha, mutated in critical serine or threonine residues required for inducer-mediated (S32A, S36A) and/or constitutive (S283A, T291A, T299A) phosphorylation of I kappa B alpha were tested for their capacity to block HIV-1 LTR transactivation. I kappa B alpha molecules mutated in the N-terminal sites were not degraded following inducer-mediated stimulation (t1/2, > 4 h) and were able to efficiently block HIV-1 LTR transactivation. Strikingly, the I kappa B alpha (S32A, S36A) transdominant mutant was at least five times as effective as wild-type I kappa B alpha in inhibiting synergistic induction of the HIV-1 LTR. This mutant also effectively inhibited HIV-1 multiplication in a single-cycle infection model in Cos-1 cells, as measured by Northern (RNA) blot analysis of viral mRNA species and viral protein production. These experiments suggest a strategy that may contribute to inhibition of HIV-1 gene expression by interfering with the NF-kappa B/Rel signaling pathway.

MRNA export correlates with activation of transcription in human subgroup C adenovirus-infected cells

To investigate the mechanisms by which viral mRNA species are distinguished from their cellular counterparts for export to the cytoplasm during the late phase of subgroup C adenovirus infection, we have examined the metabolism of several cellular and viral mRNAs in human cells productively infected by adenovirus type 5 (Ad5). Several cellular mRNAs that were refractory to, or could escape from, adenovirus-induced inhibition of export of mRNA from the nucleus have been identified. This group includes Hsp70 mRNAs synthesized upon heat shock of Ad5-infected 293 or HeLa cells during the late phase of infection. However, successful export in Ad5-infected cells is not a specific response to heat shock, for beta-tubulin and interferon-inducible mRNAs were also refractory to virus-induced export inhibition. The export of these cellular mRNAs, like that of viral late mRNAs, required the E1B 55-kDa protein. Export to the cytoplasm during the late phase of Ad5 infection of several cellular mRNAs, including members of the Hsp70 family whose export was inhibited under some, but not other, conditions, indicates that viral mRNA species cannot be selectively exported by virtue of specific sequence or structural features. Cellular and viral late mRNAs that can be exported from the nucleus to the cytoplasm were expressed from genes whose transcription was induced or activated during the late phase of Ad5 infection. Consistent with the possibility that successful export is governed by transcriptional activation in the late phase of adenovirus infection, newly synthesized viral early E1A mRNA was subject to export inhibition during the late phase of infection.

Human Papillomavirus Type 11 E1 up angle E4 and L1 Proteins Colocalize in the Mouse Xenograft System at Multiple Time Points

The most abundant viral mRNA species in HPV 11-infected tissue consists of two exons, joining a segment of open reading frame (ORF) E1 to ORF E4, potentially encoding the E1 up angle E4 protein. The L1 ORF encodes the major capsid protein of HPV. Our previous studies demonstrated colocalization of the HPV 11 E1 up angle E4 and L1 proteins within the same cells of HPV 11-infected human foreskin implants grown in athymic mice (the mouse xenograft system) and removed 12 weeks after implantation. Prior studies have demonstrated E1 up angle E4 transcripts early in infection and throughout the HPV 11-infected epithelium, while L1 transcripts are detected later, and in a subset of E1 up angle E4 mRNA-positive differentiated epithelial cells. Therefore, E1 up angle E4 protein may be produced at an earlier time point or in less differentiated cells than the L1 protein. To study these questions, athymic mice were implanted with HPV 11-infected human foreskin fragments. Mice were sacrificed at 1-week intervals beginning 2 weeks after implantation of tissue. The E1 up angle E4 and L1 proteins colocalized to the same differentiated epithelial cells or to tight clusters of cells in differentiated epithelial layers of HPV 11-infected implants. The E1 up angle E4 and L1 proteins were first detected 4 weeks after implantation. E1 up angle E4 protein was detected in the region of the cell membrane and cytoplasm, and never in the nucleus. L1 protein was only detected in the nucleus. Both proteins were detected in implants containing high viral copy numbers. No specific histologic changes were uniformly associated with detection of these proteins. The tight coupling of the E1 up angle E4 and L1 proteins at multiple time points suggests that expression of both proteins is necessary to complete the virus life cycle.

