Skip to main content
NCBI home page
The Journal of General Virology logoLink to The Journal of General Virology
. 2009 May;90(Pt 5):1270–1280. doi: 10.1099/vir.0.009613-0

Analysis of transcripts from predicted open reading frames of Musca domestica salivary gland hypertrophy virus

Tamer Z Salem 1,2, Alejandra Garcia-Maruniak 1, Verena-U Lietze 1, James E Maruniak 1, Drion G Boucias 1
PMCID: PMC2887563  PMID: 19264592

Abstract

The Musca domestica salivary gland hypertrophy virus (MdSGHV) is a large dsDNA virus that infects and sterilizes adult houseflies. The transcriptome of this newly described virus was analysed by rapid amplification of cDNA 3′-ends (3′-RACE) and RT-PCR. Direct sequencing of 3′-RACE products revealed 78 poly(A) transcripts containing 95 of the 108 putative ORFs. An additional six ORFs not amplified by 3′-RACE were detected by RT-PCR. Only seven of the 108 putative ORFs were not amplified by either 3′-RACE or RT-PCR. A series of 5′-RACE reactions were conducted on selected ORFs that were identified by 3′-RACE to be transcribed in tandem (tandem transcripts). In the majority of cases, the downstream ORFs were detected as single transcripts as well as components of the tandem transcripts, whereas the upstream ORFs were found only in tandem transcripts. The only exception was the upstream ORF MdSGHV084, which was differentially transcribed as a single transcript at 1 and 2 days post-infection (days p.i.) and as a tandem transcript (MdSGHV084/085) at 2 days p.i. Transcriptome analysis of MdSGHV detected splicing in the 3′ untranslated region (3′-UTR) and extensive heterogeneity in the polyadenylation signals and cleavage sites. In addition, 23 overlapping antisense transcripts were found. In conclusion, sequencing the 3′-RACE products without cloning served as an alternative approach to detect both 3′-UTRs and transcript variants of this large DNA virus.

INTRODUCTION

Salivary gland hypertrophy viruses (SGHVs) have been detected in several dipterans, including the house fly (Musca domestica), tsetse fly (Glossina spp.), and the narcissus bulb fly (Merodon equestris) (Amargier et al., 1979; Coler et al., 1993; Gouteux, 1987; Jaenson, 1978; Minter-Goedbloed & Minter, 1989; Otieno et al., 1980; Shaw & Moloo, 1993). Infection by these viruses both induces SGH and reduces reproductive fitness (Lietze et al., 2007; Sang et al., 1998, 1999). Currently, our lab is using the M. domestica SGHV (MdSGHV) to study the replicative pathway and mode of action of this unique virus group. Significantly, MdSGHV is capable of pervasive development in adult salivary glands; 100 % of the glands display SGH and release copious levels of infectious virus between 48 and 72 h post-injection (V. U. Lietze & D. G. Boucias, unpublished results). The synchronized infection displayed by this virus and the access to virus-free house fly colonies provides an inexpensive model to elucidate the in vivo biology of this dsDNA animal virus.

The MdSGHV genome is 124 279 bp long and has a total of 108 putative ORFs (Garcia-Maruniak et al., 2008). Comparative analysis of the genome sequences has shown that the MdSGHV is closely related to the tsetse fly virus, Glossina pallidipes SGHV (GpSGHV): both SGHVs form a monophyletic clade distinct from other circular dsDNA insect viruses (Garcia-Maruniak et al., 2009). To date, no transcriptional studies have been published on this group of viruses. Prior to initiating detailed studies on the functional aspects of the MdSGHV transcriptome, it is necessary to determine which of the putative MdSGHV ORFs are transcribed in the infected house fly. Therefore, the major goals of this study were to validate the 108 putative ORFs and to analyse the 3′-untranslated regions (3′-UTRs) of the MdSGHV transcripts. In other viruses, the 3′-UTR influences the translation and stability of mRNA (Barksdale & Baker, 1995; Goraczniak & Gunderson, 2008; Graham, 2008; Schwartz, 2008; van Oers et al., 1999; Weil & Beemon, 2006). The analysis of the 3′-UTRs of the MdSGHV transcripts may serve as a framework to assess complexity of the 3′-UTRs of other animal viruses.

METHODS

House fly infection and tissue preparation.

House fly pupae were obtained from the insecticide-susceptible ‘Orlando Normal’ colony maintained at the Center for Medical, Agricultural, and Veterinary Entomology (CMAVE), United States Department of Agriculture–Agricultural Research Service (USDA–ARS) in Gainesville, Florida. Pupae were transferred to 72-ounce plastic jars provisioned with deionized water and maintained under constant conditions (26 °C, 12 h light : 12 h dark, 40 % relative humidity). Newly emerged adults received injections of filter-sterilized viraemic salivary gland homogenate, as described by Lietze et al. (2007). Briefly, one infected-gland-pair equivalent (IGE) was dissected from an infected house fly, homogenized in 0.5 ml sterile saline solution (0.85 % NaCl), filtered through a 0.45 μm filter unit, and stored in aliquots at −35 °C until used for injection. Each fly received 2.5 μl viral inoculum at a final dosage of 2.5×10−5 IGE per fly by injection. Flies were maintained under constant conditions and provided with food (a 6 : 6 : 1 mixture of powdered milk, sucrose and dried egg) and water ad libitum. At different times post-injection, house flies were immobilized on ice and salivary glands were dissected into Tri-Reagent (Sigma) and either processed immediately or frozen at −70 °C.

RNA extraction from glands.

