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Journal of Virology, September 1998, p. 7294-7301, Vol. 72, No. 9
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Alternative Splicing of the Latency-Related
Transcript of Bovine Herpesvirus 1 Yields RNAs Containing Unique
Open Reading Frames
Laxminarayana R.
Devireddy and
Clinton
Jones*
Center for Biotechnology, Department of
Veterinary and Biomedical Sciences, University of
Nebraska
Lincoln, Lincoln, Nebraska 68583-0905
Received 2 April 1998/Accepted 27 May 1998
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ABSTRACT |
The latency-related transcript (LRT) of bovine herpesvirus 1 (BHV-1) is the only abundant viral RNA detected during latency. A
previous study (A. Hossain, L. M. Schang, and C. Jones, J. Virol. 69:5345-5352, 1995) concluded that splicing of polyadenylated [poly(A)+] and splicing of nonpolyadenylated
[poly(A)
] LRT are different. In this study, splice
junction sites of LRT were identified. In trigeminal ganglia of acutely
infected calves (1, 7, or 15 days postinfection [p.i.]) or in
latently infected calves (60 days p.i.), alternative splicing of
poly(A)+ LRT occurred. Productive viral gene expression in
trigeminal ganglia is readily detected from 2 to 7 days p.i. but not at
15 days p.i. (L. M. Schang and C. Jones, J. Virol.
71:6786-6795, 1997), suggesting that certain aspects of a lytic
infection occur in neurons and that these factors influence LRT
splicing. Splicing of poly(A) LRT was also detected in
transfected COS-7 cells or infected MDBK cells. DNA sequence analysis
of spliced LRT cDNAs, poly(A)+ or poly(A),
revealed nonconsensus splice signals at exon/intron and intron/exon boundaries. The GC-AG splicing signal utilized by the herpes simplex virus type 1 latency-associated transcript in latently infected mice is
also used by LRT in latently infected calves. Taken together, these
results led us to hypothesize that (i) poly(A)+ LRT is
spliced in trigeminal ganglia by neuron-specific factors, (ii) viral or
virus-induced factors participate in splicing, and (iii) alternative
splicing of LRT may result in protein isoforms which have novel
biological properties.
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INTRODUCTION |
All members of the alphaherpesvirus
subfamily establish and maintain a latent infection in the peripheral
nervous system of their natural hosts. Bovine herpesvirus 1 (BHV-1), a
member of the alphaherpesvirus subfamily, is an important pathogen of
cattle and establishes latent infection in sensory ganglia of infected cattle (reviewed in references 57 and
58). Since neurons are terminally differentiated
cells, it may not be necessary for the virus to replicate in these
cells to maintain latency. Viral gene expression in latently infected
neurons is restricted to the latency-related transcript (LRT). By using
in situ hybridization, LRT was detected in trigeminal ganglia (TG) of
BHV-1-infected rabbits (55, 56) or cattle (41).
These studies mapped the approximate 5' and 3' ends of LRT and
estimated its length to be 1.15 kb. LRT is also expressed during the
late stages of productively infected bovine cells (56). A
41-kDa protein is encoded by the LR (latency-related) gene in
transiently transfected cells or infected bovine cells (35).
LR gene products inhibit entry of cells into S phase, suggesting that
the LR gene regulates some aspect of latency (65).
The latency-associated transcript (LAT) of herpes simplex virus type 1 (HSV-1) has been the subject of intense scrutiny (reviewed in
references 4, 9
24, 34, and
80). It is not known if HSV-1 LAT encodes a protein
even though LAT is associated with polysomes (28). LAT is a
stable 2.0-kb intron (22, 40, 59, 83), and the 1.5- or
1.45-kb transcript is derived from the 2.0-kb LAT by further splicing
(71). The splicing event that generates the 1.5-kb LAT
utilizes a novel splice donor that is GC instead of GT (71,
74), and this splicing event requires neuron-specific splicing
factors (44). Polyadenylation of the spliced 1.5-kb LAT is
controversial (18, 50, 52, 70, 79). Disruption of splice
donor or acceptor sites prevents synthesis of the 2-kb LAT in
productively infected nonneuronal cells but not in latently infected
neurons (3).
