INTRODUCTION

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One of the major distinguishing characteristics of viruses that have segmented double-stranded RNA (dsRNA) genomes is that transcription of the genome occurs within the structurally intact core of the virion (reviewed in reference 8). Genome transcription occurs through the concerted action of multiple enzyme activities housed within the viral core, and mature transcripts are released through channels penetrating the viral capsid. In the largest known family of segmented dsRNA viruses, the Reoviridae, whose members all infect higher eukaryotes, the viral core contains all of the enzyme activities needed to synthesize not only the mRNA transcripts themselves but also a properly guanylated and methylated cap structure on the 5' end, as required by the eukaryotic translation initiation machinery. Rotaviruses, because of their medical importance as the leading cause of life-threatening gastroenteritis in young children (12), have been studied extensively using biochemical and structural techniques and represent a good model system in which to investigate the molecular and chronological events of genome transcription in segmented dsRNA viruses.

Architecturally, the mature rotavirus particle consists of three concentric icosahedrally ordered protein capsid layers which are assembled around a genome of 11 segments of dsRNA (reviewed in reference 22). At the start of the replication cycle, rotavirus attaches to the host cell as a triple-layered particle (TLP). Upon cell entry, the outermost capsid layer, consisting of a shell protein (VP7) and a spike protein (VP4), is removed, and the resulting double-layered particle (DLP) becomes transcriptionally competent. The production of mature mRNA transcripts may be considered to occur in three stages (reviewed in reference 15). Transcription starts with initiation, at which time nucleotidyl transfer begins and capping of the nascent transcript occurs; this is followed by a transition to processive elongation, during which the transcript begins to separate from the genomic template and nucleotidyl transfer continues as the transcription bubble progresses through the template. Elongation at the polymerase active site is followed by translocation of the growing transcript through channels which penetrate the inner VP2 and outer VP6 capsid layers at the icosahedral vertices (13).

The enzymatic steps of transcription, nucleotide incorporation, and cap formation, are performed by two proteins, VP1 and VP3, both of which are housed within the viral core (reviewed in reference (6)). VP1 is the viral RNA-dependent RNA polymerase (28), and VP3 is the enzyme implicated in cap formation (4, 16, 20, 21). Mature rotavirus transcripts are capped with the sequence ^m7GpppG^(m) at the 5' end (11, 18). The formation of such a cap structure requires the activity of a guanylyltransferase enzyme, which uses GTP as a substrate, as well as a methyltransferase activity, which uses S-adenosylmethionine (SAM) as a substrate (10). Rotavirus VP3 has been shown to possess both guanylyltransferase and methyltransferase activity. Structural studies suggest that the rotavirus core contains 12 copies of VP1 and VP3, which appear to be incorporated as heterodimers anchored to the inner surface of the capsid at each of the icosahedral vertices (24).

Although previous biochemical studies have provided a characterization of the enzymatic reactions which occur during transcription, and structural studies of actively transcribing DLPs have provided a framework in which to understand how mRNA is translocated through the capsid, less is known about how the upstream events of initiation and elongation unfold from a mechanistic perspective. In particular, it is not known what events constitute the point of transition between initiation and processive elongation when this transition takes place following initiation and what structural factors may influence the ability of the endogenous transcription apparatus to make this transition successfully.

To gain insight into the early molecular events in transcription, we used high-resolution denaturing polyacrylamide gel electrophoresis to investigate the length distribution of transcription products at various times following initiation, both in particles which are capable of producing full-length transcription products and in those which are incapable of undergoing the transition from initiation to processive elongation. We observed that, under normal transcription conditions, initiation and capping appear to be followed by a brief pause in elongation after 5 to 6 nucleotides have been transcribed. We propose that this pause site constitutes the point of transition between initiation and a commitment to proceed with full-length elongation. Our results also suggest that the ability of the transcription apparatus to progress successfully from initiation to processive elongation is determined primarily by the extent to which the enzymatic efficiency of the polymerase is modulated by the conformational states of other proteins in the virus.

	MATERIALS AND METHODS

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Viruses. Rotavirus strain SA11-4F was propagated in MA-104 cells according to standard procedures and purified as previously described (23). Purified TLPs were stored in TNC buffer (10 mM Tris [pH 7.4], 140 mM NaCl, 10 mM CaCl₂), and DLPs were stored in Tris-buffered saline.

