Major Late Transcription Unit Research Articles

RNA polymerase II-dependent positional effects on mRNA 3' end processing in the adenovirus major late transcription unit.

During the early phase of adenovirus infection, the promoter-proximal L1 poly(A) site in the major late transcription unit is used preferentially despite the fact that the distal L3 poly(A) site is stronger (i.e. it competes better for processing factors and is cleaved at a faster rate, in vitro). Previous work had established that this was due at least in part to the stable binding of the processing factor, cleavage and polyadenylation specificity factor, to the L1 poly(A) site as mediated by specific regulatory sequences. It is now demonstrated that in addition, the L1 poly(A) site has a positional advantage because of its 5' location in the transcription unit. We also show that preferential processing of a particular poly(A) site in a complex transcription unit is dependent on RNA polymerase II. Our results are consistent with recent reports demonstrating that the processing factors cleavage and polyadenylation specificity factor and cleavage stimulatory factor are associated with the RNA polymerase II holoenzyme; thus, processing at a weak poly(A) site like L1 can be enhanced by virtue of its being the first site to be transcribed.

The Role of the L4 33K Gene in Adenovirus Infection

The late phase of adenovirus infection is characterized not only by the synthesis of late proteins and the assembly of new virions, but also by the inhibition of early gene expression and host cell translation. Previous work has demonstrated that both of these inhibitory effects depend upon expression from the major late transcription unit (MLTU), controlled by the major late promoter (MLP). Furthermore, the repression of early gene expression has been shown to be mediated in trans, suggesting a role for one or more MLTU-encoded soluble factor(s). A possible candidate for such a factor is the L4-encoded 33K gene product, a protein conserved throughout the Mastadenoviridae, but of no known function. To test the role of this protein in viral infection, a stop codon was placed at the 20th position of the 33K ORF. Viable virus with genomes containing the mutation were recovered in an overlap recombination assay. Phenotypic analysis revealed that the mutant virus had a significant deficiency in both kinetics of replication and final yield, as compared to the wild-type virus. Detailed analysis of infected cells showed that there was no detectable change in the regulation of expression of several early genes and the pIX gene. This suggests either that 33K is not involved in this late phase phenomenon or that this function is replaceable by another late protein(s). Late protein synthesis and accumulation were similar to those in wild-type-infected cells. However, the reduced yield of infectious mutant virus could be accounted for by a marked deficiency in the accumulation of intermediate particles and completed capsids, suggesting a role for 33K in the process of assembly. In addition there was a small but reproducible deficiency in the shutoff of host cell translation. These results show that the 33K protein plays an important, although apparently not essential, function in the late phase of virus infection.

Control of Adenovirus Early Gene Expression during the Late Phase of Infection

The adenovirus gene regulatory program occurs in two distinct phases, as defined by the onset of DNA replication. During the early phase, the E1A, E1B, E2, E3, and E4 genes are maximally expressed, while the major late promoter (MLP) is minimally expressed and transcription is attenuated. After the onset of DNA replication, the IVa2 and pIX genes are expressed at high levels, transcription from the MLP is unattenuated and fully activated, and early gene expression is repressed. Although the cis elements and trans-acting factors responsible for the late-phase activation of the MLP have been identified and characterized and the role of DNA replication in activation has been established, the mechanism(s) underlying the commensurate decrease in early gene expression has yet to be elucidated. The results of this study demonstrate that this decrease depends on a fully functional MLP. Specifically, virus mutants with severely deficient transcription from the MLP exhibit a marked increase in expression of the E1A, E1B, and E2 early genes. These increases were observed at the level of transcription initiation, mRNA accumulation, and protein production. In addition, expression from the late gene pIX, which is not contained within the major late transcription unit (MLTU), is also markedly increased. To begin the analysis of the mechanisms underlying these late-phase effects, mixed-infection experiments with mutant and wild-type viruses were performed. The results show that the effects on early gene expression, as measured both at the protein and RNA levels, are mediated in trans and not in cis. These observations are consistent either with a model in which one or more late protein products encoded by the MLTU acts as a repressor of early gene expression or with one in which the wild-type MLP competes with early promoters for limiting transcription factors.