Salivary glands (100 mg) were dissected from MdSGHV-infected house flies at 5 days post-infection (days p.i.). Gland preparations were homogenized in 1 ml aliquots of Tri-Reagent and processed to extract total RNA, according to the manufacturer's protocol. Ethanol-precipitated RNA pellets were suspended in 100 μl DEPC-treated water. Samples (5 μg) of the extracted total RNA were digested with 3 U RQ1 RNase-Free DNase (Promega). Enzymic activity was stopped by adding 2 μl 20 mM EGTA, pH 8.0 and incubation at 65 °C for 10 min. The presence of contaminating DNA was assessed using RT-PCR with the universal 28S primers (forward primer 5′-GTTAAGCCCGATGAACCTGA-3′, reverse primer 5′-GACTCCTTGGTCCGTGTTTC-3′). The reaction mix was divided into two 0.2 ml PCR tubes. To one tube, avian myeloblastosis virus (AMV) reverse transcriptase (2 U) was added and the second tube received water instead (negative control). The RT-PCR program was: 45 °C for 1 h, 70 °C for 15 min and 94 °C for 3 min, then 35 cycles of 94 °C for 1 min, 55 °C for 1 min and 72 °C for 2 min and an extension step at 72 °C for 7 min. Products were electrophoresed on agarose gels and stained with ethidium bromide to check for the 28S product to determine the presence or absence of DNA contamination in the RNA preparations.

3′-RACE.

cDNA preparations were synthesized from the mRNA of MdSGHV-infected house fly salivary glands using the oligo-dT-based primer (AP) from the 3′-RACE System for Rapid Amplification of cDNA Ends (Invitrogen). For a 20 μl reaction, 5 μg DNase-treated infected gland RNA and 10 μM AP were heated to 70 °C for 10 min, incubated on ice, and centrifuged briefly. To this mixture was added 2 μl 10× PCR buffer [200 mM Tris/HCl (pH 8.4), 500 mM KCl], 2 μl 25 mM MgCl2, 1 μl 10 mM dNTP mix, and 2 μl 0.1 M DTT. Samples were incubated at 42 °C for 5 min, after which 1 μl (200 U μl−1) SuperScript II reverse transcriptase was added, and the reaction was again incubated at 42 °C for 55 min. The cDNA was amplified using the Abridged Universal Amplification Primer (AUAP) in combination with forward-ORF-specific primers designed for each of the 108 MdSGHV putative ORF sequences. The forward primers were designed to anneal approximately 400 bp upstream of the stop codon (see Supplementary Table S1, available with the online version of this paper). PCR was performed in a total volume of 20 μl by mixing 13 μl ddH2O, 1.2 μl 25 mM MgCl2, 0.4 μl 10 mM dNTPs, 0.2 μl of the first strand cDNA synthesis reaction, 0.5 μl 10 μM AUAP, 0.5 μl 10 μM ORF-specific forward primer, 4 μl 5× GoTaq Flexi buffer, and 0.2 μl GoTaq Hot Start polymerase (5 U μl−1; Promega). A touchdown PCR program with the following settings was used: 94 °C for 5 min, and 35 cycles of 94 °C for 1 min, 60 °C for 1 min (decreasing by 0.5 °C every three cycles) and 72 °C for 2 min, followed by 72 °C for 7 min. The 3′-RACE products from samples showing discrete bands on ethidium bromide-stained agarose gels were purified with the QIAquick PCR Purification kit (Qiagen) and subjected to Sanger sequencing with the forward primers.

5′-RACE.

5′-RACE reactions were conducted on selected ORFs that were identified by 3′-RACE to be transcribed in tandem. First-strand cDNA synthesis was performed using DNase-treated RNA according to the manual of the SMART RACE cDNA amplification kit (Clontech). A series of reactions were conducted with Advantage 2 polymerase mix (Clontech) using the UPM primer (Clontech manual) in combination with an ORF-specific reverse primer (∼300 bp downstream of the start codon, see Supplementary Table S2, available with the online version of this paper). A PCR program with the following settings was used: 94 °C for 3 min, and 30 cycles of 94 °C for 30 s, 68 °C for 30 s and 72 °C for 3 min, followed by 72 °C for 7 min. The PCR products were purified and sequenced using their respective gene-specific reverse primers.

RT-PCR.

Reactions were conducted following the instruction manual of the Access RT-PCR kit (Promega). Each reaction was performed in 20 μl total volume with 12.2 μl DEPC-treated water, 0.4 μl 10 mM dNTPs, 4 μl AMV/Tfl 5× reaction buffer, 0.5 μl 10 μM of each forward and reverse gene-specific primer (see Supplementary Table S3, available with the online version of this paper), 1.2 μl 25 mM MgSO4, 0.4 μl Tfl DNA polymerase (5 U μl−1), 0.4 μl DNase-treated total RNA (∼0.1 μg), and 0.4 μl AMV reverse transcriptase (5 U μl−1). The program used was: 45 °C for 1 h, 70 °C for 15 min and 94 °C for 3 min, then 35 cycles of 94 °C for 1 min, 60 °C for 1 min (decreasing by 0.5 °C every three cycles), and 72 °C for 2 min. This was followed by 72 °C for 7 min. Control reactions targeted the 28S gene of M. domestica and the viral ORF MdSGHV037. All products were sequenced with each of the gene-specific primers used for the RT-PCR amplifications.

Data analysis.

The sequences obtained from the 3′-RACE products were aligned against the MdSGHV genome sequence using the SeqMan program (dnastar, Lasergene). All 3′-RACE sequences were trimmed after the first A of the poly(A) tail. Examination of sequencing chromatograms showed one or more possible terminations for most of the transcripts. A library of sequencing files of the trimmed 3′-RACE sequences and each of the 108 putative ORF sequences described for MdSGHV (Garcia-Maruniak et al., 2008) was generated using the EditSeq program (dnastar, Lasergene), and sequences were aligned along with the complete MdSGHV genome sequence to detect the 3′-UTR of each transcript. Each 3′-RACE sequence was opened with Artemis v. 10 software (Rutherford et al., 2000). Frames displaying transcripts of viral ORFs were compared with the DNA sequence of those same ORFs. If a particular ORF of the 3′-RACE sequence matched the viral ORF sequence, that ORF was considered validated, and its sequence, including the 3′-UTR, was added as a separate data file for further analysis. In few cases, the 3′-RACE sequences showed possible alternative stop codons different from the ones in the putative ORFs. In these cases, the whole original ORF sequence, plus 200 bp upstream of the putative start codon, and the 3′-RACE sequence were aligned and examined again with ORF Finder to determine if there was an alternate ORF in that location.

