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.

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.

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.

Effects of human cytomegalovirus major immediate-early proteins in controlling the cell cycle and inhibiting apoptosis: studies with ts13 cells.

The major immediate-early (MIE) gene of human cytomegalovirus (HCMV) encodes several MIE proteins (MIEPs) produced as a result of alternative splicing and polyadenylation of the primary transcript. Previously we demonstrated that the HCMV MIEPs expressed from the entire MIE gene could rescue the temperature-sensitive (ts) transcriptional defect in the ts13 cell line. This defect is caused by a ts mutation in TAFII250, the 250-kDa TATA binding protein-associated factor (TAF). These and other data suggested that the MIEPs perform a TAF-like function in complex with the basal transcription factor TFIID. In addition to the transcriptional defect, the ts mutation in ts13 cells results in a defect in cell cycle progression which ultimately leads to apoptosis. Since all of these defects can be rescued by wild-type TAFII250, we asked whether the MIEPs could rescue the cell cycle defect and/or affect the progression to apoptosis. We have found that the MIEPs, expressed from the entire MIE gene, do not rescue the cell cycle block in ts13 cells grown at the nonpermissive temperature. However, despite the maintenance of the cell cycle block, the ts13 cells which express the MIEPs are resistant to apoptosis. MIEP mutants, which have previously been shown to be defective in rescuing the ts transcriptional defect, maintained the ability to inhibit apoptosis. Hence, the MIEP functions which affect transcription appear to be separable from the functions which inhibit apoptosis. We discuss these data in the light of the HCMV life cycle and the possibility that the MIEPs promote cellular transformation by a "hit-and-run" mechanism.

The snRNP-free U1A (SF-A) complex(es): identification of the largest subunit as PSF, the polypyrimidine-tract binding protein-associated splicing factor.

We have previously shown that a specific monoclonal antibody prepared against the U1A protein, MAb 12E12, is unique in its ability to recognize a form of U1A which is not associated with the U1snRNP. This unique form of U1A, termed snRNP-free U1A or SF-A, was found to be complexed with a novel set of non-snRNP proteins (O'Connor et al., 1997, RNA 3:1444-1455). Here we demonstrate that the largest protein in these SF-A complex(es), p105, is the polypyrimidine-tract binding protein-associated factor (PSF), an auxiliary splicing factor. We show that PSF copurifies and co-immunoprecipitates with SF-A from 293T cell nucleoplasm and that it interacts with SF-A in vitro. In addition, we show that MAb 12E12 inhibits both splicing and polyadenylation in an in vitro coupled splicing and polyadenylation reaction. This suggests that SF-A and/or the SF-A complex(es) perform an important function in both processing reactions and possibly in last exon definition.

Phosphorylation of the human cytomegalovirus 86-kilodalton immediate-early protein IE2.

We have investigated the phosphorylation state of the human cytomegalovirus 86-kDa immediate-early (IE) protein IEP86 from transfected and infected cells. We show that multiple domains of IEP86 are phosphorylated by cellular kinases, both in vitro and in vivo. Our data suggest that serum-inducible kinases play a significant role in cell-mediated IE protein phosphorylation and that a member of the mitogen-activated protein (MAP) kinase (MAPK) family, extracellular regulated kinase 2 (ERK2), phosphorylates several domains of IEP86 in vitro. Alanine substitution mutagenesis was performed on specific serines or threonines (T27, S144, T233/S234, and T555) found in consensus MAP kinase motifs. Analysis of these mutations showed that T27 and T233/S234 are the major sites for serum-inducible kinases and are the major ERK2 sites in vitro. S144 appeared to be phosphorylated in a serum-independent manner in vitro. All of the mutations except T555 eliminated specific phosphorylation in vivo. In transient transfection analyses, IEP86 isoforms containing mutations in S144 and, especially, T233/S234 displayed increased transcriptional activation relative to the wild type, suggesting that phosphorylation at these sites in wild-type IEP86 may result in reduction of its transcriptional activation ability.

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.

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.

Identification of a novel, non-snRNP protein complex containing U1A protein.

