Modulation of host cell stress responses by human cytomegalovirus.

Human cytomegalovirus (HCMV) induces cellular stress responses during infection due to nutrient depletion, energy depletion, hypoxia and synthetic stress, e.g., endoplasmic reticulum (ER) stress. Cellular stress responses initiate processes that allow the cell to survive the stress; some of these may be beneficial to HCMV replication while others are not. Several studies show that HCMV manipulates stress response signaling in order to maintain beneficial effects while inhibiting detrimental effects. The inhibition of translation is the most common effect of stress responses that would be detrimental to HCMV infection. This chapter will focus on the mechanisms by which cap-dependent translation is maintained during HCMV infection through alterations of the phosphatidylinositol-3' kinase (PI3K)-Akt-tuberous sclerosis complex (TSC)-mammalian target of rapamycin (mTOR) signaling pathway. The emerging picture is that HCMV affects this pathway in multiple ways, thus ensuring that cap-dependent translation is maintained despite the induction of stress responses that would normally inhibit it. Such dramatic alterations of this pathway lead to questions of what other beneficial effects the virus might gain from these changes and how these changes may contribute to HCMV pathogenesis.

Transient gene expression control: effects of transfected DNA stability and trans-activation by viral early proteins.

The effects of trans-acting factors and transfected DNA stability on promoter activity were examined with chloramphenicol acetyl transferase (CAT) transient expression analysis. With cotransfection into CV-1P and HeLa cells, simian virus 40 T antigen, adenovirus E1a, and herpes-virus IE proteins were compared for their ability to trans-activate a variety of eucaryotic promoters constructed into CAT plasmids. T antigen and the IE protein were promiscuous activators of all the promoters tested [the simian virus 40 late promoter, the adenovirus E3 promoter, the alpha 2(I) collagen promoter, and the promoter of the Rous sarcoma virus long terminal repeat]. Conversely the E1a protein was specific, activating only the adenovirus E3 promoter and suppressing the basal activity of the other promoters. This specificity of activation by E1a contrasted with the high activity generated by all of the promoter-CAT plasmids when transfected into 293 cells, which endogenously produce E1a protein. Examination of transfected 293 cells determined that they stabilized much greater amounts of plasmid DNA than any other cells tested (CV-1P, COS, NIH-3T3, KB). Thus the high activity of nonadenovirus promoter-CAT plasmids in 293 cells results from the cumulative effect of basal promoter activity from a very large number of gene copies, not from E1a activation. This conclusion was supported by similar transfection analysis of KB cell lines which endogenously produce E1a protein. These cells stabilize plasmid DNA at a level comparable to that of CV-1P cells and, in agreement with the CV-1P cotransfection results, did not activate a nonadenovirus promoter-CAT plasmid. These results indicate that the stability of plasmid DNA must be considered when transient gene expression is being compared between cell lines. The use of relative plasmid copy numbers for the standardization of transient expression results is discussed.

Analysis of an activatable promoter: sequences in the simian virus 40 late promoter required for T-antigen-mediated trans activation.

The late promoter of simian virus 40 (SV40) is activated in trans by the viral early gene product, T antigen. We inserted the wild-type late-promoter region, and deletion mutants of it, into chloramphenicol acetyltransferase transient expression vectors to identify promoter sequences which are active in the presence of T antigen. We defined two promoter activities. One activity was mediated by a promoter element within simian virus 40 nucleotides 200 to 270. The activity of this element was detectable only in the presence of an intact, functioning origin of replication and accounted for 25 to 35% of the wild-type late-promoter activity in the presence of T antigen. The other activity was mediated by an element located within a 33-base-pair sequence (simian virus nucleotides 168 to 200) which spans the junction of the 72-base-pair repeats. This element functioned in the absence of both the origin of replication and the T-antigen-binding sites and appeared to be responsible for trans-activated gene expression. When inserted into an essentially promoterless plasmid, the 33-base-pair element functioned in an orientation-dependent manner. Under wild-type conditions in the presence of T antigen, the activity of this element accounted for 65 to 75% of the late-promoter activity. The roles of the 33-base-pair element and T antigen in trans-activation are discussed.

Sequences on the 3' side of hexanucleotide AAUAAA affect efficiency of cleavage at the polyadenylation site.

