Lepidopteran wing scales contain abundant cross-linked film-forming histidine-rich cuticular proteins.

Scales are symbolic characteristic of Lepidoptera; however, nothing is known about the contribution of cuticular proteins (CPs) to the complex patterning of lepidopteran scales. This is because scales are resistant to solubilization, thus hindering molecular studies. Here we succeeded in dissolving developing wing scales from Bombyx mori, allowing analysis of their protein composition. We identified a distinctive class of histidine rich (His-rich) CPs (6%-45%) from developing lepidopteran scales by LC-MS/MS. Functional studies using RNAi revealed CPs with different histidine content play distinct and critical roles in constructing the microstructure of the scale surface. Moreover, we successfully synthesized films in vitro by crosslinking a 45% His-rich CP (BmorCPR152) with laccase2 using N-acetyl- dopamine or N-ß-alanyl-dopamine as the substrate. This molecular study of scales provides fundamental information about how such a fine microstructure is constructed and insights into the potential application of CPs as new biomaterials.

Expression map of a complete set of gustatory receptor genes in chemosensory organs of Bombyx mori.

Most lepidopteran species are herbivores, and interaction with host plants affects their gene expression and behavior as well as their genome evolution. Gustatory receptors (Grs) are expected to mediate host plant selection, feeding, oviposition and courtship behavior. However, due to their high diversity, sequence divergence and extremely low level of expression it has been difficult to identify precisely a complete set of Grs in Lepidoptera. By manual annotation and BAC sequencing, we improved annotation of 43 gene sequences compared with previously reported Grs in the most studied lepidopteran model, the silkworm, Bombyx mori, and identified 7 new tandem copies of BmGr30 on chromosome 7, bringing the total number of BmGrs to 76. Among these, we mapped 68 genes to chromosomes in a newly constructed chromosome distribution map and 8 genes to scaffolds; we also found new evidence for large clusters of BmGrs, especially from the bitter receptor family. RNA-seq analysis of diverse BmGr expression patterns in chemosensory organs of larvae and adults enabled us to draw a precise organ specific map of BmGr expression. Interestingly, most of the clustered genes were expressed in the same tissues and more than half of the genes were expressed in larval maxillae, larval thoracic legs and adult legs. For example, BmGr63 showed high expression levels in all organs in both larval and adult stages. By contrast, some genes showed expression limited to specific developmental stages or organs and tissues. BmGr19 was highly expressed in larval chemosensory organs (especially antennae and thoracic legs), the single exon genes BmGr53 and BmGr67 were expressed exclusively in larval tissues, the BmGr27-BmGr31 gene cluster on chr7 displayed a high expression level limited to adult legs and the candidate CO2 receptor BmGr2 was highly expressed in adult antennae, where few other Grs were expressed. Transcriptional analysis of the Grs in B. mori provides a valuable new reference for finding genes involved in plant-insect interactions in Lepidoptera and establishing correlations between these genes and vital insect behaviors like host plant selection and courtship for mating.

Construction, complete sequence, and annotation of a BAC contig covering the silkworm chorion locus.

The silkmoth chorion was studied extensively by F.C. Kafatos' group for almost 40 years. However, the complete structure of the chorion locus was not obtained in the genome sequence of Bombyx mori published in 2008 due to repetitive sequences, resulting in gaps and an incomplete view of the locus. To obtain the complete sequence of the chorion locus, expressed sequence tags (ESTs) derived from follicular epithelium cells were used as probes to screen a bacterial artificial chromosome (BAC) library. Seven BACs were selected to construct a contig which covered the whole chorion locus. By Sanger sequencing, we successfully obtained complete sequences of the chorion locus spanning 871,711 base pairs on chromosome 2, where we annotated 127 chorion genes. The dataset reported here will recruit more researchers to revisit one of the oldest model systems which has been used to study developmentally regulated gene expression. It also provides insights into egg development and fertilization mechanisms and is relevant to applications related to improvements in breeding procedures and transgenesis.

A comprehensive analysis of the chorion locus in silkmoth.