Mouse monoclonal antibodies (MAbs) were generated against Escherichia coli-produced U1snRNP-A (U1A) protein. U1A-specific MAbs as well as MAbs that reacted with both U1A and U2snRNP-B" (U2B") were isolated. MAb 12E12 was unique among the characterized MAbs because it failed to immunoprecipitate U1A protein produced by in vitro transcription and translation using rabbit reticulocyte lysates. However, when U1A protein was made using a wheat germ extract, MAb 12E12 could immunoprecipitate U1A quite readily, as did the other MAbs. These data suggest that the MAb 12E12 epitope is masked when U1A is prepared in reticulocyte lysate. Further studies showed that MAb 12E12 recognizes an epitope that is masked when U1A protein is bound to U1 RNA. The unique nature of MAb 12E12 was used to demonstrate that U1A could be immunoprecipitated from whole-cell extracts in a form that was free of U1 RNA and other snRNP components. However, this snRNP-free U1A (SF-A) was found to co-immunoprecipitate with a unique set of non-snRNP proteins. In order to confirm that U1A exists in at least two distinct complexes (snRNP bound and snRNP free), [35S]-labeled nucleoplasmic extracts were analyzed by sucrose density gradient fractionation and immunoprecipitation. MAb 12E12 specifically immunoprecipitated SF-A, which migrated in a novel non-snRNP complex. Specifically, proteins of approximately 58, 59, 63, 65, and 105 kDa co-sedimented and co-immunoprecipitated with SF-A. Our data show that a significant portion of the cellular U1A (at least 3% or approximately 30,000 molecules) exists in the nucleoplasm in one or more novel complexes. Our previous studies have demonstrated an effect of purified U1A on polyadenylation of pre-mRNAs and, consistent with this finding, purified antibodies to SF-A significantly diminish polyadenylation in vitro.

TAF-like functions of human cytomegalovirus immediate-early proteins.

The human cytomegalovirus (HCMV) major immediate-early (IE) proteins IEP86 (IE2(579aa)) and IEP72 (IE1(491aa)) can transcriptionally activate a variety of simple promoters containing a TATA element and one upstream transcription factor binding site. In our previous studies, transcriptional activation was shown to correlate with IEP86 binding to both the TATA-box binding protein (TBP) and the transcription factor bound upstream. IEP72 often synergistically affects the activation by IEP86, although it has not previously been shown to directly interact in vitro with IEP86, TBP, or transcription factors (e.g., Sp1 and Tef-1) bound by IEP86. We report biochemical and genetic evidence suggesting that the major IE proteins may perform a function similar to that of the TBP-associated factors (TAFs) which make up TFIID. Consistent with this model, we found that the major IE proteins interact with a number of TAFs. In vitro, IEP86 bound with drosophila TAF(II)110 (dTAF(II)110) and human TAF(II)130 (hTAF(II)130), while IEP72 bound dTAF(II)40, dTAF(II)110, and hTAF(II)130. Regions on major IE proteins which mediate binding have been defined. In addition, our data indicate that both IEP72 and IEP86 can bind simultaneously to hTAF(II)130, suggesting that this TAF may provide bridging interactions between the two proteins for transcriptional activation and synergy. In agreement, a transcriptional activation mutant of IEP72 is unable to participate in bridging. Confirmation that these in vitro interactions were relevant was provided by data showing that both IEP72 and IEP86 copurify with TFIID and coimmunoprecipitate with purified TFIID derived from infected cell nuclei. To further support a TAF-like function of the IE proteins, we have found that the IE proteins expressed from the intact major IE gene, and to a lesser extent IEP86 alone, can rescue the temperature-sensitive (ts) transcriptional defect in TAF(II)250 in the BHK-21 cell line ts13. Analyses of mutations in the major IE region show that IEP86 is essential for rescue and that IEP72 augments its effect, and that mutations which affect TAF interactions are debilitated in rescue. Our data, showing that the IE proteins can bind with TFIID and rescue a ts transcriptional defect in TAF(II)250, support the model that the IE proteins perform a TAF-like function as components of TFIID.

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.

TAF-like function of SV40 large T antigen.

The simian virus 40 (SV40) early gene product large T antigen promiscuously activates simple promoters containing a TATA box or initiator element and at least one upstream transcription factor-binding site. Previous studies have suggested that promoter activation requires that large T antigen interacts with both the basal transcription complex and the upstream-bound factor. This mechanism of activation is similar to that proposed for TBP-associated factors (TAFs). We report genetic and biochemical evidence suggesting that large T antigen performs a TAF-like function. In the ts13 cell line, large T antigen can rescue the temperature-sensitive (ts) defect in TAF(II)250. In contrast, neither E1a, small t antigen, nor mutants of large T antigen defective in transcriptional activation were able to rescue the ts defect. These data suggest that transcriptional activation by large T antigen is attributable, at least in part, to an ability to augment or replace a function of TAF(II)250. In addition, we show that large T antigen interacts in vitro with the Drosophila TAFs (dTAFs) dTAF(II)150, dTAF(II)110, and dTAF(II)40, as well as TBP. The relevance of these in vitro results was established in coimmunoprecipitation experiments using extracts of SV40-infected alpha3 cells that express an epitope-tagged TBP. Large T antigen was coimmunoprecipitated by antibodies to epitope-tagged TBP, endogenous TBP, hTAF(II)100, hTAF(II)130, and hTAF(II)250, under conditions where holo-TFIID would be precipitated. In addition, large T antigen copurified and coimmunoprecipitated with phosphocellulose-purified TFIID from SV40-infected alpha3 cells. Large T antigen also coprecipitated with anti-TBP antibody from extracts of ts13 cells expressing wild-type large T antigen under conditions where the ts defect in TAF(II)250 was rescued. In contrast, a transactivation mutant of large T antigen, which was unable to rescue the ts defect, failed to coprecipitate. We conclude from these data that transcriptional activation of many promoters by large T antigen results from its performing a TAF-like function in a complex with TFIID.

