Prior to the purification of the viral RNAP to homogeneity, partial purifications were able to transcribe late/very late genes (3, 18, 76). Separation of the polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that the complex had less than 10 main polypeptides (3, 77). However, the identification of the subunits forming the RNAP complex remained unknown.
Guarino and colleagues purified the viral RNAP to homogeneity from AcMNPV-infected cells (26). The purified protein complex specifically transcribed a late and a very late promoter template but not an early promoter template in vitro. The complex was estimated to have a molecular mass of 560 kDa. Separation of polypetides by SDS-PAGE identified four polypeptides of relative molecular masses of 98, 55, 53, and 46 kDa present in equimolar concentrations, suggesting that the active complex contained two molecules of each polypeptide.
The composition of this RNAP represents the simplest eukaryotic RNAP described to date (26). In addition, these results are in agreement with in vitro transcription system assays in which a preincubation step of template and factors was not required for efficient subunit assembly preceding transcription initiation and elongation (76). Lack of a requirement for a preincubation step suggests that the transcribing complex has a simple composition (76).
In addition to its simple composition, the AcMNPV RNAP is unique to DNA-containing viruses that replicate in the nucleus of cells. In general, viruses with DNA genomes utilize host RNAPs. The only two families of DNA-containing viruses that do not conform are Poxviridae and Asfarviridae. Members of both of these families encode an RNAP, however, they replicate in the cytoplasm of cells, making it impractical to access the nuclear RNAP Ⅱ encoded by the host.
The individual subunits of the RNAP complex were identified by sequencing their N termini, peptide fingerprinting, and immunoblotting. Four subunits encoded by previously characterized genes were identified and their predicted sizes roughly corresponded to the sizes of the four purified polypeptides: LEF-8 (98 kDa), LEF-9 (55 kDa), LEF-4 (46 kDa), and P47 (46 kDa). The genes that encode the four RNAP subunits are present in all baculoviruses sequenced to date, and they are the most conserved LEFs (4). The percent similarities between specific RNAP subunits encoded by Group Ⅰ baculoviruses and those of Group Ⅱ, granuloviruses, and baculoviruses isolated from non-lepidopteran insects averages 62% (Table 1) in contrast to that of other LEFs which ranges from 36 to 55% and averages 46% (4).
Table 1. Average percent identities and similarities among RNAP subunits within different baculovirus groupsa
Table 2. Known or predicted activities of the baculovirus RNAP subunits
The lef-8 gene was first identified in a functional transient assay screen for late gene transacting factors and proposed to be a potential RNAP subunit due to the presence of a short invariable motif encoded by well-defined RNAPs (55). The lef-8 gene was required for expression from two late and two very late promoters but not from an early one (55, 71). Since its identification in 1993, only two functional studies focusing on lef-8 have been published; one study characterized a temperature sensitive mutation within the Bombyx mori NPV (BmNPV) lef-8 (62), and another described a detailed function-structure analysis (70). Thus, information about the specific mechanisms governing its role in late gene tran-scription is scant.
A virus with a temperature sensitive mutation within the BmNPV lef-8 replicated normally at early times and had normal viral DNA accumulation, but lacked processes that either required late gene expression and/or occurred at late times, including occluded virus production, late gene expression, host protein shutoff, and budded virus production. Furthermore, infection of larvae with this virus at the nonpermissive temperature killed only approximately 34% of the infected larvae (62). However, the experiment was not controlled for infection of larvae with wild-type BmNPV at the non-permissive temperature. It is possible that the combination of virus infection and high temperature resulted in different mortality rates.
A detailed functional-structure analysis of lef-8 was conducted to define the domains of lef-8 required for late gene promoter activation (70). The sequence of lef-8 predicts a polypeptide of 102 kDa, the largest subunit of the viral RNAP and one of the largest predictted AcMNPV gene products. However, its overall sequence does not reveal obvious homology to other RNAPs (55). Towards the C terminus of LEF-8, LEF-4RNA 5'-triphosphatase Nucleoside triphosphatase (22, 25, 30) there is a 13-amino acid motif that is similar to that found in the second largest subunits of both prokaryotic (region H) and eukaryotic RNAPs. Mutations within this motif in lef-8 abolish late gene expression, indicating the importance of this motif in late gene expression (70). In addition, site-directed mutations and deletions throughout lef-8 resulted in late gene activity reductions, implying that regions throughout the polypeptide were necessary for interactions with other subunits, accessory factors, or nucleic acids (70). Alternatively, alterations or deletions of approximately 50 amino acids within the sequence of lef-8 had drastic effects on the stability of the polypeptide. It has been suggested that the lef-8 mRNA may be unstable (1), and we have had difficulty expressing lef-8 transiently (13).
As with lef-8, lef-9 was also identified in a subtractive complementation transient late gene expression assay (41) but remarkably, 12 years after its identification, no other reports on its functional characterization are available. LEF-9 has an invariable heptapeptide motif also encoded in the largest subunits of other RNAPs (41). Furthermore, this motif has three aspartic acid residues that bind Mg2+ at the active center of the enzyme complex, and disruption of these residues obliterates catalytic activity in other RNAPs (78).
The Escherichia coli RNAP β' subunit, the largest subunits of eukaryotic RNAP Ⅰ, Ⅱ, and Ⅲ, and the largest RNAP subunits from cytoplasmic DNA viruses all contain the conserved NADFDGD catalytic center motif (63) also present in LEF-9. Moreover, these subunits also have a well-conserved sequence, RQPT/SLH, between 26 and 31 amino acids upstream of the NADFDGD motif. Although the function of this region is not clear, a similar motif, RHPNIS, can be found 32 residues upstream of the AcMNPV NADFDGD-like LEF-9 sequence. Furthermore, this motif upstream of NADFDGD is conserved in all of the LEF-9 homologs from baculoviruses sequenced to date (Fig. 1). The conservation of these sequences stresses the evolutionary relationships among different RNAPs.
LEF-4 was identified as a product required for viral late gene expression in transient assays (53) and localizes to the nucleus of infected cells (15, 60). Viruses with mutations within lef-4 also have defects in late processes (7, 33, 52).
Baculovirus mRNAs are known to be capped (32). The enzyme that may be responsible for processing the 5' ends of viral mRNAs is LEF-4, which is associated with the viral RNAP. Its role in capping was discovered by tracking guanylyltransferase activity of the viral RNAP (25) and by sequence and biochemical analyses (22). LEF-4 also has metaldependent RNA triphosphatase activity (22, 25, 28). Although its role in capping mRNAs in vivo has not been established, its close association with the tran-scription machinery suggests a role in capping viral mRNAs in vivo.
Baculoviruses also encode a non-essential RNA triphosphatase and diphosphatase, but the enzyme has no associated guanylyltransferase activity (21, 39, 69). However, when fused to a functional guanylyltransferase module, it can form mRNA caps in vivo (44).
Some baculoviruses encode an RNA cap (nucleoside-2'O)-methyltransferase, but a mutant virus was viable (75). In transient expression assays, this gene stimulates late gene expression (38), thus, its specific role during virus infection and RNA processing is not clear.
The least characterized of the RNAP subunits is P47. Its sequence does not reveal any obvious motifs or homology to other known proteins. Its role in late gene expression was discovered by characterizing a virus with a temperature sensitive mutation that mapped to p47 (8, 52) and in transient late gene expression assays (72). The temperature sensitive virus was defective in late gene expression and budded virus production at non-permissive temperatures (52). Its product localizes to the nucleus of infected cells (8), commensurate with its role in transcription. In transient late gene expression assays, it was found that p47 was required for late gene expression of viral late and very late gene promoters (71, 72).