Regulated RNA Processing and RNA Transport during Adenovirus Infection

Adenovirus (Ad) gene expression involves regulation both of RNA processing and RNA transport. The major late transcription unit (MLTU) in particular requires complex differential splicing and polyadenylation to produce its full array of mRNA and the pattern of processing changes as infection proceeds. Many MLTU mRNAs cannot reach the cytoplasm effectively without viral functions, probably because they are recognized by host nuclear retention mechanisms. Infection produces changes to the activity of cellular splicing factors and polyadenylation factors so altering the relative recognition of competing RNA processing sites as infection proceeds. In addition, three Ad proteins, E1B 55K, E4 Orf3, and E4 Orf6 are involved in posttranscriptional regulation. These directly or indirectly alter the pattern of MLTU splicing, facilitate late RNA export, and alter the distribution of cellular antigens within the nucleus.

Sequence-Mediated Regulation of Adenovirus Gene Expression by Repression of mRNA Accumulation

Gene expression in complex transcription units can be regulated at virtually every step in the production of mature cytoplasmic mRNA, including transcription initiation, elongation, termination, pre-mRNA processing, nucleus-to-cytoplasm mRNA transport, and alterations in mRNA stability. We have been characterizing alternative poly(A) site usage in the adenovirus major late transcription unit (MLTU) as a model for regulation at the level of pre-mRNA 3'-end processing. The MLTU contains five polyadenylation sites (L1 through L5). The promoter proximal site (L1) functions as the dominant poly(A) site during the early stage of adenovirus infection and in plasmid transfections when multiple poly(A) sites are present at the 3' end of a reporter plasmid. In contrast, stable mRNA processed at all five poly(A) sites is found during the late stage of adenovirus infection, after viral DNA replication has begun. Despite its dominance during early infection, L1 is a comparatively poor substrate for 3'-end RNA processing both in vivo and in vitro. In this study we have investigated the basis for the early L1 dominance. We have found that mRNA containing an unprocessed L1 poly(A) site is compromised in its ability to enter the steady-state pool of stable mRNA. This inhibition, which affects either the nuclear stability or nucleus-to-cytoplasm transport of the pre-mRNA, requires a cis-acting sequence located upstream of the L1 poly(A) site.

Spliced exons of adenovirus late RNAs colocalize with snRNP in a specific nuclear domain.

Posttranscriptional steps in the production of mRNA include well characterized polyadenylation and splicing reactions, but it is also necessary to understand how RNA is transported within the nucleus from the site of its transcription to the nuclear pore, where it is translocated to the cytoplasmic compartment. Determining the localization of RNA within the nucleus is an important aspect of understanding RNA production and may provide clues for investigating the trafficking of RNA within the nucleus and the mechanism for its export to the cytoplasm. We have previously shown that late phase adenovirus-infected cells contain large clusters of snRNP and non-snRNP splicing factors; the presence of these structures is correlated with high levels of viral late gene transcription. The snRNP clusters correspond to enlarged interchromatin granules present in late phase infected cells. Here we show that polyadenylated RNA and spliced tripartite leader exons from the viral major late transcription unit are present in these same late phase snRNP-containing structures. We find that the majority of the steady state viral RNA present in the nucleus is spliced at the tripartite leader exons. Tripartite leader exons are efficiently exported from the nucleus at a time when we detect their accumulation in interchromatin granule clusters. Since the enlarged interchromatin granules contain spliced and polyadenylated RNA, we suggest that viral RNA may accumulate in this late phase structure during an intranuclear step in RNA transport.

Sequences regulating poly(A) site selection within the adenovirus major late transcription unit influence the interaction of constitutive processing factors with the pre-mRNA