Interaction between the U1 snRNP-A protein and the 160-kD subunit of cleavage-polyadenylation specificity factor increases polyadenylation efficiency in vitro.

We have previously shown that the U1 snRNP-A protein (U1A) interacts with elements in SV40 late polyadenylation signal and that this association increases polyadenylation efficiency. It was postulated that this interaction occurs to facilitate protein-protein association between components of the U1 snRNP and proteins of the polyadenylation complex. We have now used GST fusion protein experiments, coimmunoprecipitations and Far Western blot analyses to demonstrate direct binding between U1A and the 160-kD subunit of cleavage-polyadenylation specificity factor (CPSF). In addition, Western blot analyses of fractions from various stages of CPSF purification indicated that U1A copurified with CPSF to a point but could be separated in the highly purified fractions. These data suggest that U1A protein is not an integral component of CPSF but may be able to interact and affect its activity. In this regard, the addition of purified, recombinant U1A to polyadenylation reactions containing CPSF, poly(A) polymerase, and a precleaved RNA substrate resulted in concentration-dependent increases in both the level of polyadenylation and poly(A) tail length. In agreement with the increase in polyadenylation efficiency caused by U1A, recombinant U1A stabilized the interaction of CPSF with the AAUAAA-containing substrate RNA in electrophoretic mobility shift experiments. These findings suggest that, in addition to its function in splicing, U1A plays a more global role in RNA processing through effects on polyadenylation.

Simian virus 40 large T antigen affects the Saccharomyces cerevisiae cell cycle and interacts with p34CDC28.

Simian virus 40 tumor (T) antigen, an established viral oncoprotein, causes alterations in cell growth control through interacting with, and altering the function of, cellular proteins. To examine the effects of T antigen on cell growth control, and to identify the cellular proteins with which it may functionally interact, T antigen was expressed in the budding yeast Saccharomyces cerevisiae. The yeast cells expressing T antigen showed morphological alterations as well as growth inhibition attributable, at least in part, to a lag in progression from G1 to S. This point in the cell cycle is also known to be affected by T antigen in mammalian cells. Both p34CDC28 and p34CDC2Hs were shown to bind to a chimeric T antigen-glutathione S-transferase fusion protein, indicating that T antigen interacts directly with cell cycle proteins which control the G1 to S transition. This interaction was confirmed by in vivo cross-linking experiments, in which T antigen and p34CDC28 were coimmunoprecipitated from extracts of T-antigen-expressing yeast cells. These immunoprecipitated complexes could phosphorylate histone H1, indicating that kinase activity was retained. In addition, in autophosphorylation reactions, the complexes phosphorylated a novel 60-kDa protein which appeared to be underphosphorylated (or underrepresented) in p34CDC28-containing complexes from cells which did not express T antigen. These results suggest that T antigen interacts with p34CDC28 and alters the kinase function of p34CDC28-containing complexes. These events correlate with alterations in the yeast cell cycle at the G1 to S transition.

Transcriptional activation by the human cytomegalovirus immediate-early proteins: requirements for simple promoter structures and interactions with multiple components of the transcription complex.