Despite more than 40 years of intense study, essential features of the silkmoth chorion (eggshell) are still not fully understood. To determine the precise structure of the chorion locus, we performed extensive EST analysis, constructed a bacterial artificial chromosome (BAC) contig, and obtained a continuous genomic sequence of 871,711 base pairs. We annotated 127 chorion genes in two segments interrupted by a 164 kb region with 5 non-chorion genes, orthologs of which were on chorion bearing scaffolds in 4 ditrysian families. Detailed transcriptome analysis revealed expression throughout choriogenesis of most chorion genes originally categorized as "middle", and evidence for diverse regulatory mechanisms including cis-elements, alternative splicing and promoter utilization, and antisense RNA. Phylogenetic analysis revealed multigene family associations and faster evolution of early chorion genes and transcriptionally active pseudogenes. Proteomics analysis identified 99 chorion proteins in the eggshell and micropyle localization of 1 early and 6 Hc chorion proteins.

Transcriptional activation of a moderately expressed tRNA gene by a positioned nucleosome.

All of the members of a tRNA1(Gly) multigene family from the mulberry silkworm, Bombyx mori, have identical coding regions and consequently identical internal promoter elements, but are transcribed at different levels. A moderately expressed copy, tRNA1(Gly)-4 from within this multigene family, which was transcribed to 30-50% of the highly transcribed gene copies harboured two typical TATAA box sequences in the 5' upstream region at positions -27 nt and -154 nt with respect to the +1 nt of mature tRNA. Deletion of the distal TATAA sequence at -154 nt brought down the transcription more than 70%, whereas mutation of the proximal element did not affect transcription. tRNA1(Gly)-4 could be readily assembled into chromatin, with a positioned nucleosome in the upstream region, and the assembled nucleosome formed stable complexes with the transcription factors TFIIIC and TFIIIB. Organization of the gene into nucleosomes also enhanced transcription significantly above that of the naked DNA, reaching transcription levels comparable with those of the highly transcribed copies. This nucleosome-mediated enhancement in transcription was absent when the distal TATAA sequences were deleted, whereas mutation of the proximal TATAA element showed no effect. In the absence of the distal TATAA sequences, assembly into the nucleosome inhibited transcription of tRNA1(Gly)-4. TFIIIB bound directly through the distal TATAA sequence at -154 nt and the positioned nucleosome facilitated its interaction with TFIIIC. The direct binding of TFIIIB to the DNA provided anchoring of the factor to the template DNA which conferred a higher stability on the TFIIIB-TFIIIC-DNA complex. We have proposed a novel mechanism for the nucleosome-mediated stimulation of pol III (RNA polymerase III) transcription of tRNA genes, a model not presented previously.

Transcription of individual tRNA1Gly genes from within a multigene family is regulated by transcription factor TFIIIB.

Members of a multigene family from the silkworm Bombyx mori have been classified based on their transcriptions in homologous nuclear extracts, into three groups of highly, moderately and poorly transcribed genes. Because all these gene copies have identical coding sequences and consequently identical promoter elements (the A and B boxes), the flanking sequences modulate their expression levels. Here we demonstrate the interaction of transcription factor TFIIIB with these genes and its role in regulating differential transcriptions. The binding of TFIIIB to the poorly transcribed gene -6,7 was less stable compared with binding of TFIIIB to the highly expressed copy, -1. The presence of a 5' upstream TATA sequence closer to the coding region in -6,7 suggested that the initial binding of TFIIIC to the A and B boxes sterically hindered anchoring of TFIIIB via direct interactions, leading to lower stability of TFIIIC-B-DNA complexes. Also, the multiple TATATAA sequences present in the flanking regions of this poorly transcribed gene successfully competed for TFIIIB reducing transcription. The transcription level could be enhanced to some extent by supplementation of TFIIIB but not by TATA box binding protein. The poor transcription of -6,7 was thus attributed both to the formation of a less stable transcription complex and the sequestration of TFIIIB. Availability of the transcription factor TFIIIB in excess could serve as a general mechanism to initiate transcription from all the individual members of the gene family as per the developmental needs within the tissue.

Modulation of differential transcription of tRNA genes through chromatin organization.