RESULTS AND DISCUSSION

Validation of MdSGHV ORFs

In order to identify transcripts and examine the expression of each of the ORFs in the MdSGHV genome, forward primers (∼400 bp upstream of the putative stop codon) specific to each of the 108 predicted ORFs were synthesized. Each primer was used in combination with the AUAP primer, which binds to the adaptor–primer region introduced during first-strand cDNA synthesis, to amplify the 3′-end of each ORF-specific cDNA. Ethidium bromide-stained gels containing different 3′-RACE reactions produced a range of intense to undetectable bands. This variation may reflect either differences in the efficiency of the PCRs, or differences in the spatial and temporal viral transcription events. The absence of 3′-RACE products suggests that the putative ORFs predicted by Garcia-Maruniak et al. (2008) were not expressed, were expressed at different times other than at 5 days p.i., or were degraded. The majority of the putative MdSGHV ORFs were validated by directly sequencing their respective 3′-RACE products (see Supplementary Fig. S1, available with the online version of this paper). However, examination of the chromatograms demonstrated that 31 3′-RACE sequences (indicated in Table 1) displayed more than one cleavage site (CS), generating transcripts with different lengths.

Table 1.

Heterogeneity of polyadenylation signals (PS) and cleavage sites (CS) found in MdSGHV

graphic file with name 1270tbl1.jpg

Open in a new tab

*ORF numbers were taken from Garcia-Maruniak et al. (2008); numbers in parentheses correspond to contiguous ORF(s) transcribed in tandem.

†Commas separate information originating from more than one transcript variant; the underlined number indicates the cleavage site (CS) of the most abundant transcript variant.

‡Underlining indicates PS fusion; the number in parentheses corresponds to the number of similar PSs detected; bold type indicates the non-canonical PS; shaded areas indicate which PS was used to measure the distance between PS and CS whenever the first PS was not used.

§Bold type indicates the last nucleotide in the transcript, before the poly(A) tail, homologous to the MdSGHV genome sequence.

||3′-UTR length of all possible transcript variants detected.

¶Indicates transcripts with 3′-UTRs that have an AUUUA motif (reported to increase mRNA degradation).

#Indicates transcripts with 3′-UTRs that have a UGUA motif (CFIm binding motif).

**Indicates a stop codon located within the polyadenylation signal (PS).

††Indicates a stop codon located downstream of the PS.

Sequencing the 3′-RACE products identified a total of 78 poly(A) transcripts that contained both single and tandem combinations of 95 ORFs of the 108 putative MdSGHV ORFs (Fig. 1; Garcia-Maruniak et al., 2008). Two 3′-RACE sequences (MdSGHV075 and MdSGHV076) produced non-resolvable sequences at their 3′-ends; however, they displayed poly(A) tails and sequences identical to their corresponding genomic ORFs, therefore, they were considered to be validated ORFs. Of the 13 ORFs not detected by 3′-RACE, six were validated by RT-PCR using internal gene-specific primers. Seven putative ORFs (MdSGHV006, 007, 009, 041, 059, 060 and 080) were not detected by 3′-RACE or by RT-PCR (Fig. 1). Six of the non-detected ORFs were relatively short ORFs and, among them, five were adjacent to regions containing direct repeats.

Fig. 1.

Fig. 1.

Open in a new tab

MdSGHV genome representation [modified from Garcia-Maruniak et al. (2008)] showing location, size and transcriptional direction of 108 putative ORFs. Black arrows indicate ORFs validated by sequencing the 3′-RACE products for which the 3′-UTRs were analysed. Dotted arrows indicate ORFs validated only by RT-PCR of their transcripts and for which no 3′-UTR analysis was possible. White arrows indicate ORFs with no detectable transcripts in infected salivary glands at 5 days p.i. ORFs that were detected as tandem transcripts are inside boxes.

MdSGHV ORFs transcribed in tandem

A total of 34 putative ORFs were transcribed in tandem. The 3′-RACE sequences of the respective upstream and downstream ORFs showed that both transcripts co-terminated at the same 3′-end. Fourteen transcripts contained two adjacent ORFs and two transcripts contained three adjacent ORFs (Fig. 2). Sequence analysis of selected 5′-RACE products (see Supplementary Fig. S2, available with the online version of this paper) of the upstream and/or downstream ORFs transcribed in tandem (MdSGHV004, MdSGHV024 + MdSGHV025 + MdSGHV026, MdSGHV032, MdSGHV054, MdSGHV055 + MdSGHV056, MdSGHV058, MdSGHV070 + MdSGHV071, MdSGHV076, MdSGHV084 + MdSGHV085, MdSGHV088 + MdSGHV089, MdSGHV091, MdSGHV100, and MdSGHV102) showed that the downstream ORFs (underlined) were transcribed both individually and in tandem with their respective upstream ORFs. Notably, in the majority of the tandem transcripts, the upstream ORFs (except for MdSGHV084) were not detected as single transcripts. The 5′-UTRs were identified as the sequence between the start codon and the SMART II A oligonucleotide anchor (Clontech). Upstream of this 5′-anchor region were sequences identical to the upstream ORFs of the tandem transcripts. The 5′-UTRs ranged from three to 205 bases (Supplementary Fig. S2). Previously, adjacent genes transcribed in tandem have been detected in several insect baculoviruses (Friesen & Miller, 1985; Gross & Rohrmann, 1993; Passarelli & Guarino, 2007). In these reported cases, the downstream ORFs are transcribed either individually or in tandem with their upstream ORFs, whereas the upstream ORFs are only transcribed in tandem. However, the downstream ORFs are translated only from the individual transcripts and not from the tandem transcript. Kaposi's sarcoma-associated herpesvirus (KSHV) also transcribes the downstream ORFs 57 and 58 individually and in tandem (Graham, 2008; Majerciak et al., 2006; Schwartz, 2008; Taniguchi & Yasumoto, 1990; Zheng & Baker, 2006). However, in KSHV, the downstream ORFs of the tandem transcript are translated using mechanisms such as alternative transcriptional start sites, alternative splicing, and internal ribosome entry sites (IRES) (Bieleski & Talbot, 2001; Dittmer et al., 1998; Lin et al., 1999). In MdSGHV, it is not known if the downstream ORFs are translated from only their individual transcripts.

Fig. 2.

Fig. 2.