In vitro transcription. In vitro transcription was carried out using a basic transcription reaction buffer containing 100 mM Tris-HCl (pH 8); 10 mM magnesium acetate; 1 mM ATP; a 100 µM concentration (each) of GTP, CTP, and UTP; and 1 µCi of [-³²P]GTP/ml (3,000 Ci/mmol; Amersham Pharmacia Biotech). Reaction mixture also contained 0.5 mM SAM unless otherwise indicated. Reactions requiring radiolabeled SAM contained 0.5 mM S-adenosyl-L-[methyl-³H]methionine (500 mCi/mmol; Amersham-Pharmacia Biotech), and [-³²P]GTP was omitted. Transcription reactions were incubated at 37°C as described in the text. The quantity of virus particles present in each reaction is specified in the text. Because TLP preparations typically also contain a small number of DLPs (usually less than 1%), TLP transcription reactions were supplemented with the anti-VP6 Fab 2A11/E9 at 25 µg/ml when radiolabeled GTP was included in the reaction mixture in order to suppress full-length mRNA production from contaminating DLPs (14).

High-resolution denaturing polyacrylamide gel electrophoresis (PAGE). The products of in vitro transcription were resolved by electrophoresis on 20% polyacrylamide gels (0.4 mm thickness) containing 7 M urea. Electrophoresis was typically carried out at 400 V until the bromophenol blue dye front migrated 20 cm (between 22 and 24 h). Following electrophoresis, both the gel and one supporting glass plate were wrapped in a thin plastic film and autoradiographed for an appropriate period of time as needed to visualize the transcription products of interest.

Quantitation of ³²P-labeled transcription products. To quantitate the amount of radioactivity associated with specific transcription products resolved by denaturing PAGE, regions of the gel containing the product of interest were placed in contact with a Molecular Dynamics Phosphor Storage Screen. Following exposure, the signal stored in the phosphor screen was quantitated on a Molecular Dynamics Storm 860 PhosphorImager operated at a 200-µm scanning interval, and image analysis was carried out using the ImageQuant software package (Molecular Dynamics). Quantitation was performed on the desired bands by drawing a rectangular mask around the edge of each band and noting the total number of counts present within the mask. Background correction was performed by subtracting the counts obtained within similar-sized masks drawn around corresponding areas from a negative control reaction.

Quantitation of ³H-labeled transcription products. For reactions in which the 5' cap was labeled using S-adenosyl-L-[methyl-³H]methionine, regions of the gel containing the transcription products of interest were excised, crushed, and mixed with ~3 volumes of a standard nucleotide elution buffer containing 0.5 M ammonium acetate and 10 mM magnesium acetate (pH7). The gel slices were incubated with the elution buffer for 36 h at 40°C with rapid agitation. Following elution of the nucleic acid, the supernatant (~200 µl) was withdrawn and mixed with 5 ml of CytoScint ES scintillation fluid (ICN Biomedicals). Radioactivity was quantitated using a Beckman 1010LS liquid scintillation counter operating within the tritium counting window.

Pulse-chase analysis of transcription in TLPs and structurally converted DLPs. TLPs were added to a standard transcription reaction buffer containing SAM and supplemented with [-³²P]GTP. The reaction was incubated at 37°C for 5 min. After this initial pulse incubation, both the TLP reaction, as well as a negative control reaction containing [-³²P]GTP but no particles, were passed successively through two Micro Bio Spin P-30 columns equilibrated in 10 mM Tris-HCl buffer (pH 8.6) and 10 mM magnesium acetate to remove unincorporated radiolabeled nucleotides. Particles eluting in the void volume were divided into three equal portions. One portion was set aside as a sample for gel electrophoretic analysis. The second portion was added to a standard transcription reaction mixture without radiolabeled nucleotides and incubated for 10 min at 37°C. A third portion was incubated for 5 min at room temperature with 5 mM BAPTA [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid] (Sigma) to remove the outermost capsid layer (VP7 capsid plus VP4 spikes) without substantially affecting the Mg²⁺ concentration in the buffer. The resulting DLPs were then added to a standard transcription reaction mixture without radionucleotides and incubated for 10 min at 37 °C. Following incubation, these two chase reactions were used directly as samples for gel electrophoresis.

	RESULTS

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Short oligonucleotides are produced during transcription. As with most transcription systems, the production of mature, full-length mRNA transcripts in rotavirus is believed to occur in several stages, beginning with initiation and followed by a transition to processive elongation and, finally, translocation out of the particle interior. To visualize the manner in which growing transcripts progress from initiation to elongation, we examined the length distribution of transcription products as a function of time in both native DLPs, the viral form in which transcription ordinarily occurs, and mature TLPs, a viral form in which full-length transcripts cannot be made. Virus particles were incubated in a standard in vitro transcription reaction mixture, and, at various time points, aliquots of the reaction mixture were removed and analyzed by high-resolution denaturing PAGE.