The hexanucleotide AAUAAA has been demonstrated to be part of the signal for cleavage and polyadenylation at appropriate sites on eucaryotic mRNA precursors. Since this sequence is not unique to polyadenylation sites, it cannot be the entire signal for the cleavage event. We have extended the definition of the polyadenylation cleavage signal by examining the cleavage event at the site of polyadenylation for the simian virus 40 late mRNAs. Using viable mutants, we have determined that deletion of sequences between 3 and 60 nucleotides on the 3' side of the AAUAAA decreases the efficiency of utilization of the normal polyadenylation site. These data strongly indicate a second major element of the polyadenylation signal. The phenotype of these deletion mutants is an enrichment of viral late transcripts longer than the normally polyadenylated RNA in infected cells. These extended transcripts appear to have an increased half-life due to the less efficient cleavage at the normal polyadenylation site. The enriched levels of extended transcripts in cells infected with the deletion mutants allowed us to examine regions of the late transcript which normally are difficult to study. The extended transcripts have several discrete 3' ends which we have analyzed in relation to polyadenylation and other RNA processing events. Two of these ends map to nucleotides 2794 and 2848, which lie within a region of extensive secondary structure which marks the putative processing signal for the formation of the simian virus 40-associated small RNA. A third specific 3' end reveals a cryptic polyadenylation site at approximately nucleotides 2980 to 2985, more than 300 nucleotides beyond the normal polyadenylation site. This site appears to be utilized only in mutants with debilitated normal sites. The significance of sequences on the 3' side of an AAUAAA for efficient polyadenylation at a specific site is discussed.

Definition of the upstream efficiency element of the simian virus 40 late polyadenylation signal by using in vitro analyses.

The polyadenylation signal for the late mRNAs of simian virus 40 is known to have sequence elements located both upstream and downstream of the AAUAAA which affect efficiency of utilization of the signal. The upstream efficiency element has been previously characterized by using deletion mutations and transfection analyses. Those studies suggested that the upstream element lies between 13 and 48 nucleotides upstream of the AAUAAA. We have utilized in vitro cleavage and polyadenylation reactions to further define the upstream element. 32P-labeled substrate RNAs were prepared by in vitro transcription from wild-type templates as well as from mutant templates having deletions and linker substitutions in the upstream region. Analysis of these substrates defined the upstream region as sequences between 13 and 51 nucleotides upstream of the AAUAAA, in good agreement with the in vivo results. Within this region, three core elements with the consensus sequence AUUUGURA were identified and were specifically mutated by linker substitution. These core elements were found to contain the active components of the upstream efficiency element. Using substrates with both single and double linker substitution mutations of core elements, we observed that the core elements function in a distance-dependent manner. In mutants containing only one core element, the effect on efficiency increases as the distance between the element and the AAUAAA decreases. In addition, when core elements are present in multiple copies, the effect is additive. The core element consensus sequence, which bears homology to the Sm protein complex-binding site in human U1 RNA, is also found within the upstream elements of the ground squirrel hepatitis B and cauliflower mosaic virus polyadenylation signals (R. Russnak, Nucleic Acids Res. 19:6449-6456, 1991; H. Sanfacon, P. Brodmann, and T. Hohn, Genes Dev. 5:141-149, 1991), suggesting functional conservation of this element between mammals and plants.

Elements upstream of the AAUAAA within the human immunodeficiency virus polyadenylation signal are required for efficient polyadenylation in vitro.

Recent in vivo studies have identified specific sequences between 56 and 93 nucleotides upstream of a polyadenylation [poly(A)] consensus sequence, AAUAAA, in human immunodeficiency virus type 1 (HIV-1) that affect the efficiency of 3'-end processing at this site (A. Valsamakis, S. Zeichner, S. Carswell, and J. C. Alwine, Proc. Natl. Acad. Sci. USA 88:2108-2112, 1991). We have used HeLa cell nuclear extracts and precursor RNAs bearing the HIV-1 poly(A) signal to study the role of upstream sequences in vitro. Precursor RNAs containing the HIV-1 AAUAAA and necessary upstream (U3 region) and downstream (U5 region) sequences directed accurate cleavage and polyadenylation in vitro. The in vitro requirement for upstream sequences was demonstrated by using deletion and linker substitution mutations. The data showed that sequences between 56 and 93 nucleotides upstream of AAUAAA, which were required for efficient polyadenylation in vivo, were also required for efficient cleavage and polyadenylation in vitro. This is the first demonstration of the function of upstream sequences in vitro. Previous in vivo studies suggested that efficient polyadenylation at the HIV-1 poly(A) signal requires a spacing of at least 250 nucleotides between the 5' cap site and the AAUAAA. Our in vitro analyses indicated that a precursor containing the defined upstream and downstream sequences was efficiently cleaved at the polyadenylation site when the distance between the 5' cap and the AAUAAA was reduced to at least 140 nucleotides, which is less than the distance predicted from in vivo studies. This cleavage was dependent on the presence of the upstream element.