Although cis-acting sequences that regulate neuron-specific
transcription of the BHV-1 LR gene have been studied (10, 11, 17,
37), processing of LRT has not been well characterized. A
previous study concluded that LRT is spliced, but splice junctions were
not identified (35). In this study, LRT splicing patterns in
TG of infected calves were compared to those of productively infected
bovine cells or COS-7 cells transfected with a plasmid that expresses
LR gene products. We have identified three alternatively spliced
poly(A)+ LRT isoforms at 7, 15, or 60 days postinfection
(p.i.). A spliced poly(A) LRT was detected at 1 day p.i.,
suggesting LRT is expressed early in TG. LRT was spliced in the
poly(A) RNA fraction after bovine cells were infected or
after COS-7 cells were transfected with a plasmid containing the LR
gene. It is hypothesized that poly(A)+ LRT is alternatively
spliced in TG and that these spliced variants have the potential to
encode novel proteins.
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MATERIALS AND METHODS |
Virus, plasmids, and cells.
MDBK (Madin-Darby bovine kidney)
cells or COS-7 cells (American Type Culture Collection, Rockville, Md.)
were grown in Earle's modified Eagle's medium supplemented with 10%
fetal calf serum. The Cooper strain of BHV-1 was obtained from the
National Veterinary Services Laboratory, Animal and Plant Health
Inspection Services (Ames, Iowa). MDBK cells were infected with 5 PFU
of BHV-1 per cell, and RNA was extracted 24 h p.i.
Plasmid pcDNA1/LRT was constructed by inserting a 2-kb
HindIII-SalI fragment which contains the LR
gene (35) (Fig. 1) into the
mammalian expression vector pcDNA1/Amp (Invitrogen). A SalI site was inserted into the unique XbaI site of pcDNA1/Amp
prior to insertion of the LR gene. COS-7 cells were transfected with pcDNA1/LRT by calcium phosphate precipitation (19), and
total RNA was prepared 48 h after transfection.
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FIG. 1.
Schematic of the LR promoter, locations of 5' termini of
LR transcripts, and partial restriction enzyme map of the LR gene. The
5' ends of LRT were mapped by RACE (rapid amplification of cDNA ends)
PCR or primer extension (11, 35). DNA sequences within the
LR promoter which are bound by neuron-specific proteins (NSB) were
identified by electrophoretic mobility shift assays and exonuclease III
footprinting (17). DNA sequences within the LR promoter
which cis activate a minimal tk promoter in
neuronal cells are designated as a neuron-specific transcriptional
activator (NSTA) (10). Splicing of LR RNA occurs in LRT;
this is designated by the dashed lines (35). The transcripts
(IE2.9/E2.6) antisense to LRT are indicated by a solid black line, and
the circle at the 3' end of the transcript is the position of the stop
codons for the protein encoded by IE2.9/E2.6. The primers P1, P2, P3,
and P4 used for amplification of LRT are indicated by small black
rectangles. The number below each primer indicates the position of the
5' terminus. The predicted sizes of the PCR products that can be
amplified by these primers are also indicated. The small hatched
rectangle indicates the probe used in Southern blot analysis to detect
the PCR product. Except for the boxes depicting P1, P2, P3, P4, and the
probe, the line map is drawn to scale. Plasmid pcDNA1/LRT contains the
2-kb HindIII-SalI fragment cloned into
pcDNA1/Amp as described in Materials and Methods.
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Infection of cattle and preparation of tissue samples.
BHV-1-free calves were divided into six groups of two each and then
infected with 108.8 50% tissue culture infective doses
intranasally and intraocularly as described previously (66).
Two animals were used as controls. TG were collected 1, 2, 4, 7, 15, or
60 days p.i. (two calves per time point) or from two uninfected calves.
TG were frozen in an ethanol-dry ice bath and stored at 
120°C
(66). Some of the RNA samples from the cattle described by
Schang and Jones (66) were used for these studies.
Preparation of RNA.