The adenovirus major late transcription unit (MLTU) encodes five families of mRNAs, L1 to L5, each distinguished by a unique poly(A) site. Use of the promoter-proximal L1 poly(A) site predominates during early infection, whereas poly(A) site choice shifts to the promoter-distal sites during late infection. A mini-MLTU containing only the L1 and L3 poly(A) sites has been shown to reproduce this processing switch. In vivo analysis has revealed that sequences extending 5' and 3' of the L1 core poly(A) site are required for efficient processing as well as for regulated expression. By replacement of the L1 core poly(A) site with that of the ground squirrel hepatitis virus poly(A) site, we now demonstrate that the L1 flanking sequences can enhance the processing of a heterologous poly(A). Upon recombination of the chimeric L1-ground squirrel hepatitis virus poly(A) site onto the viral chromosome, the L1 flanking sequences were also found to be sufficient to reproduce the processing switch during the course of viral infection. Subsequent in vitro analysis has shown that the L1 flanking sequences function to enhance the stability of binding of cleavage and polyadenylation specificity factor to the core poly(A) site. The impact of L1 flanking sequences on the binding of cleavage and polyadenylation specificity factor suggests that the regulation of the MLTU poly(A) site selection is mediated by the interaction of constitutive processing factors.

A late adenovirus factor induces eIF-4E dephosphorylation and inhibition of cell protein synthesis

Adenovirus prevents host cell protein synthesis during its late phase of replication in large part by causing the underphosphorylation of translation initiation factor eIF-4E, a component of initiation factor eIF-4F (cap-binding protein complex). Late adenovirus mRNAs are preferentially translated because they possess a reduced requirement for eIF-4F. This study continues the characterization of the mechanism by which adenovirus inhibits cellular protein synthesis. First it is shown that adenovirus blocks the addition of phosphate to eIF-4E rather than enhancing its removal, establishing that the virus impairs a signalling pathway or protein kinase activity involved in eIF-4E phosphorylation. It is then shown that shutoff of cell protein synthesis and translation of late viral mRNAs are uncoupled, in that shutoff actually occurs a short time (1 to 3 h) after late adenovirus mRNAs are already undergoing translation. Finally, by using a variety of genetic mutants stalled at different stages in the viral life cycle, it was found that dephosphorylation of eIF-4E and inhibition of cell translation are not caused by early adenovirus gene products acting at late times or by events related to viral DNA replication. Instead, it is shown that inhibition of eIF-4E phosphorylation and cell translation are mediated upon activation of the viral major late transcription unit. These and other results presented indicate that the adenovirus signal which induces eIF-4E dephosphorylation and shutoff of cell protein synthesis is linked either to an activity of one or more late viral polypeptides, to double-stranded RNA produced by opposition of the early and late viral transcription units, or to both.

Sequence Elements Upstream of the 3′ Cleavage Site Confer Substrate Strength to the Adenovirus L1 and L3 Polyadenylation Sites

The adenovirus major late transcription unit is a well-characterized transcription unit which relies heavily on alternative pre-mRNA processing to generate distinct populations of mRNA during the early and late stages of viral infection. In the early stage of infection, two major late transcription unit mRNA transcripts are generated through use of the first (L1) of five available poly(A) sites (L1 through L5). This contrasts with the late stage of infection when as many as 45 distinct mRNAs are generated, with each of the five poly(A) sites being used. In previous work characterizing elements involved in alternative poly(A) site use, we showed that the L1 poly(A) site is processed less efficiently than the L3 poly(A) site both in vitro and in vivo. Because of the dramatic difference in processing efficiency and the role processing efficiency plays in production of steady-state levels of mRNA, we have identified the sequence elements that account for the differences in L1 and L3 poly(A) site processing efficiency. We have found that the element most likely to be responsible for poly(A) site strength, the GU/U-rich downstream element, plays a minor role in the different processing efficiencies observed for the L1 and L3 poly(A) sites. The sequence element most responsible for inefficient processing of the L1 poly(A) site includes the L1 AAUAAA consensus sequence and those sequences which immediately surround the consensus hexanucleotide. This region of the L1 poly(A) site contributes to an inability to form a stable processing complex with the downstream GU/U-rich element. In contrast to the L1 element, the L3 poly(A) site has a consensus hexanucleotide and surrounding sequences which can form a stable processing complex in cooperation with the downstream GU/U-rich element. The L3 poly(A) site is also aided by the presence of sequences upstream of the hexanucleotide which facilitate processing efficiency. The sequence UUCUUUUU, present in the L3 upstream region, is shown to enhance processing efficiency as well as stable complex formation (shown by increased binding of the 64-kDa cleavage stimulatory factor subunit) and acts as a binding site for heterogeneous nuclear ribonucleoprotein C proteins.