Simian virus 40 large T antigen interacts with human TFIIB-related factor and small nuclear RNA-activating protein complex for transcriptional activation of TATA-containing polymerase III promoters.

The TATA-binding protein (TBP) is common to the basal transcription factors of all three RNA polymerases, being associated with polymerase-specific TBP-associated factors (TAFs). Simian virus 40 large T antigen has previously been shown to interact with the TBP-TAFII complexes, TFIID (B. Damania and J. C. Alwine, Genes Dev. 10:1369-1381, 1996), and the TBP-TAFI complex, SL1 (W. Zhai, J. Tuan, and L. Comai, Genes Dev. 11: 1605-1617, 1997), and in both cases these interactions are critical for transcriptional activation. We show a similar mechanism for activation of the class 3 polymerase III (pol III) promoter for the U6 RNA gene. Large T antigen can activate this promoter, which contains a TATA box and an upstream proximal sequence element but cannot activate the TATA-less, intragenic VAI promoter (a class 2, pol III promoter). Mutants of large T antigen that cannot activate pol II promoters also fail to activate the U6 promoter. We provide evidence that large T antigen can interact with the TBP-containing pol III transcription factor human TFIIB-related factor (hBRF), as well as with at least two of the three TAFs in the pol III-specific small nuclear RNA-activating protein complex (SNAPc). In addition, we demonstrate that large T antigen can cofractionate and coimmunoprecipitate with the hBRF-containing complex TFIIIB derived from HeLa cells infected with a recombinant adenovirus which expresses large T antigen. Hence, similar to its function with pol I and pol II promoters, large T antigen interacts with TBP-containing, basal pol III transcription factors and appears to perform a TAF-like function.

Functionally significant secondary structure of the simian virus 40 late polyadenylation signal.

The structure of the highly efficient simian virus 40 late polyadenylation signal (LPA signal) is more complex than those of most known mammalian polyadenylation signals. It contains efficiency elements both upstream and downstream of the AAUAAA region, and the downstream region contains three defined elements (two U-rich elements and one G-rich element) instead of the single U- or GU-rich element found in most polyadenylation signals. Since many reports have indicated that the secondary structure in RNA may play a significant role in RNA processing, we have used nuclease structure analysis techniques to determine the secondary structure of the LPA signal. We find that the LPA signal has a functionally significant secondary structure. Much of the region upstream of AAUAAA is sensitive to single-strand-specific nucleases. The region downstream of AAUAAA has both double- and single-stranded characteristics. Both U-rich elements are predominately sensitive to the double-strand-specific nuclease RNase V(1), while the G-rich element is primarily single stranded. The U-rich element closest to AAUAAA contains four distinct RNase V(1)-sensitive regions, which we have designated structural region 1 (SR1), SR2, SR3, and SR4. Linker scanning mutants in the downstream region were analyzed both for structure and for function by in vitro cleavage analyses. These data show that the ability of the downstream region, particularly SR3, to form double-stranded structures correlates with efficient in vitro cleavage. We discuss the possibility that secondary structure downstream of the AAUAAA may be important for the functions of polyadenylation signals in general.

Efficiency of utilization of the simian virus 40 late polyadenylation site: effects of upstream sequences.