We have utilized a number of well-defined, simple, synthetic promoters (upstream factor binding sites and TATA elements) to analyze the activation mechanisms of the human cytomegalovirus immediate-early (IE) proteins. We found that the 86-kDa IE protein (known as IEP86, IE2(559aa), or ppUL122a) can recognize and activate a variety of simple promoters, in agreement with the observation that it is a promiscuous activator. However, in the comparison of otherwise identical promoters IEP86 does have preferences for specific TATA elements (hsp70 > adenovirus E2 > simian virus 40 early) and specific upstream transcription factor binding sites (CAAT > SP1 approximately Tef-1 > ATF; no activation with AP1 or OCT). In contrast, the 72-kDa IE protein (known as IEP72, IE1(491aa), or ppUL123) alone did not significantly activate the simple promoters under our experimental conditions. However, each promoter activated by IEP86 was synergistically affected by the addition of IEP72. In addition, the 55-kDa IE protein (IEP55, a splice variant form of IE2, IE2(425aa), or ppUL122b) repeatedly had a negative effect, downregulating the activation of promoters caused by IEP86 and the synergy of IEP86 and IEP72. We show that the ability of IEP86 to activate many simple promoters correlates not only with its previously described ability to interact with the TATA-binding protein (TBP) (B. A. Furnari, E. Poma, T. F. Kowalik, S.-M. Huong, and E.-S. Huang, J. Virol. 67:4981-4991, 1993; C. Hagemeier, S. Walker, R. Caswell, T. Kouzarides, and J. Sinclair, J. Virol. 66:4452-4456, 1992; R. Jupp, S. Hoffman, R. M. Stenberg, J. A. Nelson, and P. Ghazal, J. Virol. 67:7539-7546, 1993) but also with its ability to interact with the transcription factors which bind to the upstream element of promoters it activated (e.g., SP1 and Tef-1 but not Oct-1). This ability to have multiple interactions with the promoter complex may be crucial for transcriptional activation, since the IE proteins cannot activate promoters having only a TATA element or only an upstream transcription factor binding site. In addition, we show that proteins which bind IEP86 also bind to IEP55. Thus, the negative effect on transcription noted with IEP55 may be the result of competition with IEP86 for interaction with the promoter complex. The synergy caused by IEP72 appears to be mediated by a more indirect mechanism. This is suggested by our observation that IEP72 could not bind to any of the proteins tested (TBP, Tef-1, or Oct-1) or to IEP86.

Direct interaction of the U1 snRNP-A protein with the upstream efficiency element of the SV40 late polyadenylation signal.

An integral component of the splicing machinery, the U1 snRNP, is here implicated in the efficient polyadenylation of SV40 late mRNAs. This occurs as a result of an interaction between U1 snRNP-A protein and the upstream efficiency element (USE) of the polyadenylation signal. UV cross-linking and immunoprecipitation demonstrate that this interaction can occur while U1 snRNP-A protein is simultaneously bound to U1 RNA as part of the snRNP. The target RNA of the first RRM (RRM1) has been shown previously to be the second stem-loop of U1 RNA. We have found that a target for the second RRM (RRM2) is within the AUUUGURA motifs of the USE of the SV40 late polyadenylation signal. RNA substrates containing the wild-type USE efficiently bind to U1 snRNP-A protein, whereas substrates fail to bind when motifs of the USE were replaced by linker sequences. The addition of an oligoribonucleotide containing a USE motif to an in vitro polyadenylation reaction inhibits polyadenylation of a substrate representing the SV40 late polyadenylation signal, whereas a mutant oligoribonucleotide, a nonspecific oligoribonucleotide, and an oligoribonucleotide containing the U1 RNA-binding site had much reduced or no inhibitory effects. In addition, antibodies to bacterially produced, purified U1 snRNP-A protein specifically inhibit in vitro polyadenylation of the SV40 late substrate. These data suggest that the U1 snRNP-A protein performs an important role in polyadenylation through interaction with the USE. Because this interaction can occur when U1 snRNP-A protein is part of the U1 snRNP, our data provide evidence to support a link between the processes of splicing and polyadenylation, as suggested by the exon definition model.

Transcriptional activation by simian virus 40 large T antigen: requirements for simple promoter structures containing either TATA or initiator elements with variable upstream factor binding sites.