Sequence elements upstream of the 3' cleavage site confer substrate strength to the adenovirus L1 and L3 polyadenylation sites.

The adenovirus major late transcription unit is a well-characterized transcription unit which relies heavily on alternative pre-mRNA processing to generate distinct populations of mRNA during the early and late stages of viral infection. In the early stage of infection, two major late transcription unit mRNA transcripts are generated through use of the first (L1) of five available poly(A) sites (L1 through L5). This contrasts with the late stage of infection when as many as 45 distinct mRNAs are generated, with each of the five poly(A) sites being used. In previous work characterizing elements involved in alternative poly(A) site use, we showed that the L1 poly(A) site is processed less efficiently than the L3 poly(A) site both in vitro and in vivo. Because of the dramatic difference in processing efficiency and the role processing efficiency plays in production of steady-state levels of mRNA, we have identified the sequence elements that account for the differences in L1 and L3 poly(A) site processing efficiency. We have found that the element most likely to be responsible for poly(A) site strength, the GU/U-rich downstream element, plays a minor role in the different processing efficiencies observed for the L1 and L3 poly(A) sites. The sequence element most responsible for inefficient processing of the L1 poly(A) site includes the L1 AAUAAA consensus sequence and those sequences which immediately surround the consensus hexanucleotide. This region of the L1 poly(A) site contributes to an inability to form a stable processing complex with the downstream GU/U-rich element. In contrast to the L1 element, the L3 poly(A) site has a consensus hexanucleotide and surrounding sequences which can form a stable processing complex in cooperation with the downstream GU/U-rich element. The L3 poly(A) site is also aided by the presence of sequences upstream of the hexanucleotide which facilitate processing efficiency. The sequence UUCUUUUU, present in the L3 upstream region, is shown to enhance processing efficiency as well as stable complex formation (shown by increased binding of the 64-kDa cleavage stimulatory factor subunit) and acts as a binding site for heterogeneous nuclear ribonucleoprotein C proteins.

Human adenovirus encodes two proteins which have opposite effects on accumulation of alternatively spliced mRNAs.

All mRNAs expressed from the adenovirus major late transcription unit have a common, 201-nucleotide-long 5' leader sequence, which consists of three short exons (the tripartite leader). This leader has two variants, either with or without the i-leader exon, which, when present, is spliced between the second and the third exons of the tripartite leader. Previous studies have shown that adenovirus early region 4 (E4) encodes two proteins, E4 open reading frame 3 (E4-ORF3) and E4-ORF6, which are required for efficient expression of mRNAs from the major late transcription unit. These two E4 proteins appear to have redundant activities, and expression of one has been shown to be sufficient for efficient major late mRNA accumulation during a lytic virus infection. In this report, we provide evidence that E4-ORF3 and E4-ORF6 both regulate major late mRNA accumulation by stimulating constitutive splicing. Moreover, we show that the two proteins have different effects on accumulation of alternatively spliced tripartite leader exons. In a DNA transfection assay, E4-ORF3 was shown to facilitate i-leader exon inclusion, while E4-ORF6 preferentially favored i-leader exon skipping. In addition, E4-ORF3 and E4-ORF6 had the same effects on accumulation of alternatively spliced chimeric beta-globin transcripts. This finding suggests that the activities of the two proteins may be of more general relevance and not restricted to splicing of major late tripartite leader-containing pre-mRNAs. Interestingly, E4-ORF6 expression was also shown to stimulate i-leader exon skipping during a lytic virus infection.

Human adenovirus encodes two proteins which have opposite effects on accumulation of alternatively spliced mRNAs.