In higher eukaryotes, tRNA multigene families comprise several copies encoding the same tRNA isoacceptor species. Of the 11 copies of a tRNA1Gly family from the mulberry silkworm Bombyx mori, individual members are differentially transcribed in vivo in the B. mori-derived BmN cell lines and in vitro in silk gland nuclear extracts. These genes have identical coding regions and hence harbour identical internal control sequences (the A and B boxes), but differ significantly in their 5' and 3' flanking regions. In the present study, we demonstrate the role of chromatin structure in the down-regulation of the poorly expressed copy, tRNA1Gly-6,7. Distinct footprints in the 5'-upstream region of the poorly transcribed gene in vitro as well as in vivo suggested the presence of nucleosomes. A theoretical analysis of the immediate upstream sequence of this gene copy also revealed a high propensity of nucleosome formation. The low transcription of tRNA1Gly-6,7 DNA was further impaired on assembly into chromatin and this inhibition was relieved by externally supplemented TFIIIC with an associated histone acetyltransferase activity. The inhibition due to nucleosome assembly was absent when the 5'-upstream region beyond -53 nt was deleted or entirely swapped with the 5'-upstream region of the highly transcribed gene copy, which does not position a nucleosome. Footprinting of the in vitro assembled tRNA1Gly-6,7 chromatin confirmed the presence of a nucleosome in the immediate upstream region potentially masking TFIIIB binding. Addition of TFIIIC unmasked the footprints present on account of the nucleosome. Our studies provide the first evidence for nucleosomal repression leading to differential expression of individual members from within a tRNA multigene family.

Characterization of a cyclin homolog from Bombyx mori nucleopolyhedrovirus.

We have identified and characterized a cyclin homolog from Bombyx mori nucleopolyhedrovirus (BmNPV), encoding a 34 kDa protein (ORF 120) with 48% homology to the host Bombyx mori cellular cyclin B. The expression of the viral cyclin (v-cyc) was detected from 12 h following virus infection and the maximum transcript levels were seen at 24-36 h. The transcription start site mapping of v-cyc revealed the presence of a transcript initiating from a TAAG motif located 13 nucleotide (nt) upstream of the ORF as well as longer transcripts initiating from farther upstream region and encompassing the preceding ORF 119. The transcription was terminated at 15 nt downstream of the ORF 120. The expression of the host cellular cyclin B declined following virus infection and the transcript disappeared almost completely by 24 h even as the expression of v-cyc reached high levels. The synthesis of the viral cyclin was detected at 36-48 h post-infection. The viral cyclin in association with other host or viral proteins catalysed phosphorylation of histone H1. The host cells were arrested in G2/M phase following virus infection and thus, the virus cyclin in association with other proteins maintains the host cells at the G2/M phase while permitting the virus DNA replication.

Comparative analysis of the development of the mandibular salivary glands and the labial silk glands in the mulberry silkworm, Bombyx mori.

The mulberry silkworm, Bombyx mori has a pair of salivary glands arising from the mandibular segment, in addition to the labial silk glands which are generally considered as modified salivary glands. Here we report the characterization of salivary glands and the comparative gene expression profiling of the silk and salivary glands. The two independent salivary glands made up by 330 cells, grow about 1000 fold during larval development. These individual glands extend up to the T(1) thoracic segment unlike silk glands with fused anterior ends and extending up to the caudal region. The salivary glands also undergo endomitosis resembling the silk glands. The B. mori homologue of the homeotic gene Deformed (BmDfd) was expressed in the mandibular and maxillary segments in stage 17 embryo and got localized to the centre of the mandibular segment at stage 18 to form the salivary gland placodes. The expression was also seen in the distal ends of the leg appendages after blastokinesis (stage 22). Only low variations in BmDfd expression ranging from 1.6 to 2.1 fold were apparent during embryonic development. BmDfd expression was observed in the salivary glands all through the larval instars but not in the silk glands. The transcription factor, Forkhead and the segment polarity gene, Wingless were expressed throughout the salivary glands, the latter confirming the absence of physiological compartmentation within these glands unlike the silk glands. The expression of Amylase and Fibrohexamerin was restricted to the salivary and silk glands, respectively and therefore, served as molecular markers for these tissues.

Systemic and in vitro infection process of Bombyx mori nucleopolyhedrovirus.