Colocalization of Human Papillomavirus Type 11 E1^E4 and L1 Proteins in Human Foreskin Implants Grown in Athymic Mice

The most abundant viral mRNA species in tissues infected with HPV 11 consists of two exons, joining a short segment of open reading frame (ORF) E1 to ORF E4, potentially encoding an E1^E4 protein of 10 kDa. E4 gene products have previously been identified by immunohistochemistry in human tissues infected with HPV 1 and HPV 16, and in HPV 11-infected raft cultures. The E1^E4 mRNA is produced in abundance in HPV 11-infected human foreskin implants grown in athymic mice. In contrast, the L1 mRNA is present at low levels and appears late in the course of infection. To characterize the relationship of these proteins, polyclonal rabbit antisera were produced against bacterially expressed HPV 11 trpE/E1^E4 and trpE/L1 fusion proteins and tested in an immunohistochemical assay of paraffin-embedded sections of HPV 11-infected human foreskin tissue fixed with 10% buffered formalin phosphate or zinc formalin. In sections fixed with either fixative, the anti-L1 serum stained nuclei of cells in the upper spinous and granular layers. In contrast, the anti-E1^E4 serum stained the cell membrane and, to a lesser degree, the cytoplasm of cells in the upper spinous and granular layers of tissue fixed with zinc formalin, but not 10% buffered formalin phosphate. In sections treated with both the E1^E4 and L1 antisera, cell membrane staining occurred in the same cells that exhibited nuclear staining The HPV 11 E1^E4 protein appears to be a cell membrane-associated protein. Expression of the HPV 11 E1^E4 and L1 proteins may be influenced by similar factors in differentiating cells.

Rev and the Fate of Pre-mRNA in the Nucleus: Implications for the Regulation of RNA Processing in Eukaryotes

Although a great deal is known about the regulation of gene expression in terms of transcription, relatively little is known about the modulation of pre-mRNA processing. In this study, we exploited a genetically regulated system, human immunodeficiency virus type 1 (HIV-1) and its trans-activator Rev, to examine events that occur between the synthesis of pre-mRNA in the nucleus and the translation of mRNA in the cytoplasm. Unlike the majority of eukaryotic pre-mRNAs whose introns are efficiently recognized and spliced prior to nucleocytoplasmic transport, HIV-1 mRNAs containing functional introns must be exported to the cytoplasm for the expression of many viral proteins. Using human T cells containing stably integrated proviruses, we demonstrate that such incompletely spliced viral mRNAs are exported to the cytoplasm only in the presence of the Rev trans-activator. In the absence of Rev, these intron-containing RNAs are sequestered in the T-cell nucleus and either spliced or, more commonly, degraded. Because Rev does not inhibit the expression of fully spliced viral mRNA species in T cells, we propose that Rev, rather than inhibiting viral pre-mRNA splicing, is acting here both to prevent the nuclear degradation of HIV-1 pre-mRNAs and to induce their translocation to the cytoplasm. Taken together, these findings indicate that the cellular factors responsible for the nuclear retention of unspliced pre-mRNAs, although most probably splicing factors, do not invariably commit these RNAs to productive splicing and can, instead, program such transcripts for degradation.

Rev and the fate of pre-mRNA in the nucleus: implications for the regulation of RNA processing in eukaryotes.

Although a great deal is known about the regulation of gene expression in terms of transcription, relatively little is known about the modulation of pre-mRNA processing. In this study, we exploited a genetically regulated system, human immunodeficiency virus type 1 (HIV-1) and its trans-activator Rev, to examine events that occur between the synthesis of pre-mRNA in the nucleus and the translation of mRNA in the cytoplasm. Unlike the majority of eukaryotic pre-mRNAs whose introns are efficiently recognized and spliced prior to nucleocytoplasmic transport, HIV-1 mRNAs containing functional introns must be exported to the cytoplasm for the expression of many viral proteins. Using human T cells containing stably integrated proviruses, we demonstrate that such incompletely spliced viral mRNAs are exported to the cytoplasm only in the presence of the Rev trans-activator. In the absence of Rev, these intron-containing RNAs are sequestered in the T-cell nucleus and either spliced or, more commonly, degraded. Because Rev does not inhibit the expression of fully spliced viral mRNA species in T cells, we propose that Rev, rather than inhibiting viral pre-mRNA splicing, is acting here both to prevent the nuclear degradation of HIV-1 pre-mRNAs and to induce their translocation to the cytoplasm. Taken together, these findings indicate that the cellular factors responsible for the nuclear retention of unspliced pre-mRNAs, although most probably splicing factors, do not invariably commit these RNAs to productive splicing and can, instead, program such transcripts for degradation.