Total RNA was extracted from TG as
described previously (35). Total RNA from infected or
transfected cells was extracted by using the RNAgents Total RNA
Isolation system (catalog no. Z5110; Promega) according to the
manufacturer's instructions. RNA concentrations were measured in a
spectrophotometer at 260 nm.
Preparation of poly(A)+ and poly(A) LRT
RNA by oligo(dT) chromatography.
Poly(A)+ RNA was
prepared by using an mRNA purification kit (catalog no. 27-9258;
Pharmacia) according to manufacturer's instructions. Total RNA was
passed through an oligo(dT)-cellulose column twice, and the column was
washed with a buffer supplied in the kit. Bound RNA was recovered by
incubating the column with 250 µl of elution buffer at 65°C, and
RNA was subsequently precipitated with ethanol. RNA in the flowthrough
fraction of the oligo(dT) column [designated poly(A)]
was recovered and precipitated with ethanol.
Primers for RT-PCR.
Figure 1 shows the design of primers
used for reverse transcriptase (RT)-mediated PCR (RT-PCR) and location
of the hybridization probe. LRT was detected by using primers P1 (same
sense as LRT 5'-AGGCTGGGGGTCGCAAATACACGGC-3') and P2
(antisense to LRT 5'-GGCCCGCCGGAGAAGAAGGACAGAGT-3'), which
amplify a 757-bp fragment. With primers P3 (same sense as LRT
5'-CCCCAGGAGGCTTTCTCGCACC-3') and primer P4 (antisense to LRT 5'-CACAGTGATAGACCTGACGGCGAACG-3'), spliced LRT was
amplified. The probe used for detection of LRT cDNA was from
nucleotides (nt) 1092 to 1117 (5'-GCGCACCGAAATGGAAGTGGCCGCC-3').
The numbering system for the LR gene was described previously
(41) and is based on the Cooper strain of BHV-1. The 3'
termini of primers P1, P2, P3, and P4 correspond to positions 872, 1629, 1068, and 1523, respectively. The primers were designed based on
the following criteria: (i) the oligonucleotides are located adjacent
to an AT-rich region, (ii) the oligonucleotides have a GC content
greater than 50%, (iii) there is no significant similarity to other
viral genes, and (iv) PCR product size is more than 300 bp.
RT-PCR and sequence analysis of cDNAs.
The method for RT-PCR
was described previously (14). Prior to cDNA synthesis, RNA
was treated with 1 U of DNase I (GIBCO-BRL) to eliminate contaminating
DNA (20). Five hundred nanograms of DNase I-treated
poly(A)+ or 3 µg of poly(A) RNA was
denatured at 65°C for 7.5 min, incubated at room temperature with 1 µg of oligo(dT) or random primers (Invitrogen) for 15 min, and then
incubated on ice. This mixture was incubated with 200 U of Moloney
murine leukemia virus RT (GIBCO-BRL) in the presence of 20 U of RNase
inhibitor (Promega) according to the manufacturer's instructions for
synthesis of cDNA. RT was inactivated by heating at 95°C for 5 min.
Amplification of cDNA was conducted with 2.5 U of Taq DNA
polymerase and 100 µM deoxynucleoside triphosphates in a 50-µl
reaction. Forty cycles of amplification were carried out with primers
P1 and P2 (200 ng of each) in the presence of 10% glycerol to improve
denaturation of GC-rich DNA and to enhance the extension through
secondary structures (68) on a DNA thermal cycler (Hybaid).
The following conditions were used for amplification: 1 min at 94°C
(denaturation), 2 min at 55°C (annealing), 2 min at 72°C
(polymerization), and 7 min at 72°C to complete the extension. The
PCR products were then reamplified with primers P3 and P4 (200 ng of
each) under the same conditions. To avoid contamination, PCR was
performed in a separate room, gloves were changed frequently, all
reagents were used exclusively for these studies, and numerous other
precautions were taken to avoid contamination (32).