The simian virus 40 large T antigen is a promiscuous transcriptional activator of many viral and cellular promoters. We show that the promoter structure necessary for T antigen-mediated transcriptional activation is very simple. A TATA or initiator element is required, in addition to an upstream factor-binding site, which can be quite variable. We found that promoters containing an SP1-, ATF-, AP1-, or TEF-I-binding site, in conjunction with a TATA element, can all be activated in the presence of T antigen. In addition, preference for specific TATA elements was indicated. Promoters containing the HSP70 TATA element functioned better than those with the adenovirus E2 TATA element, while promoters containing the simian virus 40 (SV40) early TATA element failed to be activated. In addition, simple promoters containing the initiator element from the terminal deoxynucleotidyltransferase gene could be activated by T antigen. The SV40 late promoter, a primary target for T antigen transcriptional activation, conforms to this simple promoter structure. The region from which most late transcripts initiate contains a cluster of initiator-like elements (SV40 nucleotides [nt] 250 to 335) forming an initiator region (IR). This lies downstream of the previously described octamer-TEF element (SV40 nt 199 to 218) which contains the TEF-I-binding sites shown to be necessary for T antigen-mediated transcriptional activation of the late promoter. We show that a simple late promoter made up of IR sequences and octamer-TEF element-containing sequences is transcriptionally activated by T antigen. These experiments also showed that specific sequences in the IR, SV40 nt 272 to 294, are particularly important for late promoter activation. Previous findings (M. C. Gruda, J. M. Zablotny, J. H. Xiao, I. Davidson, and J. C. Alwine, Mol. Cell. Biol. 13:961-969, 1993) suggested that T antigen could mediate transcriptional activation through interaction with the TATA-binding protein, as well as upstream bound transcription factors. Our present data are predicted by this model and suggest that at least one mechanism by which the T antigen manifests promiscuous transcriptional activation is its ability to interact with numerous transcription factors in a simple promoter context.

Sodium butyrate treatment of cells latently infected with HIV-1 results in the expression of unspliced viral RNA.

To investigate potential mechanisms for HIV-1 proviral latency, we generated a set of chronically HIV-1 infected and stably long terminal repeat-chloramphenicol acetyl transferase (LTR-CAT)-transfected TE671/RD cells, and studied both their virus production and LTR-driven reporter gene expression. Established tissue culture models of retroviral latency in lymphoid and monocytoid cell lines have demonstrated that the induction of virus production is associated with a shift in HIV-1-specific mRNA from a predominance of singly and multiply spliced mRNA's to the production of full-length HIV-1 RNA. We found a similar pattern in TE671/RD cells, but in contrast to U1 and ACH2 cells, could not induce viral replication by exposure to phorbol myristate acetate (PMA) alone. We demonstrated instead that production of full-length viral RNA, viral replication, and LTR-driven CAT expression could be induced by exposure to sodium butyrate. The most proximate effect of sodium butyrate is inhibition of cellular histone deacetylase(s) which results in disruption of nucleosomes relieving one level of restriction to gene expression. Consistent with this mechanism of action, we further found that sodium butyrate's effects: (i) act synergistically with PMA and TNF-alpha; (ii) are independent of protein synthesis; (iii) do not affect the constitutively expressed creatine phosphokinase gene; (iv) do not map to a discrete sequence motif in the viral LTR; and (v) are not blocked by N-acetyl cysteine but (vi) are blocked by novobiocin, an inhibitor of cellular topoisomerase II. These data show that a similar pattern of restricted viral RNA expression exists in this nonlymphoid cellular model of HIV-1 latency. In contrast however, these results suggest that in these cells there is an additional block to viral gene expression, which is overcome with sodium butyrate. These results are discussed in the context of histone-mediated repression of HIV-1 gene expression.

Replication of type 1 human immunodeficiency viruses containing linker substitution mutations in the -201 to -130 region of the long terminal repeat.

In previous transfection analyses using the chloramphenicol acetyltransferase reporter gene system, we determined that linker substitution (LS) mutations between -201 and -130 (relative to the transcription start site) of the human immunodeficiency virus type 1 long terminal repeat (LTR) caused moderate decreases in LTR transcriptional activity in a T-cell line (S. L. Zeichner, J. Y. H. Kim, and J. C. Alwine, J. Virol. 65:2436-2444, 1991). In order to confirm the significance of this region in the context of viral replication, we constructed several of these LS mutations (-201 to -184, -183 to -166, -165 to -148, and -148 to -130) in proviruses and prepared viral stocks by cocultivation of transfected RD cells with CEMx174 cells. In addition, two mutations between -93 and -76 and between -75 and -58 were utilized, since they affect the nuclear factor kappa B (NF-kappa B)- and Sp1-binding sites and were expected to diminish viral replication. Our results suggest that while transfection analyses offer an adequate approximation of the effects of the LS mutations, the analysis of viral replication using a mutant viral stock presents a more accurate picture, which is sometimes at variance with the transfection results. Three mutants (-201/-184 NXS, -165/-148 NXS, and -147/-130 NXS) had effects on viral replication that were much more severe than the effects predicted from their performance in transfection analyses, and the effects of two LS mutations (-201/-184 NXS and -183/-166 NXS) were not predicted by their effects in transfection. In addition, we observed cell type-specific permissiveness to replication of some mutant viruses. In the cell types tested, the LS mutations indicated an apparent requirement not only for the intact NF-kappa B and SP1-binding sites but also for several regions between -201 and -130 not previously associated with viral infectivity.

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)