The late polyadenylation signal of simian virus 40 functions with greater efficiency than the early polyadenylation signal, in turn affecting steady-state mRNA levels. Two chloramphenicol acetyltransferase (CAT) transient expression vectors, pL-EPA and pL-LPA, that differ only in their polyadenylation signals were constructed by using the early and late polyadenylation signals, respectively. In transfections of Cos, CV-1P, or HeLa cells and subsequent Northern blot analysis of CAT-specific RNA, approximately five times more steady-state CAT mRNA was produced in transfections with pL-LPA than with pL-EPA. The basis for this difference was not related to the specific promoter used or to RNA stability. Overall, the difference in steady-state mRNA levels derived from the two plasmids appeared to be attributable to intrinsic properties of the two polyadenylation signals, resulting in distinctly different cleavage and polyadenylation efficiencies. Additionally, we found that the utilization of the late polyadenylation site was dramatically reduced by deletion of sequences between 48 and 29 nucleotides 5' of the AAUAAA hexanucleotide. This reduction of mRNA levels was shown not to be caused by altered stability of mutant precursor RNAs or mRNAs, suggesting that these upstream sequences constitute an element of the late polyadenylation signal and may cause, at least to some extent, the greater efficiency of utilization of the late polyadenylation site.

Simian virus 40 large T antigen stabilizes the TATA-binding protein-TFIIA complex on the TATA element.

Large T antigen (T antigen), the early gene product of simian virus 40 (SV40), is a potent transcriptional activator of both cellular and viral genes. Recently we have shown that T antigen is tightly associated with TFIID and, in this position, performs a TATA-binding protein (TBP)-associated factor (TAF)-like function. Based on this observation, we asked whether T antigen affected steps in preinitiation complex assembly. Using purified components in in vitro complex assembly assays, we found that T antigen specifically enhances the formation of the TBP-TFIIA complex on the TATA element. T antigen accomplishes this by increasing the rate of formation of the TBP-TFIIA complex on the TATA element and by stabilizing the complexes after they are formed on the promoter. In addition, DNA immunoprecipitation experiments indicate that T antigen is associated with the stabilized TBP-TFIIA complexes bound to the DNA. In this regard, it has previously been shown that T antigen interacts with TBP; in the present study, we show that T antigen also interacts with TFIIA in vitro. In testing the ability of T antigen to stabilize the TBP-TFIIA complex, we found that stabilization is highly sensitive to the specific sequence context of the TATA element. Previous studies showed that T antigen could activate simple promoters containing the TATA elements from the hsp70 and c-fos gene promoters but failed to significantly activate similar promoters containing the TATA elements from the promoters of the SV40 early and adenovirus E2a genes. We find that the ability to stabilize the TBP-TFIIA complex on the hsp70 and c-fos TATA elements, and not on the SV40 early and E2A TATA elements, correlates with the ability or inability to activate promoters containing these TATA elements.

The cap and the 3' splice site similarly affect polyadenylation efficiency.

The 5' cap of a mammalian pre-mRNA has been shown to interact with splicing components at the adjacent 5' splice site for processing of the first exon and the removal of the first intron (E. Izaurralde, J. Lewis, C. McGuigan, M. Jankowska, E. Darzynkiewicz, and I.W. Mattaj, Cell 78:657-668, 1994). Likewise, it has been shown that processing of the last exon and removal of the last intron involve interaction between splicing components at the 3' splice site and the polyadenylation complex at the polyadenylation signal (M. Niwa, S. D. Rose, and S.M. Berget, Genes Dev. 4:1552-1559, 1990; M. Niwa and S. M. Berget, Genes Dev. 5:2086-2095, 1991). These findings suggest that the cap provides a function in first exon processing which is similar to the function of the 3' splice site at last exon processing. To determine whether caps and 3' splice sites function similarly, we compared the effects of the cap and the 3' splice site on the in vitro utilization of the simian virus 40 late polyadenylation signal. We show that the presence of a m7GpppG cap, but not a cap analog, can positively affect the efficiency of polyadenylation of a polyadenylation-only substrate. Cap analogs do not stimulate polyadenylation because they fail to bind titratable cap-binding factors. The failure of cap analogs to stimulate polyadenylation can be overcome if a 3' splice site is present upstream of the polyadenylation signal. These data indicate that factors interacting with the cap or the 3' splice site function similarly to affect polyadenylation signal, along with m7GpppG cap, is inhibitory to polyadenylation. This finding suggests that the interaction between the cap-binding complexes and splicing components at the 5' splice site may form a complex which is inhibitory to further processing if splicing of an adjacent intron is not achieved.

Utilization of splicing elements and polyadenylation signal elements in the coupling of polyadenylation and last-intron removal.