Open in a new tab

Intergenic sequences between ORFs transcribed in tandem. The termination codons (UAA, UGA or UAG) of the first ORF and the initiation codon (AUG) of the second ORF are presented in bold type with the intergenic region underlined. In several cases (MdSGHV011–010, MdSGHV025–026 and MdSGHV054–053) adjacent ORFs overlapped, and the start codon (italicized AUG) of the second ORF was located upstream of the termination codon of the first ORF. The size of these regions ranged from 3 to 125 bp (indicated in parentheses after the downstream ORF). For MdSGHV025 and MdSGHV073, the numbers in parentheses embedded in the sequence indicate the size of the intergenic regions. Negative values denote the size of overlap occurring with MdSGHV011–010, MdSGHV025–026 and MdSGHV054–053. The ORFs are presented in their transcriptional orientation.

Examination of MdSGHV084 and MdSGHV085 transcripts

Analysis of the 3′-RACE products of MdSGHV084 detected the presence of two transcripts with different lengths (Fig. 3a). In this case, both the single upstream MdSGHV084 and the tandem MdSGHV084–085 transcript variants were polyadenylated, indicating that the polyadenylation signal of MdSGHV084 was read-through when the MdSGHV084–085 was transcribed (Fig. 3b). Sequencing the 5′-RACE products showed that the downstream MdSGHV085 was also transcribed as a single transcript. Hence, MdSGHV084 and MdSGHV085 were transcribed both individually and together, in tandem. Consequently, the presence of single MdSGHV084 and tandem MdSGHV084–085 transcripts was examined in DNase-treated RNA extracted from infected salivary glands dissected at 1 and 2 days p.i. using multiplex RT-PCR and RT-PCR (Fig. 3c and d). RT-PCR detected MdSGHV084–085 at 2 days p.i., but not at 1 day p.i., while both MdSGHV084 and MdSGHV085 were detected at 1 and 2 days p.i. (Fig. 3c and d). Detailed sequence analysis of the 3′-UTR of both MdSGHV084 and MdSGHV084–085 transcripts revealed that they had different polyadenylation signals (PS) (Fig. 3e). The single MdSGHV084 transcript contained a rare mammalian PS (AAUAUA), while the tandem MdSGHV084–085 transcript had the canonical PS (AAUAAA). A second PS (ATTAAA), detected less frequently in mammalian cells, was found downstream of the expected MdSGHV084 cleavage site (CS). Similarly, a downstream element (DSE) motif TTTTTGTTT followed by an ATTTA motif, reported to increase mRNA degradation (Norris et al., 1995), was predicted from the MdSGHV genome sequence, but not from the 3′-UTR sequence, downstream from the detected CS of MdSGHV084–085 (Fig. 3e). Unlike KSHV, which only transcribes the downstream ORFs of tandem pairs with adjacent ORFs (Majerciak et al., 2006), our analysis demonstrated that both MdSGHV084 and MdSGHV085 were transcribed individually in salivary glands of infected houseflies at 1 and 2 days p.i. The tandem transcript, MdSGHV084–085, was faint at 1 day p.i., but was easily detected at 2 days p.i. These observations suggest temporal expression of the MdSGHV084–085 in the infected flies.

Fig. 3.

Fig. 3.

Open in a new tab

Temporal transcription of MdSGHV084 and MdSGHV084–085. Two different polyadenylated 3′-RACE products of MdSGHV084 were detected: a short transcript corresponding to ORF 84 and a long transcript that included MdSGHV084 and MdSGHV085 (a). The relative location of the primers used to conduct the multiplex and RT-PCRs are indicated by arrows (b). Multiplex RT-PCR combining the 28S forward (F) and reverse (R) primers (controls to normalize the reaction) with primers 84F and 84R showed differential amplification of MdSGHV084; the reaction produced a faint product at 1 day p.i. and a stronger product at 2 days p.i. A second multiplex reaction with 84F, 85F and 85R primers indicated that MdSGHV084–085 was mainly transcribed at 2 days p.i. (c). RT-PCR confirmed the differential transcription of MdSGHV084 and MdSGHV084–085 at 1 and 2 days p.i. (d). Sequences of the 3′-UTRs of MdSGHV084 and MdSGHV084–085 possessed different polyadenylation signals (underlined) that overlapped their respective termination codons (bold type). A second polyadenylation signal was detected downstream of the cleavage site in MdSGHV084. The defined downstream element (DSE) motif (double underlined) and a motif related to mRNA degradation (dashed underlining) were present in MdSGHV084–085 (e). The first nucleotide in the poly(A) site is indicated by a bold capital letter.

3′-UTR splicing of ORF 45

Comparison of the MdSGHV045 3′-RACE sequence with the genomic MdSGHV045 sequence revealed that the initial 335 nt matched perfectly the last 322 bases of the genomic MdSGHV045, plus an additional 13 bases downstream of the stop codon. However, the following 262 bases of the MdSGHV045 3′-RACE product aligned perfectly with the beginning of MdSGHV043, an ORF located 847 bases downstream of MdSGHV045 (Fig. 4). The sequence obtained from the MdSGHV045 3′-RACE product indicated that the majority of MdSGHV044 had been spliced out from the MdSGHV045 mRNA, and that the splicing occurred in the 3′-UTR (Fig. 4). Sequence analysis of the spliced ends revealed the presence of the dinucleotides GU at the 5′ and AG at the 3′ splice sites, which were homologous to the consensus splice sites (Mount, 1982). However, the region flanking these specific sites differed by 1 and 2 nt at the 5′ and 3′ splice sites, respectively (Fig. 4).

Fig. 4.

Fig. 4.

Open in a new tab

Schematic representation of the splicing found in the 3′-UTR of MdSGHV045. The spliced region was mapped between the first 13 bases of the 3′-UTR and base one of the coding region. Sequence analysis of the 275 bases of the 3′-UTR indicated a splicing process that removed the majority of MdSGHV044. The nucleotides either flanking or at the ends of the spliced region were very similar or identical, respectively, to the sequences found in the U2 5′-splicing site. Black boxes represent the 3′-UTRs and white boxes the coding regions. Nucleotides in bold type and underlined indicate differences from the U2 splicing site. Nucleotides in boxes show the common intron borders (5′-GU/AG-3′). Vertical dotted lines indicate the location between the imaginary exons and the intron, although the splicing occurred in the 3′-UTR.