To analyse the systemic progression of infection by Bombyx mori nucleopolyhedrovirus (BmNPV) through oral ingestion by the silkworm larvae, a recombinant virus (vBmp10GFP) expressing the green fluorescent protein (GFP) under the control of the very late, viral p10 promoter (which still forms the polyhedral occlusion bodies) was constructed. Infection of B. mori derived BmN cells with the recombinant virus resulted in the expression of GFP from 12 h post infection (hpi), with maximal accumulation of the expressed protein by 60 hpi. B. mori larvae that ingested the polyhedra containing vBmp10GFP showed localized expression of GFP in the midgut epithelial cells within 24 hpi, indicating virus replication. The primary spread of the virus infection occurred through the tracheae. Viral multiplication was subsequently detected in nearly all the larval tissues including the neurons and regions of silk-glands that were in contact with the tracheae. Infection in fat bodies was widespread by 48 hpi, by which time the haemocytes also showed infection. In vitro infection of isolated organs/tissues from B. mori with the budded virions (BV) of vBmp10GFP also showed viral multiplication in the cells that were associated with the tracheae, confirming the role of tracheae in spreading the infection.

Baculovirus display of fusion protein of Peste des petits ruminants virus and hemagglutination protein of Rinderpest virus and immunogenicity of the displayed proteins in mouse model.

Recombinant Bombyx mori nucleopolyhedroviruses (BmNPV) displaying the immunodominant ectodomains of fusion glycoprotein (F) of Peste des petitis ruminants virus (PPRV) and the hemagglutinin protein (H) of Rinderpest virus (RPV), on the budded virions as well as the surface of the infected host cells have been constructed. The F and H protein sequences were inserted in-frame within the amino-terminal region of BmNPV envelope glycoprotein GP64 expressing under the strong viral polyhedrin (polh) promoter. We improved the recombinant virus selection in BmNPV by incorporating the green fluorescent protein gene (gfp) as selection marker under a separate promoter within the transfer cassette harboring the desired genes. Following infection of the insect larvae or the host-derived BmN cells with these recombinant BmNPVs, the expressed GP64 fusion proteins were displayed on the host cell surface and the budded virions. The antigenic epitopes of the recombinant proteins were properly displayed and the recombinant virus particles induced immune response in mice against PPRV or RPV.

Bombyx mori nucleopolyhedrovirus-based surface display system for recombinant proteins.

We describe here the development of a 'eukaryotic display system' for heterologous proteins on the viral and host cell surfaces using Bombyx mori nucleopolyhedrovirus (BmNPV). The reporter gene gfp (green fluorescent protein) was fused to either the gp64 gene encoding the full-length BmNPV envelope protein GP64 or to its 5' region encoding only the N-terminal domain harbouring the signal sequence, and recombinant viruses expressing the corresponding fusion proteins under the strong viral polyhedrin promoter were generated. On infection of the host insect B. mori or the host-derived BmN cells with the full-length GP64-GFP virus, abundant expression of the recombinant protein and its display on the cell surface were achieved. The fusion protein was also a component of the budded virions. Thus, the BmNPV-based display system provides an alternative to the previously established Autographa californica multinucleocapsid nucleopolyhedrovirus display system. The recombinant virus expressing GFP has also been used in preliminary pathological investigations on virus infection in B. mori and provides a simple method for screening for antiviral agents.

Characterization of the gene encoding the envelope fusion glycoprotein GP64 from Bombyx mori nucleopolyhedrovirus.

We describe here the characterization of the gene gp64 encoding the envelope fusion protein GP64 (open reading frame) ORF 105 from Bombyx mori nucleopolyhedrovirus (BmNPV). gp64 was transcribed from the early to late stages of infection and the transcripts were seen from 6 to 72 h post infection (hpi). The early transcripts initiated from a consensus CAGT motif while the late transcripts arose from three conserved TAAG motifs, all of which were located in the near upstream region of the coding sequence. Both early and late transcripts terminated at a run of T residues following the second polyadenylation signal located 31 nt downstream of the translation termination codon. BmGP64 protein was detectable from 6 hpi and was present in larger quantities throughout the infection process from 12 hpi, in BmNPV-infected BmN cells. The persistent presence of GP64 in BmN cells differed from the protein expression pattern of GP64 in Autographa californica multinucleocapsid nucleopolyhedrovirus infection, where the protein levels decreased significantly by late times (48 hpi). BmGP64 was located in the membrane and cytoplasm of the infected host cells and as a component of the budded virions. The production of infectious budded virus and the fusion activity were reduced when glycosylation of GP64 was inhibited.

Analysis of host specificity of two closely related baculoviruses in permissive and nonpermissive cell lines.

