We have used the polymerase chain reaction technique to clone the small multiply spliced mRNA species produced after infection of human cells by a molecular clone of human immunodeficiency virus type 1 (HIV-1). We identified six Rev-expressing mRNAs, which were generated by the use of two splice acceptors located immediately upstream of the rev AUG. The class of small mRNAs included 12 mRNAs expressing Tat, Rev, and Nef. In addition, HIV-1 produced other multiply spliced mRNAs that used alternative splice sites identified by cloning and sequencing. All of these mRNAs were found in the cytoplasm and should be able to produce additional proteins. The coding capacity of the tat, rev, and nef mRNAs was analyzed by transfection of the cloned cDNAs into human cells. The tat mRNAs produced high levels of Tat, but very low levels of Rev and Nef. All the rev mRNAs expressed high levels of both Rev and Nef and were essential for the production of sufficient amounts of Rev. Therefore, HIV-1 uses both alternatively spliced and bicistronic mRNAs for the production of Tat, Rev, and Nef proteins.

We have previously identified cis-acting RNA sequences in the human papillomavirus type 16 (HPV-16) L1 coding region which inhibit expression of L1 from eukaryotic expression plasmids. Here we have determined the function of one of these RNA elements, and we provide evidence that this RNA element is a splicing silencer which suppresses the use of the 3 splice site located immediately upstream of the L1 AUG. We also show that this splice site is inefficiently utilized as a result of a suboptimal polypyrimidine tract. Introduction of point mutations in the L1 coding region that altered the RNA sequence without affecting the L1 protein sequence resulted in the inactivation of the splicing silencer and induced splicing to the L1 3 splice site. These mutations also prevented the interaction of the RNA silencer with a 35-kDa cellular protein identified here as hnRNP A1. The splicing silencer in L1 inhibits splicing in vitro, and splicing can be restored by the addition of RNAs containing an hnRNP A1 binding site to the reaction, demonstrating that hnRNP A1 inhibits splicing of the late HPV-16 mRNAs through the splicing silencer sequence. While we show that one role of the splicing silencer is to determine the ratio between partially spliced L2/L1 mRNAs and spliced L1 mRNAs, we also demonstrate that it inhibits splicing from the major 5 splice site in the early region to the L1 3 splice site, thereby playing an essential role in preventing late gene expression at an early stage of the viral life cycle. We speculate that the activity of the splicing silencer and possibly the concentration of hnRNP A1 in the HPV-16-infected cell determines the ability of the virus to establish a persistent infection which remains undetected by the host immune surveillance.Human papillomaviruses (HPVs) are a group of nonenveloped, double-stranded DNA tumor viruses with tropism for epithelial cells (21, 62). HPV type 16 (HPV-16) is one of the most common sexually transmitted HPV types and is also the HPV type most frequently detected in cervical cancers (43). While many of the early HPV gene products are present in all layers of the squamous epithelium, expression of the late mRNAs encoding the L1 and L2 capsid proteins is restricted to the terminally differentiated cells in the upper layers of the epithelium (21). For this reason, it has been difficult to grow HPVs in vitro, and only organotypic raft cultures in which infected or transfected keratinocytes are grown in the airliquid interface to induce terminal cell differentiation produce HPV virions (27). Expression of the late genes is regulated at the levels of transcription and RNA processing (1,38,39,42,55). Members of our group and others have previously shown that the late HPV-16 mRNAs contain cis-acting regulatory RNA elements (23, 24, 51), both in the 3Ј untranslated region (UTR) and in the L1 and L2 coding regions (9,33,46,51). It is likely that these elements are involved in the regulation of HPV late gene expression. Inhibitory RNA elements in the late 3Ј UTR have been identif...

Human papillomaviruses (HPVs) are small DNA tumour viruses that are present in more than 99% of all cervical cancers. The ability of these viruses to cause disease is partly attributed to the strict coordination of viral gene expression with the differentiation stage of the infected cell. HPV gene expression is regulated temporally at the level of RNA splicing and polyadenylation, and a dysregulated gene expression programme allows some HPV types to establish long-term persistence, which is a risk factor for cancer. In this Review, we summarize the role of splicing and polyadenylation in the regulation of HPV gene expression and discuss the viral and cellular factors that control these processes.