In general, most of the splicing events found in dsDNA viruses are located in exon/intron borders or at 5′ ends (Berget et al., 1977; Chisholm & Henner, 1988; Nasseri et al., 1982; Rixon & Clements, 1982). Although some of the reported viral 3′-UTRs possess a sequence homologous to the 5′ splice sites, none of these viral transcripts show deletions in their 3′-UTRs (Barksdale & Baker, 1995; Furth & Baker, 1991; Furth et al., 1994). In adenoviruses, known to undergo intensive splicing, the numbers and types of spliced mRNAs vary with the time of infection (reviewed by Akusjarvi, 2008). We have found that unspliced mRNA of MdSGHV045, which terminates in the middle of MdSGHV044, was also produced (data not shown). This result indicates that this splicing event may be selectively produced based on temporal or spatial conditions during MdSGHV infection.

Heterogeneity of cleavage sites (CS) and polyadenylation signals (PS)

MdSGHV transcripts showed multiple types of heterogeneity in their CSs and PSs. Approximately 40 % of the analysed MdSGHV transcripts (31 out of 77) showed CS heterogeneity. Twenty-six and four 3′-RACE products displayed variants that used two and three CSs, respectively. Only the 3′-RACE product of MdSGHV108 presented four potential variants (CSs, Table 1). Sixty-four MdSGHV transcripts had a 3′-UTR shorter than 100 bases, and three had a 3′-UTR longer than 400 bases. Eighteen transcripts, possessing relatively short 3′-UTRs, had stop codons located within their PS. Seven transcripts, also having short 3′-UTRs, had their stop codons downstream of the PS. Fifty-three transcripts had multiple PSs and 24 transcripts had a single PS. Of the 3′-UTRs analysed, 66 had a canonical PS (AAUAAA) (Table 1). The canonical PS (AAUAAA) was found 76 times in the 3′-UTRs of 66 transcripts. Less frequent and rare PSs were detected, including: AUUAAA (11 times), AAUAUA (11), AAUACA (10), AAGAAA (11), UAUAAA (8), AAUGAA (6), AGUAAA (4), CAUAAA (4), GAUAAA (4), ACUAAA (3), AAUAGA (3), and AUGAAA (1) on a total of 51 transcripts. More details about MdSGHV 3′-UTRs can be found in Table 1 and Supplementary Fig. S1.

We found it difficult to compare the MdSGHV PS/CS sequences with those of other viruses, since most of the published viral transcripts have the canonical PS (AAUAAA). However, some viral transcripts are known to use other PS variants such as AGUAAA, UAUAAA and CAUAAA (Andrews & Dimaio, 1993; Hilger et al., 1991; Klemenz et al., 1981; Nasseri et al., 1987; Silver Key & Pagano, 1997; Simonsen & Levinson, 1983). Most of the available data on PS/CS heterogeneity come from human and mammalian systems (Proudfoot, 1991; Zhao et al., 1999). Interestingly, at least half of the human genes, based on analyses of EST libraries, have multiple PSs and show variant PSs (Beaudoing et al., 2000; Iseli et al., 2002). Unlike the human 3′-UTRs that may extend for kilobases, most of the MdSGHV 3′-UTRs were short.

Sense–antisense gene pairs

Sets of transcript pairs, distributed throughout the MdSGHV genome, were found to overlap either in a unidirectional, convergent, or divergent pattern. The most common pattern was the convergent one (3′–3′ overlap), followed by unidirectional, and then the divergent (5′–5′ overlap) pattern (Table 2). Nineteen convergent pairs overlapped mainly in their 3′-UTR and ranged from two to 478 bases in length. Eleven transcript pairs were aligned in the unidirectional pattern and overlapped by 1–264 bases. The divergent pattern, detected in four transcript pairs, had 52, 71, 105, and 147 bases of overlapping ORF sequences.

Table 2.

Number of overlapping bases found between adjacent MdSGHV transcripts

The bias in unidirectional and convergent overlaps reflects the increased 3′-UTR sequence abundance generated by 3′-RACE.

ORF numbers Unidirectional Convergent Divergent
1 2 478 bases
2 *3/4 264 bases
1 *3/4 13 bases
17 18 111 bases
21 22 32 bases
22 23 120 bases
23 *24/25/26 171 bases
*24/25/26 27 9 bases
28 29 80 bases
29 30 64 bases
30 *31/32 4 bases
34 35 2 bases
37 38 45 bases
42 43 102 bases
43 44 52 bases
48 49 9 bases
49 50 147 bases
50 51 101 bases
52 *53/54 35 bases
*53/54 *55/56 43 bases
*61/62 63 23 bases
66 67 105 bases
69 *70/71 105 bases
77 78 1 base
79 81 25 bases
82 83 5 bases
86 87 2 bases
*88/89 90 65 bases
90 *91/92 71 bases
94 95 85 bases
98 99 68 bases
*100/101 *102/103 128 bases
104 105 450 bases
107 108 25 bases
Open in a new tab

*Indicates MdSGHV ORFs transcribed in tandem.

The presence of overlapping ORFs in the MdSGHV genome may in part reflect the close proximity of the putative ORFs (Garcia-Maruniak et al., 2008). Historically, gene overlap, or ‘overprinting’, was thought to be a mechanism for maximizing the coding information within genomes of limited size (Fukuda et al., 2003; Lamb & Horvath, 1991). In the MdSGHV genome, the most frequent pattern was 3′–3′ overlap of adjacent genes, a feature common to eukaryotes (Lehner et al., 2002; Shendure & Church, 2002; Veeramachaneni et al., 2004). This bias may be due to either the lack of intensive 5′-UTR sequence information or a preference for 3′–3′ overlapping sense–antisense pairs. Sun et al. (2005), in their comparative analysis of mouse and human cis-encoded natural antisense transcripts (NATs), reported that 3′–3′ sense–antisense pairs were both more frequently detected and more highly conserved than 5′–5′ pairs. The presence of both divergent and convergent gene pairs in MdSGHV suggests the potential involvement of cis-NATs in gene expression. In other systems, the production of cis-NATs has been postulated to regulate gene expression via (i) transcriptional interference or RNA polymerase collision (Shearwin et al., 2005), (ii) RNA masking (Lavorgna et al., 2005), and (iii) dsRNA-dependent mechanisms resulting in RNA interference (Carlile et al., 2008). It should be pointed out that 9 out of 23 of the 3′–3′ and 5′–5′ overlapped regions in MdSGHV extended more than 100 bases. In addition to the cis-NATs identified in this study, several pre-miRNA sequences have been predicted in the MdSGHV genome (Garcia-Maruniak et al., 2009). Potentially, both the cis and trans antisense transcripts could influence both host and/or viral gene regulation via RNA interference, methylation, RNA masking, alternative splicing, genomic imprinting, X-inactivation, RNA editing, and/or gene silencing (Carlile et al., 2008; Hastings et al., 1997, 2000; Kumar & Carmichael, 1998; Lavorgna et al., 2004; Munroe & Lazar, 1991; Vanhée-Brossollet & Vaquero, 1998).