View larger version (39K): [in this window] [in a new window]   FIG. 1.   Analysis of transcription products in DLP and TLP. (a) Visualization of transcription products by 20% denaturing PAGE. Lanes 1 to 3, DLP transcription products sampled after 10, 20, and 30 min of incubation at 37 °C (each reaction aliquot contained 1-µg particles); lane 4, negative-control reaction containing no particles, sampled after 30 min of incubation; lanes 5 to 7, TLP transcription products sampled after 10, 20, and 30 min of incubation. Each reaction aliquot contained 2-µg particles. RNA and DNA size standards are indicated (length in nucleotides). (b) Quantitation of full-length mRNA yield in the DLP transcription reaction. (c) Quantitation of the major and minor oligoncleotide species in the DLP and TLP reactions at each of the indicated time points. Quantitation was performed as described in the Materials and Methods.

Short oligonucleotides produced in DLPs during transcription are particle associated. To determine whether these oligonucleotide transcription products are particle associated, as would be expected if they represent early intermediates in the elongation process, we used gel filtration to separate actively transcribing DLPs and elongated transcripts from free nucleotides and oligonucleotide species shorter than ~20 bases. Aliquots taken from a standard transcription reaction at various time points were passed through a centrifugal gel filtration column, and material eluting in the void volume was then analyzed by high-resolution denaturing PAGE (Fig. 2).

View larger version (38K): [in this window] [in a new window]   FIG. 2.   Analysis of particle-bound oligonucleotide transcription products in DLP. Lanes 1 to 5 contain DLP transcription products sampled after incubation at 37°C for the indicated period of time. Prior to electrophoresis, each reaction aliquot was passed through a size exclusion column to remove free nucleotides and unbound oligonucleotides shorter than 20 bases. Material eluting in the void volume was resolved on the gel. Each reaction aliquot contained 6-µg particles. Close inspection of the intensity of major and minor oligonucleotide bands in the gel indicates that the ratio of the two products varied slightly over the time course of the reaction and, additionally, was different from that observed in Fig. 1. Variations in the ratio of the major to minor oligonucleotide products were routinely observed throughout our studies and are likely to reflect subtle variations in reaction conditions. In all of our experiments, however, the quantity of the minor oligonucleotide species was consistently observed to be less than that of the major oligonucleotide species. Lane 6 contains a negative control reaction with no particles, sampled after 15 min of incubation. The intensity of the band corresponding to unincorporated nucleotides is not the same in each reaction aliquot, perhaps because of the varying efficiency with which the size exclusion columns removed free radiolabeled nucleotides after transcription. RNA and DNA size standards are indicated (length in nucleotides).

Both major and minor oligonucleotide transcription products are capped. The observation that specific, particle-associated oligonucleotide transcripts having discrete lengths and transient lifetimes can be observed at a very early point in the transcription process suggests that one or more of the initial steps in mRNA production occurs more slowly than the other steps, giving rise to a population of transcripts which are temporarily stalled at a specific point. To determine whether this delay in transcription occurs before or after the events involved in cap formation, we investigated whether or not a methylated cap can be detected on the 5' end of either of these oligonucleotide transcripts. Methylation is recognized to be the final step in cap formation and involves the transfer of a methyl group from S-adenosylmethionine (SAM) to the penultimate Gppp moiety attached by the guanylyltransferase (4, 11, 26). The rotavirus methyltransferase has also been observed to attach a methyl group to the first sequence-derived G base uniformly conserved in all rotavirus mRNA transcripts, although with less efficiency and only following the attachment and methylation of a G base on the 5' end (11).

View larger version (24K): [in this window] [in a new window]   FIG. 3.   Analysis of 3H-methyl incorporation in oligonucleotide transcription products. The quantity of tritium radioactivity present in each of the indicated RNA species is expressed with respect to background. To estimate the level of background radioactivity, an average value of 55.4 cpm (range, 49 to 63 cpm) was obtained from five independent measurements taken from regions of the polyacrylamide gel which were adjacent to those from which oligonucleotide transcription products were isolated. A series of parallel reactions run with both 3H and 32P labeling (data not shown) suggested that the variation in tritium signal strength between the major and minor oligonucleotides of DLPs and TLPs was due primarily to differences in the quantity of the oligonucleotide species present in the gel, as indicated by 32P incorporation.