The baculoviruses Bombyx mori nucleopolyhedrovirus (BmNPV) and Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) share about 90% identity at the genomic level but they have non-overlapping host range and show a high degree of host specificity. We have demonstrated here that AcMNPV undergoes DNA replication and early gene expression in Bombyx-derived BmN cells but fails to show very late gene expression or produce budded virion (BV) particles. Coinfection with BmNPV supported BV production from AcMNPV in BmN cells at low levels but not very late gene expression or polyhedral inclusion body formation. BV production and very late gene expression from BmNPV, on the contrary, were adversely affected in coinfections. In Spodoptera frugiperda-derived Sf21 cell lines, BmNPV DNA replication, BV production, and very late gene expression took place only when coinfected with AcMNPV. BmNPV exerted a less profound effect on AcMNPV multiplication and very late gene expression in permissive host cell lines. AcMNPV shuts down cellular and viral protein synthesis completely when infected alone or coinfected with BmNPV in BmN cells, whereas BmNPV infection did not affect cellular and viral protein synthesis in Sf21 cells. Overall, AcMNPV showed a more dominant effect by complementing the multiplication of BmNPV in nonpermissive host cells while inhibiting it in BmN cells.

Temporal expression profile of late gene expression factor 4 from Bombyx mori nucleopolyhedrovirus.

Temporal expression profile of lef4, the gene encoding late gene expression factor 4 (LEF4) from the baculovirus, Bombyx mori nucleopolyhedrovirus (BmNPV), has been analysed. lef4 behaved like an early gene and the transcripts were detectable from 6 h post infection (hpi) which reached maximal levels by 18-24 hpi, and declined considerably at later times. The LEF4 open reading frame was bacterially expressed as a glutathione S-transferase (GST) fusion protein which was solubilized from the inclusion bodies and purified by adsorption to the affinity matrix, GST-Sepharose. Using polyclonal antibodies raised against the bacterially expressed protein, the temporal profile of LEF4 synthesis in BmNPV-infected BmN cells was analysed. The LEF4 protein levels were also higher at 24 hpi compared to 12 or 36 hpi, correlating with the RNA patterns. The protein was predominantly localized to the nucleus of the infected BmN cell and only a small portion was present in the cytosolic fraction. Preliminary studies with antisense lef4 expression revealed substantial reduction in expression from the viral polyhedrin promoter without significantly affecting the viral DNA replication.

Functional characterization and structural modelling of late gene expression factor 4 from Bombyx mori nucleopolyhedrovirus.

Late gene expression factor 4 (LEF4), a multifunctional protein encoded by the Bombyx mori nucleopolyhedrovirus has been bacterially expressed and characterized. Sequence analyses and three-dimensional modelling of B. mori LEF4 showed that the protein is related to mRNA-capping enzymes, which are organized as two modular domains. Most of the acidic side chains in LEF4 were solvent-exposed and spread all along the fold. A region dominated by negatively charged groups, which protrudes from the larger domain was ideally suited for interactions with proteins having positively charged patches at the surface. The purified LEF4 protein exhibited different enzyme activities associated with mRNA-capping enzymes, i.e. GTP-binding, RNA triphosphatase and guanylate transferase activities. In addition, LEF4 also showed NTP-hydrolysing activity. The kinetic analysis of ATP hydrolysis revealed a sigmoidal response with two deduced binding sites for ATP, whereas the guanylate transferase activity showed a typical hyperbolic response to varying concentrations of GTP with a Km of 330+/-20 microM. Analysis of the modelled three-dimensional structure of LEF4 suggested the presence of crucial residues in sequence motifs important for the integrity of the fold. Mutation of one such conserved and buried tyrosine residue to cysteine in the motif IIIa, located close to the interlobe region of the model, resulted in a 44% loss of guanylate transferase activity of LEF4 but had no effect on the ATPase activity.

Characterization of late gene expression factors lef-9 and lef-8 from Bombyx mori nucleopolyhedrovirus.