In summary, direct and large-scale sequencing of MdSGHV 3′-RACE and RT-PCR products validated 101 of the 108 MdSGHV ORFs and provided insight into the complexity of transcription termination in this virus. In addition to identifying 78 poly(A) transcripts, we found that 16 of these transcripts contained multiple putative ORFs in tandem and detected a total of 23 pairs of cis-NATs. Furthermore, one transcript (MdSGHV045) showed splicing in the 3′-UTR. Sequencing of 3′-RACE products detected transcripts that possessed variant 3′-UTRs and many transcripts contained heterogeneity in their respective polyadenylation signals and cleavage sites. Finally, our approach of sequencing the 3′-RACE products directly without cloning demonstrated that this method is a useful approach for detection of 3′-UTRs and transcript variants of large-genome dsDNA viruses.

Supplementary Material

[Supplementary Material]
supp_90_5_1270__index.html (1.2KB, html)

Acknowledgments

We gratefully acknowledge Dr Michael E. Scharf and John Denton (University of Florida, FL, USA) for their critical comments on an earlier draft of the manuscript. We also thank Savita Shanker for her excellent technical support (University of Florida, FL, USA) and acknowledge the financial support provided by the National Institute of Health (NIAID R21 A1073501-01) and USDA/NRI (2007-35302-18127). We would like to thank Dr Chris Geden and Melissia Doyle (USDA, FL, USA) as well as Nii Sai Torto and William Kipersztok (undergraduate students, University of Florida, FL, USA) for their help in rearing the house flies.

Footnotes

Two supplementary figures, showing sequences flanking the cleavage sites of MdSGHV transcripts and the 5′-UTR sequences of selected ORFs that transcribed in tandem, and three supplementary tables, listing primers used for 3′-RACE, reverse primers used for 5′-RACE and primers used for RT-PCR, are available with the online version of this paper.