Polyadenylation (PA) is the process by which the 3' ends of most mammalian mRNAs are formed. In nature, PA is highly coordinated, or coupled, with splicing. In mammalian systems, the most compelling mechanistic model for coupling arises from data supporting exon definition (2, 34, 37). We have examined the roles of individual functional components of splicing and PA signals in the coupling process by using an in vitro splicing and PA reaction with a synthetic pre-mRNA substrate containing an adenovirus splicing cassette and the simian virus 40 late PA signal. The effects of individually mutating splicing elements and PA elements in this substrate were determined. We found that mutation of the polypyrimidine tract and the 3' splice site significantly reduced PA efficiency and that mutation of the AAUAAA and the downstream elements of the PA signal decreased splicing efficiency, suggesting that these elements are the most significant for the coupling of splicing and PA. Although mutation of the upstream elements (USEs) of the PA signal dramatically decreased PA, splicing was only modestly affected, suggesting that USEs modestly affect coupling. Mutation of the 5' splice site in the presence of a viable polypyrimidine tract and the 3' splice site had no effect on PA, suggesting no effect of this element on coupling. However, our data also suggest that a site for U1 snRNP binding (e.g., a 5' splice site) within the last exon can negatively effect both PA and splicing; hence, a 5' splice site-like sequence in this position appears to be a modulator of coupling. In addition, we show that the RNA-protein complex formed to define an exon may inhibit processing if the definition of an adjacent exon fails. This finding indicates a mechanism for monitoring the appropriate definition of exons and for allowing only pre-mRNAs with successfully defined exons to be processed.

Simian virus 40 T antigen alters the binding characteristics of specific simian DNA-binding factors.

The late promoter of simian virus 40 is transcriptionally activated, in trans, by large T antigen, the primary viral early gene product. Although large T antigen is a well-characterized DNA-binding protein, a variety of data suggest that its trans-activation function does not require direct interaction with DNA. We demonstrate that defined late promoter elements, omega (omega), tau (tau), and delta (delta), necessary for T-antigen-mediated trans-activation, are binding sites for simian cellular factors, not T antigen. Two of the late promoter elements (omega and tau) are shown to bind the same factor or family of factors. These factors bind to a site very similar to that for the HeLa cell factor AP1. We refer to these factors as the simian AP1-sequence recognition proteins (sAP1-SRPs). Compared with normal simian CV-1P cells, the sAP1-SRPs from T-antigen-producing COS cells, or from 14-h simian virus 40-infected CV-1P cells, showed altered binding patterns to both the omega and tau binding sites. In addition, the sAP1-SRPs from T-antigen-containing cells bound to the tau site more stably than did the analogous factors from normal CV-1P cells. The altered pattern of binding and the increased stability of binding correlated with the presence of T antigen in the cell. Additionally, the alteration of the binding pattern within 14 h of infection in CV-1P cells is temporally correct for late promoter activation. Overall, the data show (i) that the late promoter elements necessary for T-antigen-mediated trans-activation contain binding sites for simian cellular DNA-binding proteins; (ii) that the presence of T antigen causes alterations in the binding characteristics of specific simian cellular DNA-binding factors or families of factors; and (iii) that factors which bind to the late promoter elements required for activation have altered and more stable binding characteristics in the presence of T antigen. These points strongly suggest that T antigen mediates trans-activation indirectly through the alteration of binding of at least one specific simian cellular factor, sAP1-SRP, or through the induction of a family of sAP1-SRP factors.

Identification of a sequence element on the 3' side of AAUAAA which is necessary for simian virus 40 late mRNA 3'-end processing.

Our previous studies of the 3'-end processing of simian virus 40 late mRNAs indicated the existence of an essential element (or elements) downstream of the AAUAAA signal. We report here the use of transient expression analysis to study a functional element which we located within the sequence AGGUUUUUU, beginning 59 nucleotides downstream of the recognized signal AAUAAA. Deletion of this element resulted in (i) at least a 75% drop in 3'-end processing at the normal site and (ii) appearance of readthrough transcripts with alternate 3' ends. Some flexibility in the downstream position of this element relative to the AAUAAA was noted by deletion analysis. Using computer sequence comparison, we located homologous regions within downstream sequences of other genes, suggesting a generalized sequence element. In addition, specific complementarity is noted between the downstream element and U4 RNA. The possibility that this complementarity could participate in 3'-end site selection is discussed.