Human papillomavirus (HPV) type 16 belongs to the group of "high risk" HPV types that are frequently detected in anogenital cancers. The expression of HPV-16 late genes encoding the virus capsid proteins L1 and L2 is restricted to terminally differentiated epithelial cells in the superficial layers of the squamous epithelium. We have previously identified negative elements in the 3 end of L2 RNA that act in cis to reduce mRNA utilization without substantially affecting mRNA levels. The experiments reported here demonstrate the interaction of cellular proteins with an inhibitory sequence present in the coding region of the L2 mRNA. Using RNA gel shift assays and UV cross-linking, we have detected three cellular proteins interacting specifically with the sense strand of the L2 mRNA, two of which were identified as heterogeneous ribonucleoprotein K (hnRNP K) and the poly(rC) binding-protein (PCBP). Recombinant hnRNP K, PCBP-1, and PCBP-2 that were over expressed in bacteria and partially purified bound to the HPV-16 L2 mRNA in a sequence-specific manner. Interestingly, PCBP-1, PCBP-2, and hnRNP K specifically and efficiently inhibited translation of the HPV-16 L2 mRNA in vitro. Therefore, these proteins may play an important role in the regulation of HPV-16 late gene expression and virus production in vivo.Human papillomaviruses (HPVs) 1 are nonenveloped, epitheliotropic DNA tumor viruses with a circular double-stranded genome of approximately 8 kilobases (1). At present more than 70 different types of HPVs have been identified that can be divided into mucosal or cutaneous types on the basis of the epithelium they infect (2). The major stimulus for current interest in the HPV group originates from the discovery of the casual relationship to carcinoma of the cervix of certain HPV types, primarily types 16 and 18 (3, 4). The genomic organization of the various HPV types is very similar. All of the open reading frames are located on one strand of viral genomic DNA that consists of an early, a late, and a noncoding region (1). Early genes are expressed throughout the infected epithelium and are responsible for initiation of viral DNA replication, regulation of transcription, and transformation of cells (1, 5-9). The late genes code for the major and minor capsid proteins, L1 and L2, respectively, and their expression is thought to be regulated at both the transcriptional and the post-transcriptional level. Production of L1 and L2 protein is seen to be inhibited in dividing cells, and the L1 and L2 proteins are detected primarily in the superficial layers of terminally differentiated squamous epithelial cells (1, 5-9). Sequences with inhibitory function have been identified on HPV-1 (10), HPV-16 (11), and BPV-1 (12) late mRNAs. Inhibitory RNA elements present in the HPV-16 L1 and L2 coding regions (13-15) reduce the levels of late mRNAs and proteins, and a negative element in the late 3Ј-untranslated region (11,13,16,19) was seen to reduce RNA stability in vitro (11). The inhibitory RNA sequences on the late papilloma...

The expression of Gag, Pol, Vif, Vpr, Vpu, and Env proteins from unspliced and partially spliced human immunodeficiency virus type 1 (HIV-1) mRNAs depends on the viral protein Rev, while the production of Tat, Rev, and Nef from multiply spliced mRNAs does not require Rev. To investigate the difference between gag and tat mRNAs, we generated plasmids expressing tat-gag hybrid mRNAs. Insertion of the gag gene downstream of the tat open reading frame in the tat cDNA resulted in the inhibition of Tat production. This inhibition was caused, at least in part, by a decrease in the stability of the produced mRNA. Deletions in gag defined a 218-nucleotide inhibitory sequence named INS-1 and located at the 5' end of the gag gene. Further experiments indicated the presence of more than one inhibitory sequence in the gag-protease gene region of the viral genome. The inhibitory effect of INS-1 was counteracted by the positive effect mediated by the Rev-Revresponsive element interaction, indicating that this sequence is important for Rev-regulated gag expression. The INS-1 sequence did not contain any known HIV-1 splice sites and acted independently of splicing. It was found to have an unusually high AU content (61.5% AU), a common feature among cellular mRNAs with short half-lives. These results suggest that HIV-1 and possibly other lentiviruses have evolved to express unstable mRNAs which require additional regulatory factors for their expression. This strategy inay offer the virus several advantages, including the ability to enter a state of low or latent expression in the host. The genome of human immunodeficiency virus type 1 (HIV-1) contains the gag, pol, and env genes common to all retroviruses. In addition to these genes, the HIV-1 genome contains several open reading frames (ORFs) encoding the accessory and regulatory proteins Tat, Rev, Nef, Vpu, Vif, and Vpr. The expression of all of these ORFs from one promoter is accomplished primarily by the generation of alternatively spliced-mRNAs grouped into three size classes: full length (9 kb), intermediate (4 to 5 kb), and small, multiply spliced (2 kb). The full-length and intermediate