References

  1. Akusjarvi, G. (2008). Temporal regulation of adenovirus major late alternative RNA splicing. Front Biosci 13, 5006–5015. [DOI] [PubMed] [Google Scholar]
  2. Amargier, A., Lyon, J. P., Vago, C., Meynadier, G. & Veyrunes, J. C. (1979). Mise en évidence et purification d'un virus dans la prolifération monstrueuse glandulaire d'insectes. Etude sur Merodon equestris (Diptera, Syrphidae). Note. C R Seances Acad Sci D 289, 481–484 (in French). [PubMed] [Google Scholar]
  3. Andrews, E. M. & Dimaio, D. (1993). Hierarchy of polyadenylation site usage by bovine papillomavirus in transformed mouse cells. J Virol 67, 7705–7710. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Barksdale, S. K. & Baker, C. C. (1995). The human-immunodeficiency-virus type-1 rev protein and the rev-responsive element counteract the effect of an inhibitory 5′ splice-site in a 3′ untranslated region. Mol Cell Biol 15, 2962–2971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Beaudoing, E., Freier, S., Wyatt, J. R., Claverie, J. M. & Gautheret, D. (2000). Patterns of variant polyadenylation signal usage in human genes. Genome Res 10, 1001–1010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Berget, S. M., Moore, C. & Sharp, P. A. (1977). Spliced segments at 5′ terminus of adenovirus 2 late messenger-RNA. Proc Natl Acad Sci U S A 74, 3171–3175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bieleski, L. & Talbot, S. J. (2001). Kaposi's sarcoma-associated herpesvirus vCyclin open reading frame contains an internal ribosome entry site. J Virol 75, 1864–1869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Carlile, M., Nalbant, P., Preston-Fayers, K., McHaffie, G. S. & Werner, A. (2008). Processing of naturally occurring sense/antisense transcripts of the vertebrate Slc34a gene into short RNAs. Physiol Genomics 34, 95–100. [DOI] [PubMed] [Google Scholar]
  9. Chisholm, G. E. & Henner, D. J. (1988). Multiple early transcripts and splicing of the Autographa californica nuclear polyhedrosis virus IE-1 gene. J Virol 62, 3193–3200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Coler, R. R., Boucias, D. G., Frank, J. H., Maruniak, J. E., Garcia-Canedo, A. & Pendland, J. C. (1993). Characterization and description of a virus causing salivary gland hyperplasia in the housefly, Musca domestica. Med Vet Entomol 7, 275–282. [DOI] [PubMed] [Google Scholar]
  11. Dittmer, D., Lagunoff, M., Renne, R., Staskus, K., Haase, A. & Ganem, D. (1998). A cluster of latently expressed genes in Kaposi's sarcoma-associated herpesvirus. J Virol 72, 8309–8315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Friesen, P. D. & Miller, L. K. (1985). Temporal regulation of baculovirus RNA: overlapping early and late transcripts. J Virol 54, 392–400. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fukuda, Y., Nakayama, Y. & Tomita, M. (2003). On dynamics of overlapping genes in bacterial genomes. Gene 323, 181–187. [DOI] [PubMed] [Google Scholar]
  14. Furth, P. A. & Baker, C. C. (1991). An element in the bovine papillomavirus late 3′ untranslated region reduces polyadenylated cytoplasmic RNA levels. J Virol 65, 5806–5812. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Furth, P. A., Choe, W. T., Rex, J. H., Byrne, J. C. & Baker, C. C. (1994). Sequences homologous to 5′ splice sites are required for the inhibitory activity of papillomavirus late 3′ untranslated regions. Mol Cell Biol 14, 5278–5289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Garcia-Maruniak, A., Maruniak, J. E., Farmerie, W. & Boucias, D. G. (2008). Sequence analysis of a non-classified, non-occluded DNA virus that causes salivary gland hypertrophy of Musca domestica, MdSGHV. Virology 377, 184–196. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Garcia-Maruniak, A., Abd-Alla, A. M. M., Salem, T. Z., Parker, A. G., Lietze, V.-U., van Oers, M. M., Maruniak, J. E., Kim, W., Burand, J. P. & other authors (2009). Two viruses that cause salivary gland hypertrophy in Glossina pallidipes and Musca domestica are related and form a distinct phylogenetic clade. J Gen Virol 90, 334–346. [DOI] [PubMed] [Google Scholar]
  18. Goraczniak, R. & Gunderson, S. I. (2008). The regulatory element in the 3′-untranslated region of human papillomavirus 16 inhibits expression by binding CUG-binding protein 1. J Biol Chem 283, 2286–2296. [DOI] [PubMed] [Google Scholar]
  19. Gouteux, J. P. (1987). Prevalence of enlarged salivary glands in Glossina palpalis, G. pallicera, and G. nigrofusca (Diptera: Glossinidae) from the Vavoua area, Ivory Coast. J Med Entomol 24, 268. [DOI] [PubMed] [Google Scholar]
  20. Graham, S. V. (2008). Papillomavirus 3′ UTR regulatory elements. Front Biosci 13, 5646–5663. [DOI] [PubMed] [Google Scholar]
  21. Gross, C. H. & Rohrmann, G. F. (1993). Analysis of the role of 5′ promoter elements and 3′ flanking sequences on the expression of a baculovirus polyhedron envelope protein gene. Virology 192, 273–281. [DOI] [PubMed] [Google Scholar]
  22. Hastings, M. L., Milcarek, C., Martincic, K., Peterson, M. L. & Munroe, S. H. (1997). Expression of the thyroid hormone receptor gene, erbAα, in B lymphocytes: alternative mRNA processing is independent of differentiation but correlates with antisense RNA levels. Nucleic Acids Res 25, 4296–4300. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hastings, M. L., Ingle, H. A., Lazar, M. A. & Munroe, S. H. (2000). Post-transcriptional regulation of thyroid hormone receptor expression by cis-acting sequences and a naturally occurring antisense RNA. J Biol Chem 275, 11507–11513. [DOI] [PubMed] [Google Scholar]
  24. Hilger, C., Velhagen, I., Zentgraf, H. & Schroder, C. H. (1991). Diversity of hepatitis-B virus X gene related transcripts in hepatocellular carcinoma – a novel polyadenylation site on viral DNA. J Virol 65, 4284–4291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Iseli, C., Stevenson, B. J., de Souza, S. J., Samaia, H. B., Camargo, A. A., Buetow, K. H., Strausberg, R. L., Simpson, A. J. G., Bucher, P. & Jongeneel, C. V. (2002). Long-range heterogeneity at the 3′ ends of human mRNAs. Genome Res 12, 1068–1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Jaenson, T. G. T. (1978). Virus-like rods associated with salivary gland hyperplasia in tsetse, Glossina pallidipes. Trans R Soc Trop Med Hyg 72, 234–238. [DOI] [PubMed] [Google Scholar]
  27. Klemenz, R., Reinhardt, M. & Diggelmann, H. (1981). Sequence determination of the 3′ end of mouse mammary tumor virus RNA. Mol Biol Rep 7, 123–126. [DOI] [PubMed] [Google Scholar]
  28. Kumar, M. & Carmichael, G. G. (1998). Antisense RNA: function and fate of duplex RNA in cells of higher eukaryotes. Microbiol Mol Biol Rev 62, 1415–1434. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lamb, R. A. & Horvath, C. M. (1991). Diversity of coding strategies in influenza viruses. Trends Genet 7, 261–266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lavorgna, G., Dahary, D., Lehner, B., Sorek, R., Sanderson, C. M. & Casari, G. (2004). In search of antisense. Trends Biochem Sci 29, 88–94. [DOI] [PubMed] [Google Scholar]