Late gene expression factors, LEF-4, LEF-8, LEF-9 and P47 constitute the primary components of the Autographa californica multinucleocapsid polyhedrovirus (AcMNPV)-encoded RNA polymerase, which initiates transcription from late and very late promoters. Here, characterization of lef-9 and lef-8, which encode their corresponding counterparts, from Bombyx mori NPV is reported. Transcription of lef-9 initiated at two independent sites: from a GCACT sequence located at -38 nt and a CTCTT sequence located at -50 nt, with respect to the +1 ATG of the open reading frame. The 3' end of the transcript was mapped to a site 17 nt downstream of a canonical polyadenylation signal located 7 nt downstream of the first of the two tandem translational termination codons. Maximum synthesis of LEF-9 was seen from 36 h post-infection (p.i.). The transcription of lef-8 initiated early in infection from a GTGCAAT sequence that differed in the corresponding region from its AcMNPV counterpart (GCGCAGT), with consequent elimination of the consensus early transcription start site motif (underlined). Peak levels of lef-8 transcripts were attained by 24 h p.i. Immunocopurification analyses suggested that there was an association between LEF-8 and LEF-9 in vivo.

A novel TATA-box-binding factor from the silk glands of the mulberry silkworm, Bombyx mori.

The presence of one or more TATATAA motifs in the flanking sequences of individual members of a multi-gene tRNA(Gly)(1) family from the mulberry silkworm, Bombyx mori, negatively modulated the transcription of the gene copies. Characterization of proteins from posterior silk gland nuclear extracts, binding to the TATATAA motif, identified a novel 43 kD protein, designated here as P43 TATA-box-binding factor (TBF). The protein was purified to homogeneity. P43 TBF binding was highly sequence-specific and showed a 100-fold-higher affinity for binding than the TATA-box-binding protein (TBP). The protein also showed binding to the TATAAA sequence of the actin5C promoter. P43 TBF inhibited transcription of all the tRNA genes examined, as well as RNA polymerase II transcription from the actin5C promoter. The amino acid sequence of eleven peptides generated from P43 TBF did not share homology with proteins that bind the TATA box, such as TBP, TRF (TBP-related factor) or TLFs (TBP-like factors) reported from other sources. Inhibition of transcription of tRNA genes by P43 TBF could not be reversed by TBP. The inhibitory effect appeared to be exerted through sequestration of the associated transcription factors.

Characterization of RNA polymerase III transcription factor TFIIIC from the mulberry silkworm, Bombyx mori.

Fractionation of nuclear extracts from posterior silk glands of mulberry silkworm Bombyx mori, resolved the transcription factor TFIIIC into two components (designated here as TFIIIC and TFIIIC1) as in HeLa cell nuclear extracts. The reconstituted transcription of tRNA genes required the presence of both components. The affinity purified TFIIIC is a heteromeric complex comprising of five subunits ranging from 44 to 240 kDa. Of these, the 51-kDa subunit could be specifically crosslinked to the B box of tRNA1Gly. Purified swTFIIIC binds to the B box sequences with an affinity in the same range as of yTFIIIC or hTFIIIC2. Although an histone acetyl transferase (HAT) activity was associated with the TFIIIC fractions during the initial stages of purification, the HAT activity, unlike the human TFIIIC preparations, was separated at the final DNA affinity step. The tRNA transcription from DNA template was independent of HAT activity but the repressed transcription from chromatin template could be partially restored by external supplementation of the dissociated HAT activity. This is the first report on the purification and characterization of TFIIIC from insect systems.

Identification of an enhancer-like element in the polyhedrin gene upstream region of Bombyx mori nucleopolyhedrovirus.

A series of deletions in the upstream region of the gene encoding polyhedrin (polh) of Bombyx mori nucleopolyhedrovirus (BmNPV) were generated in plasmid constructs and tested for transcription. In transient transfection assays in Bombyx mori-derived BmN cells with firefly luciferase as the reporter gene, a 293 bp fragment located 1.0 kb upstream with respect to the +1 ATG of polh showed 10-fold enhancement in expression from the minimal promoter. This increase in reporter activity was observed only when the fragment was positioned in cis with respect to the promoter and not in trans. The stimulation of reporter gene expression was independent of the orientation of the fragment and was due to increased transcription from the promoter. When placed upstream of another promoter, the viral very late gene p10 promoter, the enhancer brought about a 2-fold increase in expression. The region encompassing the enhancer was itself transcriptionally active, and transcripts corresponding to both of the encoded ORFs (N-terminal regions of ORF453 and ORF327, located in opposite orientations) were detected. Two AP1 sites (TGACTCG) in the 293 bp fragment did not appear to contribute to the enhancer function. Since repeat motifs, the hallmark of conventional enhancer sequences, were absent from this fragment, it is designated as an enhancer-like element. The influence of this region of the polh upstream sequence on expression from strong, very late viral promoters has not been reported previously.