Oligonucleotides are not observed during transcript elongation in DLPs when the efficiency of the polymerase is reduced. To examine whether a decrease in the rate of nucleotide incorporation leads to a reduction in the number of initiated transcripts which appear to be stalled early in the elongation process, as would be characteristic of a transcription pause site, we carried out transcription in both the presence and absence of the capping reaction cofactor SAM and compared the ratio of particle-bound oligonucleotides to full-length transcripts in the two reactions. Although the precise mechanism is unknown, biochemical studies have shown that the enzymatic efficiency of the polymerase is improved (as reflected by a reduction in the apparent K_m value for each of the nucleotide triphosphates and an increase in the overall V_max for the transcription reaction) when SAM is continuously supplied in the reaction mixture (26). A similar phenomenon has also been observed in the segmented dsRNA virus cypovirus (9).

View larger version (50K): [in this window] [in a new window]   FIG. 4.   Length distribution of transcription products in the presence and absence of the capping reaction cofactor SAM. Lanes 1 and 2 contain DLP transcription products in the presence and absence of SAM. Each reaction mixture contained 5-µg particles and was incubated at 37°C for a total of 20 min. Following transcription, reaction mixtures were passed over size exclusion columns to remove unincorporated nucleotides and any free oligonucleotides shorter than 20 bases. Though identical amounts of radiolabeled GTP were included in each reaction mixture, the reason for which the intensity of the band corresponding to unincorporated nucleotides is not the same in each reaction aliquot may be due to the varying efficiency with which the size exclusion columns removed free radiolabeled nucleotides after transcription; additionally, this band may also include nucleotides which are specifically or nonspecifically bound inside the particle during gel filtration. RNA and DNA size standards are indicated (length in nucleotides).

Transcript initiation is significantly diminished in TLPs when the efficiency of the polymerase is reduced. Under normal transcription conditions, native DLPs and mature TLPs both appear to be equally capable of performing the enzymatic steps required for the early stages of transcription, including transcript initiation, cap formation, and limited elongation up to the point which, in DLPs, is the site of momentary pausing prior to processive full-length elongation. To investigate whether the limited transcription observed in the TLP is dependent upon polymerase efficiency, we carried out transcription in the presence and absence of SAM and examined the length distribution of the resulting particle-bound transcription products.

The oligonucleotide transcription products are precursors of full-length mRNA transcripts. The observation that the short oligonucleotide transcription products in the DLP reaction are capped, have a transient lifetime, and are particle-associated suggests that they are elongation intermediates, temporarily stalled while in the process of becoming full-length transcripts. To establish that these oligonucleotides are indeed precursors of full-length transcripts, we employed a pulse-chase strategy. TLPs were first allowed to begin transcription using a reaction mixture supplemented with radiolabeled GTP; following a brief incubation at 37°C, radiolabeled nucleotides were removed from the reaction mixture using two successive centrifugal gel filtration columns. The TLPs were then converted to DLPs with the addition of the calcium-specific chelator BAPTA, and finally, transcription was resumed by supplying unlabeled nucleotide triphosphates. The length distribution of transcription products before and after structural conversion was compared using high-resolution denaturing PAGE (Fig. 5).

View larger version (28K): [in this window] [in a new window]   FIG. 5.   Pulse-chase analysis of transcription products in TLPs and structurally converted DLPs. (a) Visualization of radiolabeled transcription products using high-resolution denaturing PAGE. Each lane shows transcription products arising from 1-µg particles. Lane 1, oligonucleotide transcripts synthesized in TLP following 5 min of incubation at 37°C in a reaction mixture containing radiolabeled GTP (pulse reaction); lane 2, oligonucleotide transcripts in TLP following a 5-min pulse incubation and subsequent 10-min chase incubation at 37°C; lane 3, transcription products arising from a 5-min pulse incubation in TLP followed by structural conversion to DLP and subsequent 10-min chase incubation at 37°C; lane 4, negative-control reaction containing radiolabeled GTP but no particles, incubated at 37°C for 5 min. The absence of a detectable signal from unincorporated nucleotide precursors indicates that the use of two successive size exclusion columns was sufficient to remove the radiolabeled GTP from the reaction following the pulse. RNA and DNA size standards are indicated (length in nucleotides). (b) Quantitation of the radioactivity present in the two oligonucleotide species in each reaction. (c) Quantitation of the radioactivity present in the region of the gel corresponding to elongated transcripts.