Transcriptional activation by simian virus 40 large T antigen: interactions with multiple components of the transcription complex.

Simian virus 40 (SV40) large T antigen is a potent transcriptional activator of both viral and cellular promoters. Within the SV40 late promoter, a specific upstream element necessary for T-antigen transcriptional activation is the binding site for transcription-enhancing factor 1 (TEF-1). The promoter structure necessary for T-antigen-mediated transcriptional activation appears to be simple. For example, a promoter consisting of upstream TEF-1 binding sites (or other factor-binding sites) and a downstream TATA or initiator element is efficiently activated. It has been demonstrated that transcriptional activation by T antigen does not require direct binding to the DNA; thus, the most direct effect that T antigen could have on these simple promoters would be through protein-protein interactions with either upstream-bound transcription factors, the basal transcription complex, or both. To determine whether such interactions occur, full-length T antigen or segments of it was fused to the glutathione-binding site (GST fusions) or to the Gal4 DNA-binding domain (amino acids 1 to 147) (Gal4 fusions). With the GST fusions, it was found that TEF-1 and the TATA-binding protein (TBP) bound different regions of T antigen. A GST fusion containing amino acids 5 to 172 (region T1) efficiently bound TBP. TEF-1 bound neither region T1 nor a region between amino acids 168 and 373 (region T2); however, it bound efficiently to the combined region (T5) containing amino acids 5 to 383.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of the human immunodeficiency virus long terminal repeat by in vitro transcription competition and linker scanning mutagenesis.

Previous studies designed to map the transcriptional regulatory sequences of the human immunodeficiency virus (HIV) long terminal repeat (LTR) have shown disparate results depending on the method of analysis. Experiments have shown that deletions 5' to -104 (relative to the transcription start site, +1) are not required for transcription in vitro, while other experiments have shown that various mutations in this 5' region of the HIV-1 LTR affect both reporter gene activity in transient expression systems and viral growth. To correlate in vitro and in vivo findings, we performed in vitro transcription competition studies to define minimal sequences necessary for competitive factor binding or competitive transcription complex formation. Using normal HeLa cell nuclear extracts, we found that transcription of a reporter gene run by the U3-R region was efficiently competed only by intact LTR DNA fragments representing virtually the entire U3-R region (-453 to +80). Smaller subfragments of the LTR were less effective competitors; these included fragments from -453 to -159, which had a modest competitive ability at higher competitor concentrations, -159 to +80, and -402 to -34, which were both relatively poor competitors. These findings indicate that although the U3-R region truncated to -104 is able to promote in vitro transcription, a more stable transcription complex appears to form on the entire U3-R region. Hence sequences between -453 and -104 appear to be significant in transcription complex formation. In vivo transfection competition studies confirmed these findings. Specific sequences between -453 and -104 which may affect expression or transcription complex formation were mapped using a set of linker-scanning mutants spanning the LTR.(ABSTRACT TRUNCATED AT 250 WORDS)

Hybrid selection of small RNAs by using simian virus 40 DNA: evidence that the simian virus 40-associated small RNA is synthesized by specific cleavage from large viral transcripts.