All mRNAs expressed from the adenovirus major late transcription unit have a common, 201-nucleotide-long 5' leader sequence, which consists of three short exons (the tripartite leader). This leader has two variants, either with or without the i-leader exon, which, when present, is spliced between the second and the third exons of the tripartite leader. Previous studies have shown that adenovirus early region 4 (E4) encodes two proteins, E4 open reading frame 3 (E4-ORF3) and E4-ORF6, which are required for efficient expression of mRNAs from the major late transcription unit. These two E4 proteins appear to have redundant activities, and expression of one has been shown to be sufficient for efficient major late mRNA accumulation during a lytic virus infection. In this report, we provide evidence that E4-ORF3 and E4-ORF6 both regulate major late mRNA accumulation by stimulating constitutive splicing. Moreover, we show that the two proteins have different effects on accumulation of alternatively spliced tripartite leader exons. In a DNA transfection assay, E4-ORF3 was shown to facilitate i-leader exon inclusion, while E4-ORF6 preferentially favored i-leader exon skipping. In addition, E4-ORF3 and E4-ORF6 had the same effects on accumulation of alternatively spliced chimeric beta-globin transcripts. This finding suggests that the activities of the two proteins may be of more general relevance and not restricted to splicing of major late tripartite leader-containing pre-mRNAs. Interestingly, E4-ORF6 expression was also shown to stimulate i-leader exon skipping during a lytic virus infection.

3' RNA processing efficiency plays a primary role in generating termination-competent RNA polymerase II elongation complexes.

In several mammalian transcription units, a transcription termination mechanism in which efficient termination is dependent on the presence of an intact 3' RNA processing site has been identified. The mouse beta maj-globin transcription unit is one such example, in which an intact poly(A) site is required for efficient transcription termination. It is now evident that 3' mRNA processing sites are not always processed with the same efficiency. In this study, we characterized several pre-mRNAs as substrates for the 3' mRNA processing reaction of cleavage and polyadenylation. We then determined whether poly(A) sites which vary in processing efficiency support a poly(A) site-dependent termination event. The level of processing efficiency was determined in vitro by assays measuring the efficiency of the pre-mRNA cleavage event and in vivo by the level of poly(A) site-dependent mRNA and gene product expression generated in transient transfection assays. The beta maj globin pre-mRNA is very efficiently processed. This efficient processing correlates with its function in termination assays using recombinant adenovirus termination vectors in nuclear run-on assays. When the beta maj globin poly(A) site was replaced by the L1 poly(A) site of the adenovirus major late transcription unit (Ad-ml), which is a poor processing substrate, termination efficiency decreased dramatically. When the beta maj globin poly(A) site was replaced by the Ad-ml L3 poly(A) site, which is 10- to 20-fold more efficiently processed than the Ad-ml L1 poly(A) site, termination efficiency remained high. Termination is therefore dependent on the yield of the processing event. We then tested chimeric poly(A) sites containing the L3 core AAUAAA but varied downstream GU-rich elements. The change in downstream GU-rich elements affected processing efficiency in a manner which correlated with termination efficiency. These experiments provide evidence that the efficiency of 3' processing complex formation is directly correlated to the efficiency of RNA polymerase II termination at the 3' end of a mammalian transcription unit.

3' RNA Processing Efficiency Plays a Primary Role in Generating Termination-Competent RNA Polymerase II Elongation Complexes

In several mammalian transcription units, a transcription termination mechanism in which efficient termination is dependent on the presence of an intact 3' RNA processing site has been identified. The mouse beta maj-globin transcription unit is one such example, in which an intact poly(A) site is required for efficient transcription termination. It is now evident that 3' mRNA processing sites are not always processed with the same efficiency. In this study, we characterized several pre-mRNAs as substrates for the 3' mRNA processing reaction of cleavage and polyadenylation. We then determined whether poly(A) sites which vary in processing efficiency support a poly(A) site-dependent termination event. The level of processing efficiency was determined in vitro by assays measuring the efficiency of the pre-mRNA cleavage event and in vivo by the level of poly(A) site-dependent mRNA and gene product expression generated in transient transfection assays. The beta maj globin pre-mRNA is very efficiently processed. This efficient processing correlates with its function in termination assays using recombinant adenovirus termination vectors in nuclear run-on assays. When the beta maj globin poly(A) site was replaced by the L1 poly(A) site of the adenovirus major late transcription unit (Ad-ml), which is a poor processing substrate, termination efficiency decreased dramatically. When the beta maj globin poly(A) site was replaced by the Ad-ml L3 poly(A) site, which is 10- to 20-fold more efficiently processed than the Ad-ml L1 poly(A) site, termination efficiency remained high. Termination is therefore dependent on the yield of the processing event. We then tested chimeric poly(A) sites containing the L3 core AAUAAA but varied downstream GU-rich elements. The change in downstream GU-rich elements affected processing efficiency in a manner which correlated with termination efficiency. These experiments provide evidence that the efficiency of 3' processing complex formation is directly correlated to the efficiency of RNA polymerase II termination at the 3' end of a mammalian transcription unit.