	DISCUSSION

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In order to gain an increased understanding of the molecular events occurring early in the transcription process in rotavirus, we investigated the length distribution of transcription products arising under various conditions of polymerase efficiency in particles which are capable of producing full-length transcripts (DLPs) as well as in particles which are not (TLPs). We observed that, under normal conditions, DLPs produce both full-length mRNA transcripts and two short oligonucleotide species. These oligonucleotide species, which migrate in a polyacrylamide gel with an apparent length between 5 and 7 bases, are particle associated, contain methylated cap structures, and have a transient lifetime, suggesting them to be precursors of full-length transcripts.

The production of oligonucleotides during transcription has been well documented in many diverse transcription systems. Biochemical studies of genome transcription in the related viruses mammalian orthoreovirus and insect cypovirus have reported that transcription of full-length mRNA is accompanied by the production of a very large molar excess of short oligonucleotides two to three bases in length, some with caps but most without (1, 7, 9, 30-32). These oligonucleotides were identical in sequence with the conserved 5' ends of mature mRNAs and were believed to be aborted transcripts resulting from failed initiation attempts. Indeed, abortive reinitiation has also been observed in prokaryotic and eukaryotic transcription systems as well (3, 17).

Because of the widespread nature of reiterative initiation in transcription systems, it is likely that some form of this phenomenon accompanies transcription in rotavirus as well. However, for several reasons, the oligonucleotide transcription products we describe in the current study appear to be distinct from oligonucleotides arising through abortive reinitiation. First, the major and minor oligonucleotide species appear to be longer than the largely uncapped di- and trinucleotide species ordinarily associated with the phenomenon of abortive reinitiation. Second, while the products of abortive reinitiation typically accumulate in vast molar excess of successfully elongated transcripts, we observe that the major and minor oligonucleotide species produced in the DLP do not accumulate appreciably during the reaction and indeed appear to have a finite lifetime, as would be characteristic of stalled elongation intermediates as opposed to aborted transcripts arising through reiterative initiation. Longer oligonucleotides having these characteristics have not, to our knowledge, been observed in orthoreovirus or cypovirus and indeed may arise because of fundamental architectural differences between "turreted" reoviruses, which include orthoreovirus and cypovirus, and "nonturreted" reoviruses, which include rotavirus and orbivirus. Such architectural differences are especially prominent in the vicinity of endogenous transcription apparatus and indeed may impose significant constraints on the mechanism of mRNA production and capping (15).

The observation that capped, particle-associated oligonucleotide transcripts having transient lifetimes arise at a specific point early in the transcription process suggests that the movement of the dsRNA template through the polymerase during elongation is not a continuous process but rather is temporarily interrupted shortly after initiation and cap formation. This brief pause in nucleotide incorporation appears to divide the elongation phase of transcription into two parts. Prior to stalling, the polymerase is able to elongate the initiated transcript to approximately 5 to 7 nucleotides in length; following the pause, the polymerase is able to elongate the transcript processively all the way to the end of the dsRNA template, generating full-length mRNA. This pause site may represent the point at which the transcription apparatus must carry out specific steps to enable full-length elongation to take place.

It is not known which molecular event early in the transcription process causes the polymerase to pause shortly after initiation and cap formation. Evidently, this event occurs at a rate which is lower than the basic rate of nucleotide incorporation under ordinary conditions. Under conditions in which the rate of nucleotide incorporation is reduced, as is the case when SAM is omitted from the reaction mixture (26), the transcription apparatus no longer stalls while carrying out this step, indicating that pausing at this point in elongation occurs because of a difference in the rate at which various steps involved in elongation may proceed. From a consideration of the apparent length of the paused transcripts, the circumstances under which they arise, and the events that likely must occur to facilitate processive elongation, several possibilities emerge as to the reason why the transcription apparatus stalls momentarily before proceeding with full-length elongation.

One possibility is that the brief pause in elongation may be necessary in order to allow the viral helicase to become properly engaged on the template ahead of the polymerase. In all transcription systems operating on a double-stranded template, a helicase is required to open the transcription bubble downstream of the polymerase. Such a helicase has, in fact, been positively identified in the closely related viruses bluetongue virus (26) and mammalian orthoreovirus (2, 19). It is probable that, at the start of transcription, the polymerase is already engaged on a partially unwound template and, as such, can incorporate several nucleotides before requiring the activity of the viral helicase to begin further unwinding. Following the proper engagement of the helicase, nucleotide incorporation may then proceed unhindered, giving rise to full-length transcripts without additional delays.