The simian virus 40 (SV40)-associated small RNA (SAS-RNA), approximately 64 nucleotides, is virally encoded within a region of the viral late (+) DNA strand which encodes no known protein. The SAS-RNA arises in abundance late in SV40 lytic infection. Previous data indicate that the synthesis of the SAS-RNA may be under the control of the normal late viral promoter; i.e., inhibition of transcription from the late promoter results in cessation of SAS-RNA synthesis. The synthesis of SAS-RNA was examined to determine whether the SAS-RNA is the product of cleavage from noncoding regions of nuclear late transcripts or an independent transcription product like 5S RNA, or the adenovirus VA-RNAs. The data described below suggest that SAS-RNA is cleaved from large late transcripts. In vitro transcription of DNA fragments containing the SAS-RNA coding region yielded no SAS-RNA synthesis; this result was supported by DNA sequence analysis, which indicated no promoter-like regions either within or flanking the SAS-RNA coding region. In support of a cleavage mechanism, the SAS-RNA has a 3'-phosphate end, an occurrence which is indicative of nuclease cleavage. In addition, 5'-end labeling of the SAS-RNA was possible only after calf alkaline phosphatase treatment; this indicates that the SAS-RNA is not capped. Hybrid selection analysis was used to demonstrate that separation of the SAS-RNA coding region from the normal late promoter resulted in elimination of SAS-RNA synthesis. This was demonstrated in SV40-transformed cells in which integration of a single copy of SV40 breaks the continuity of the late coding region, so that the SAS-RNA coding region is physically separated from the normal late promoter. The lack of SAS-RNA synthesis indicates that the SAS-RNA coding region cannot function as a primary transcription unit. The same result and conclusion were obtained by using a permissive cell line transformed by SV40 (COS-1 cells); here it was found that the integrated SAS-RNA coding region was not expressed even during a viable lytic infection in which the SAS-RNA could be expressed from the infecting viral genomes. The simplest conclusion drawn from the data is that the SAS-RNA is cleaved from larger late transcripts which initiate at the normal late promoter. This conclusion suggests that many of the small RNAs found in normal eucaryotic cells may be synthesized by specific cleavage rather than by primary transcription. In the course of these studies several small cellular RNAs were detected, due to their specific hybrid selection, by using SV40 DNA. Primary mapping and characterization data of these RNAs are also presented.

Evidence for simian virus 40 late transcriptional control: mixed infections of wild-type simian virus 40 and a late leader deletion mutant exhibit trans effects on late viral RNA synthesis.

Mixed infections involving equal multiplicities of wild-type simian virus 40 and viable deletion mutant dl861 resulted in decreased cytoplasmic levels of wild-type-derived male mRNA, as well as very low to undetectable levels of mutant-derived late mRNA, as compared with individual infections. The dl861 deletion removes 16 to 25 base pairs from the late leader region. This deletion was shown to be the direct cause of the mixed-infection effect; replacement of the deletion with wild-type sequences restored normal levels of late mRNAs in mixed infections. Other viral functions, e.g., early gene expression and replication, were found to be unaffected by the dl861 deletion. Further examination of the mixed-infection effect showed that the levels of unspliced nuclear precursors of late mRNA, derived from both the mutant and wild-type genomes, were decreased or undetectable, in accord with the cytoplasmic results. Thus, the effect appears to be occurring at the transcriptional level. These data demonstrate a trans-acting effect on late transcription, which is detectable due to the presence of the dl861 mutant in the mixed infection. This finding is indicative of a diffusible factor which exerts a control on simian virus 40 late gene expression at the transcriptional level. A model for positive control of simian virus 40 late gene expression is presented.

Simian virus 40 agnoprotein facilitates perinuclear-nuclear localization of VP1, the major capsid protein.

The agnoprotein of simian virus 40 (SV40) is a 61-amino-acid protein encoded in the leader of some late mRNAs. In indirect immunofluorescence studies with antisera against SV40 capsid proteins, we show that mutants which make no agnoprotein display abnormal perinuclear-nuclear localization of VP1, the major capsid protein, but not VP2 or VP3, the minor capsid proteins. In wild-type (WT) SV40-infected CV-1P cells, VP1 was found predominantly in the cytoplasm until 36 h postinfection (p.i.), approximately the time that high levels of agnoprotein became detectable under our infection conditions. Thereafter, VP1 localized rapidly to the perinuclear region and to the nucleus. In contrast, in agnoprotein-minus mutant-infected CV-1P cells, perinuclear-nuclear accumulation of VP1 occurred much less efficiently; a significantly greater fraction of cells with predominantly cytoplasmic fluorescence was observed up to 48 h p.i. At 48 and 60 h p.i., more cells with largely perinuclear and little nuclear staining were seen than in WT-infected controls. In similar analyses with stably transfected cell lines constitutively expressing the agnoprotein, VP1 localized to the nucleus before 30 h p.i., regardless of the infecting virus. Delayed nuclear entry of VP1 in a mutant which makes no agnoprotein was also overcome in a revertant which has a second site point mutation in VP1. This suggests that an alteration of VP1 can partially overcome the defect of the agnogene mutation by enhancement of the rate of its own nuclear localization. Taken together, these results indicate that at least one function of the agnoprotein is to enhance the efficiency of perinuclear-nuclear localization of VP1.