Two Adenovirus Proteins with Redundant Activities in Virus Growth Facilitates Tripartite Leader mRNA Accumulation

Most adenovirus-specific mRNAs expressed late after infection originate from a single transcription unit, the socalled major late transcription unit. All mRNAs expressed from this unit have in common a 201 nucleotide-long spliced tripartite leader segment attached to their 5′ ends. Human adenovirus mutants that carry large deletions in early region 4 (E4) are severely defective in expression of nuclear and cytoplasmic RNA derived from the major late transcription unit. We have previously shown that E4 post-transcriptionally stimulates accumulation of tripartite leader containing mRNAs by a mechanism that requires an intron in the nuclear precursor RNA. To identify the E4 products responsible for this stimulatory effect on tripartite leader mRNA accumulation, we constructed CMV expression vectors encoding single E4 open reading frames (ORFs). By comparing the activity of these plasmids in a transient DNA cotransfection assay we could show that both the E4-ORF3 and E4-ORF6 proteins individually are able to stimulate mRNA accumulation from tripartite leader intron containing transcription units. Furthermore, this stimulatory activity was not dependent on coexpresaion of other viral gene products. These results are interesting since the same two E4 proteins have been shown to have interchangeable activities in lytic infection, and expression of one has been suggested to be sufficient to substitute for the whole E4 region for virus growth. Finally, we show that the absence of E4 expression during a virus infection results in abnormalities in tripartite leader assembly. This result suggests that E4 proteins may be required for efficient tripartite splicing also during a lytic virus infection.

Alternative poly(A) site utilization during adenovirus infection coincides with a decrease in the activity of a poly(A) site processing factor.

The recognition and processing of a pre-mRNA to create a poly(A) addition site, a necessary step in mRNA biogenesis, can also be a regulatory event in instances in which the frequency of use of a poly(A) site varies. One such case is found during the course of an adenovirus infection. Five poly(A) sites are utilized within the major late transcription unit to produce more than 20 distinct mRNAs during the late phase of infection. The proximal half of the major late transcription unit is also expressed during the early phase of a viral infection. During this early phase of expression, the L1 poly(A) site is used three times more frequently than the L3 poly(A) site. In contrast, the L3 site is used three times more frequently than the L1 site during the late phase of infection. Recent experiments have suggested that the recognition of the poly(A) site GU-rich downstream element by the CF1 processing factor may be a rate-determining step in poly(A) site selection. We demonstrate that the interaction of CF1 with the L1 poly(A) site is less stable than the interaction of CF1 with the L3 poly(A) site. We also find that there is a substantial decrease in the level of CF1 activity when an adenovirus infection proceeds to the late phase. We suggest that this reduction in CF1 activity, coupled with the relative instability of the interaction with the L1 poly(A) site, contributes to the reduced use of the L1 poly(A) site during the late stage of an adenovirus infection.

Alternative poly(A) site utilization during adenovirus infection coincides with a decrease in the activity of a poly(A) site processing factor.

The recognition and processing of a pre-mRNA to create a poly(A) addition site, a necessary step in mRNA biogenesis, can also be a regulatory event in instances in which the frequency of use of a poly(A) site varies. One such case is found during the course of an adenovirus infection. Five poly(A) sites are utilized within the major late transcription unit to produce more than 20 distinct mRNAs during the late phase of infection. The proximal half of the major late transcription unit is also expressed during the early phase of a viral infection. During this early phase of expression, the L1 poly(A) site is used three times more frequently than the L3 poly(A) site. In contrast, the L3 site is used three times more frequently than the L1 site during the late phase of infection. Recent experiments have suggested that the recognition of the poly(A) site GU-rich downstream element by the CF1 processing factor may be a rate-determining step in poly(A) site selection. We demonstrate that the interaction of CF1 with the L1 poly(A) site is less stable than the interaction of CF1 with the L3 poly(A) site. We also find that there is a substantial decrease in the level of CF1 activity when an adenovirus infection proceeds to the late phase. We suggest that this reduction in CF1 activity, coupled with the relative instability of the interaction with the L1 poly(A) site, contributes to the reduced use of the L1 poly(A) site during the late stage of an adenovirus infection.