Another early event possibly requiring a brief pause in elongation involves the initial separation of the nascent transcript from the transcription bubble. Studies in other transcription systems have suggested that, during elongation, the template strand and nascent transcript are base paired for 8 to 10 nucleotides as a hybrid duplex within the transcription bubble (25, 29), and the polymerase itself is believed to take an active role in separating the growing transcript from the template (5). Although little is known about how this may occur during rotavirus transcription, it is conceivable that, following the incorporation of the first several nucleotides, a specific event involving the polymerase, or perhaps a domain of the inner capsid protein VP2, is required to promote the initial separation of the nascent transcript from the template and the subsequent closure of the transcription bubble before processive elongation may occur.

A third transcriptional event possibly requiring a brief pause early in transcript elongation involves the export of the nascent transcript from the core. During rotavirus transcription, nascent transcripts are translocated across the intact double-layered capsid through channels at the icosahedral vertices (13). At the outset of transcription, the transcripts emerging from the transcription bubbles must be threaded through the exit channels in the inner capsid layer. Though these channels are in close proximity to the transcription enzyme complexes in the viral core (24), it is possible that the threading of the transcripts into the channels may generally require a temporary pause in elongation several nucleotides downstream of initiation in order to allow sufficient time for this to occur properly.

The transcriptional behaviour of TLPs, which differ from DLPs in that they contain an additional external capsid layer, provides further insight into the nature of the events accompanying transcriptional pausing during elongation. Under ordinary transcription conditions, TLPs produce only the major and minor oligonucleotide species and no full-length transcripts. These oligonucleotides contain methylated caps and appear to be particle associated initially but are eventually released. This behavior contrasts with that observed for mature orthoreovirus, where predominantly uncapped di- and trinucleotide aborted transcription products accumulate to high levels within the particle interior and are released only when the outermost capsid layer is removed (7).

The observation that cap formation and limited nucleotide incorporation can occur in the TLP suggests that the TLP is not strictly transcriptionally incompetent; both the polymerase and the capping enzymes are functional, and capped oligonucleotide transcripts having an apparent length of 5 to 7 bases are made. However, in the presence of the outermost capsid layer, the transcription apparatus in the core nonetheless appears to be unable to perform at least one of the critical events required early in the elongation phase to facilitate the transcription of full-length mRNA. These observations also imply that the events accompanying the momentary pause in elongation are somehow susceptible to disruption from changes in the architecture of the capsid, as has been suggested from structural studies in rotavirus (14). The conformation of the viral capsid has also been suggested to play a role in inhibiting transcript elongation in mature orthoreoviruses (7).

Interestingly, in addition to preventing the transcription apparatus from carrying out the events needed to facilitate full-length elongation, the attachment of specific ligands on the viral surface also appears to alter the function of the polymerase. Although TLPs retain the ability to perform limited transcript elongation under ordinary conditions, omission of SAM appears to render the transcription apparatus incapable of initiation (see Fig. 4). The ability of SAM, a substrate for the capping enzyme, to affect polymerase function indicates that these two enzymes are likely in close contact with one another in the viral core, as has been suggested from other studies (24, 26). Were cap formation to be the only component of the transcription process affected by the omission of SAM, then TLPs should remain capable of transcript initiation even in the absence of SAM, as is seen in DLPs. However, such a result is clearly not observed, implying that the polymerase in the TLP may have an altered structure because of the presence of an additional capsid layer on the viral surface, leading to a reduction in enzymatic efficiency. In the presence of SAM, this reduction in polymerase efficiency may be partially compensated for to the extent that initiation and limited elongation may proceed. Although the precise reason for which TLPs cannot elongate beyond the pause site remains unknown, it is possible that the efficiency of the polymerase is still not adequate for the transcription apparatus to carry out the specific downstream events needed for the transition to processive elongation.

The contrasting behavior of the transcription apparatus under various conditions illustrates the degree to which polymerase function is sensitive to the architectural environment in which it is operating. The presence of SAM tends to boost polymerase function, while the presence of specific ligands on the viral surface tends to inhibit polymerase function. From the perspective of the viral replication cycle, these two opposing influences, both of which likely affect the transcription apparatus through structural interactions, serve to ensure that transcription occurs with greatest efficiency only after the virus has entered the cytoplasm and assumed the form of a DLP. Likewise, the inhibition of transcript elongation in the TLP may benefit the virus by ensuring that transcription does not begin to occur prematurely, under circumstances in which genome transcription would not be productive.