  31. Lavorgna, G., Triunfo, R., Santoni, F., Orfanelli, U., Noci, S., Bulfone, A., Zanetti, G. & Casari, G. (2005). AntiHunter 2.0: increased speed and sensitivity in searching blast output for EST antisense transcripts. Nucleic Acids Res 33, W665–W668. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lehner, B., Williams, G., Campbell, R. D. & Sanderson, C. M. (2002). Antisense transcripts in the human genome. Trends Genet 18, 63–65. [DOI] [PubMed] [Google Scholar]
  33. Lietze, V.-U., Geden, C. J., Blackburn, P. & Boucias, D. G. (2007). Effects of salivary gland hypertrophy virus on the reproductive behavior of the house fly, Musca domestica. Appl Environ Microbiol 73, 6811–6818. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Lin, S. F., Robinson, D. R., Miller, G. & Kung, H. J. (1999). Kaposi's sarcoma-associated herpesvirus encodes a bZIP protein with homology to BZLF1 of Epstein–Barr virus. J Virol 73, 1909–1917. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Majerciak, V., Yamanegi, K. & Zheng, Z. M. (2006). Gene structure and expression of Kaposi's sarcoma-associated herpesvirus ORF56, ORF57, ORF58, and ORF59. J Virol 80, 11968–11981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Minter-Goedbloed, E. & Minter, D. M. (1989). Salivary gland hyperplasia and trypanosome infection of Glossina in two areas of Kenya. Trans R Soc Trop Med Hyg 83, 640–641. [DOI] [PubMed] [Google Scholar]
  37. Mount, S. M. (1982). A catalogue of splice junction sequences. Nucleic Acids Res 10, 459–472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Munroe, S. H. & Lazar, M. A. (1991). Inhibition of c-erbA mRNA splicing by a naturally occurring antisense RNA. J Biol Chem 266, 22083–22086. [PubMed] [Google Scholar]
  39. Nasseri, M., Wettstein, F. O. & Stevens, J. G. (1982). Two colinear and spliced viral transcripts are present in non-virus-producing benign and malignant neoplasms induced by the shope (rabbit) papilloma virus. J Virol 44, 263–268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Nasseri, M., Hirochika, R., Broker, T. R. & Chow, L. T. (1987). A human papilloma virus type-11 transcript encoding an E1 or E4 protein. Virology 159, 433–439. [DOI] [PubMed] [Google Scholar]
  41. Norris, F. A., Auethavekiat, V. & Majerus, P. W. (1995). The isolation and characterization of cDNA-encoding human and rat brain inositol polyphosphate 4-phosphatase. J Biol Chem 270, 16128–16133. [DOI] [PubMed] [Google Scholar]
  42. Otieno, L. H., Kokwaro, E. D., Chimtawi, M. & Onyango, P. (1980). Prevalence of enlarged salivary glands in wild populations of Glossina pallidipes in Kenya, with a note on the ultrastructure of the affected organ. J Invertebr Pathol 36, 113–118. [Google Scholar]
  43. Passarelli, A. L. & Guarino, L. A. (2007). Baculovirus late and very late gene regulation. Curr Drug Targets 8, 1103–1115. [DOI] [PubMed] [Google Scholar]
  44. Proudfoot, N. (1991). Poly(A) signals. Cell 64, 671–674. [DOI] [PubMed] [Google Scholar]
  45. Rixon, F. J. & Clements, J. B. (1982). Detailed structural analysis of two spliced HSV-1 immediate-early messenger-RNAs. Nucleic Acids Res 10, 2241–2256. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Rutherford, K., Parkhill, J., Crook, J., Horsnell, T., Rice, P., Rajandream, M. A. & Barrell, B. (2000). Artemis: sequence visualization and annotation. Bioinformatics 16, 944–945. [DOI] [PubMed] [Google Scholar]
  47. Sang, R. C., Jura, W. G. Z. O., Otieno, L. H. & Mwangi, R. W. (1998). The effects of a DNA virus infection on the reproductive potential of female tsetse flies, Glossina morsitans centralis and Glossina morsitans morsitans (Diptera: Glossinidae). Mem Inst Oswaldo Cruz 93, 861–864. [DOI] [PubMed] [Google Scholar]
  48. Sang, R. C., Jura, W. G. Z. O., Otieno, L. H., Mwangi, R. W. & Ogaja, P. (1999). The effects of a tsetse DNA virus infection on the functions of the male accessory reproductive gland in the host fly Glossina morsitans centralis (Diptera; Glossinidae). Curr Microbiol 38, 349–354. [DOI] [PubMed] [Google Scholar]
  49. Schwartz, S. (2008). HPV-16 RNA processing. Front Biosci 13, 5880–5891. [DOI] [PubMed] [Google Scholar]
  50. Shaw, M. K. & Moloo, S. K. (1993). Virus-like particles in Rickettsia within the midgut epithelial cells of Glossina morsitans centralis and Glossina brevipalpis. J Invertebr Pathol 61, 162–166. [Google Scholar]
  51. Shearwin, K. E., Callen, B. P. & Egan, J. B. (2005). Transcriptional interference – a crash course. Trends Genet 21, 339–345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Shendure, J. & Church, G. M. (2002). Computational discovery of sense–antisense transcription in the human and mouse genomes. Genome Biol 3, RESEARCH0044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Silver Key, S. C. & Pagano, J. S. (1997). A noncanonical poly(A) signal, UAUAAA, and flanking elements in Epstein–Barr virus DNA polymerase mRNA function in cleavage and polyadenylation assays. Virology 234, 147–159. [DOI] [PubMed] [Google Scholar]
  54. Simonsen, C. C. & Levinson, A. D. (1983). Analysis of processing and polyadenylation signals of the hepatitis-B virus surface-antigen gene by using simian virus 40–hepatitis B virus chimeric plasmids. Mol Cell Biol 3, 2250–2258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Sun, M., Hurst, L. D., Carmichael, G. G. & Chen, J. J. (2005). Evidence for a preferential targeting of 3′-UTRs by cis-encoded natural antisense transcripts. Nucleic Acids Res 33, 5533–5543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Taniguchi, A. & Yasumoto, S. (1990). A major transcript of human papillomavirus type 16 in transformed NIH 3T3 cells contains polycistronic mRNA encoding E7, E5, and E1–E4 fusion gene. Virus Genes 3, 221–233. [DOI] [PubMed] [Google Scholar]
  57. Vanhée-Brossollet, C. & Vaquero, C. (1998). Do natural antisense transcripts make sense in eukaryotes? Gene 211, 1–9. [DOI] [PubMed] [Google Scholar]
  58. van Oers, M. M., Vlak, J. M., Voorma, H. O. & Thomas, A. A. (1999). Role of the 3′ untranslated region of baculovirus p10 mRNA in high-level expression of foreign genes. J Gen Virol 80, 2253–2262. [DOI] [PubMed] [Google Scholar]
  59. Veeramachaneni, V., Makalowski, W., Galdzicki, M., Sood, R. & Makalowska, I. (2004). Mammalian overlapping genes: the comparative perspective. Genome Res 14, 280–286. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Weil, J. E. & Beemon, K. L. (2006). A 3′ UTR sequence stabilizes termination codons in the unspliced RNA of Rous sarcoma virus. RNA 12, 102–110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Zhao, J., Hyman, L. & Moore, C. (1999). Formation of mRNA 3′ ends in eukaryotes: mechanism, regulation, and interrelationships with other steps in mRNA synthesis. Microbiol Mol Biol Rev 63, 405–445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Zheng, Z. M. & Baker, C. C. (2006). Papillomavirus genome structure, expression, and post-transcriptional regulation. Front Biosci 11, 2286–2302. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

[Supplementary Material]
supp_90_5_1270__index.html (1.2KB, html)
supp_90_5_1270__1.pdf (107.5KB, pdf)
supp_90_5_1270__2.pdf (134.3KB, pdf)

Articles from The Journal of General Virology are provided here courtesy of Microbiology Society

RESOURCES