The 11,600-MW protein encoded by region E3 of adenovirus is expressed early but is greatly amplified at late stages of infection.

We have reported that an 11,600-MW (11.6K) protein is coded by region E3 of adenovirus. We have now prepared two new antipeptide antisera that have allowed us to characterize this protein further. The 11.6K protein migrates as multiple diffuse bands having apparent Mws of about 14,000, 21,000, and 31,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoblotting as well as virus mutants with deletions in the 11.6K gene were used to show that the various gel bands represent forms of 11.6K. The 11.6K protein was synthesized in very low amounts during early stages of infection, from the scarce E3 mRNAs d and e which initiate from the E3 promoter. However, 11.6K was synthesized very abundantly at late stages of infection, approximately 400 times the rate at early stages, from new mRNAs termed d' and e'. Reverse transcriptase-polymerase chain reaction and RNA blot experiments indicated that mRNAs d' and e' had the same body (the coding portion) and the same middle exon (the y leader) as early E3 mRNAs d and e, but mRNAs d' and e' were spliced at their 5' termini to the major late tripartite leader which is found in all mRNAs in the major late transcription unit. mRNAs d' and e' and the 11.6K protein were the only E3 mRNAs and protein that were scarce early and were greatly amplified at late stages of infection. This suggests that specific cis- or trans-acting sequences may function to enhance the splicing of mRNAs d' and e' at late stages of infection and perhaps to suppress the splicing of mRNAs d and e at early stages of infection. We propose that the 11.6K gene be considered not only a member of region E3 but also a member of the major late transcription unit.

Control of adenovirus major late gene expression at multiple levels

Most late adenovirus (Ad) proteins are translated from mRNAs originating from the so-called major late transcription unit (MLTU). These mRNAs are grouped into five families (designated L1 to L5), where each family consists of mRNAs that have co-terminal 3′ ends. We have used mutant and wild-type Ad infections to characterize levels at which major late gene expression is regulated. Our results suggest the existence of a novel intermediate stage during a lytic infection where mRNAs from regions L1 and L4 are selectively overexpressed compared to the L2, L3 and L5 mRNAs. Our data suggest that this RNA phenotype reflects the activity of the MLTU at a transient stage immediately following initiation of viral DNA replication. Early during an Ad infection only mRNA from region L1 accumulate. To efficiently accumulate mRNA from regions L1 to L5 both viral DNA replication and late protein synthesis were required. To allow for only viral DNA replication resulted in an extensive premature transcription termination and a preferential mRNA accumulation from regions L1 and L4. The surprising production of L4 mRNA under these conditions was not due to the activation of a novel r-strand promoter located in the vicinity of region L4 or due to a control at the level of RNA transport or stability. Instead our results indicate that in the absence of efficient late protein synthesis 3′ end formation occurs preferentially at the L1 and L4 poly(A) addition sites.

Varied poly(A) site efficiency in the adenovirus major late transcription unit.

Regulation of adenovirus major late transcription unit (MLTU) mRNA biosynthesis involves poly(A) site selection between five available sites, L1 through L5. The 5' proximal site completely dominates during early infection, whereas all five sites are used during late infection with L3 being favored slightly over the others. Previous studies have shown this early to late poly(A) switch will occur in the absence of MLTU-specific splicing patterns and hinges in large part on the character of the first poly(A) site. We have used in vitro assays to characterize basic features of the L1 and L3 pre-mRNAs which may help define how processing at poly(A) sites is controlled. We have found that L1 is 5-10-fold less efficient than L3 as a substrate for RNA cleavage. A primary difference between the L1 and L3 sites lies in the kinetics of their use, with cleavage at L3 occurring at twice the rate of cleavage at L1. In addition, L1 is 20-fold less effective than L3 in competing for processing factors. To investigate the sequence elements that contribute to poly(A) site efficiency, we have used competition assays in which the competitor RNAs lack upstream or downstream elements.