Amplified products were purified either by polyacrylamide gel
electrophoresis or by selective precipitation (62). Briefly,
0.1 volume of 10× STE (1 M NaCl, 200 mM Tris-HCl [pH 7.5], 100 mM
EDTA) was added to PCR products, followed by addition of equal amounts
of 4 M ammonium acetate, and precipitated with 2.5 volumes of ethanol at room temperature. Purified PCR products were cloned into pCR-Script vector (Stratagene) according to the manufacturer's instructions. Both
strands of the inserts were sequenced by the dideoxynucleotide chain
termination method using the Fidelity DNA sequencing system (catalog
no. 57600; Oncor), which is designed for sequencing GC-rich DNA. As a
positive control, BHV-1 DNA was used. Negative controls included RNA
from TG of uninfected calves, mock-infected MDBK cells, or
mock-transfected COS-7 cells.
Southern blot analysis.
PCR products were separated on 2%
agarose gels and transferred onto Hybond N+ membrane (Amersham) by
capillary transfer according to the protocol of the manufacturer.
Hybridization was done according to the manufacturer's instructions.
The probe was prepared by end labeling with T4 polynucleotide kinase
(New England Biolabs) and [
-32P]ATP (Amersham).
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RESULTS |
Amplification of LRT splice junction sites by RT-PCR.
A
previous study (35) demonstrated that splicing of LRT
occurred, but splice junction sites were not identified. To further study splicing of LRT, RT-PCR was conducted because this approach has
been used successfully to identify alternative splicing of other
primary transcripts (15, 23, 54). To this end, total RNA
from TG of infected calves was used. Poly(A)+ RNA was
purified by oligo(dT) chromatography. No attempts were made to prove
how efficient the purification procedure was because unnecessary
manipulation of TG RNA increases the probability of degradation. To
avoid amplification of contaminating viral DNA, total RNA was treated
with DNase I. Single-stranded cDNA was synthesized by using an
oligo(dT) primer, RT, and conditions which allow for optimal
amplification of LRT. The resulting cDNA was then amplified in a nested
PCR using the primers shown in Fig. 1. The rationale for using nested
PCR is that (i) primers P1 and P2 are adjacent to the transcription
start sites (11) and the poly(A) signals (41),
(ii) primers P3 and P4 flank the region which was spliced (35), and (iii) this strategy enables detection of small
amounts of LRT. Although these primers will amplify the IE2.9/E2.6
mRNA, this region of the RNA is not spliced (82), and thus
the amplified product migrates with the same mobility as genomic DNA
(data not shown).
Poly(A)
+ LRT was detected in bovine TG at 7, 15, or 60 days
p.i. (Fig.
2A). A previous study
demonstrated that infectious virus
was detected in ocular swabs at 2, 4, or 7 days p.i. but not 15
or 60 days p.i. (
66).
Alternative splicing apparently occurred
in TG during acute infection
because amplified products detected
at 7 or 15 days p.i. were smaller
than amplified LR DNA or LRT
cDNA at 60 days p.i. Amplified products
were not detected when
RT was excluded from the cDNA synthesis (Fig.
2B) or when RNA
was prepared from TG of an uninfected calf (Fig.
2A,
lane U).
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FIG. 2.
Detection of splicing in poly(A)+ LR RNA in
TG of infected cattle. The cDNA reaction containing RT was subsequently
amplified by using the LRT-specific primers in the nested PCR (A). Lane
V, BHV-1 genomic DNA used as an unspliced target; lane U, RNA from TG
of an uninfected calf. Numbers above the lanes indicate the days p.i.
at which RNA was prepared from TG.  174 DNA which was digested with
HaeIII was used as a molecular weight marker, and the
positions of the bands are listed as base pairs. RNA samples were
incubated in the standard RT reaction, but RT was omitted and then the
nested PCR was performed (B). The lanes are labeled as in panel A. PCR
products were detected by Southern blot analysis as described in
Materials and Methods.
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The flowthrough from the oligo(dT) column [poly(A)
RNA]
was subjected to cDNA synthesis using random primers and nested PCR
to
detect LRT. A 455-bp PCR product was detected by Southern blot
analysis
using the LRT-specific probe described in Fig.
1 (Fig.
3A, lanes 2, 7, and 15). Amplified
products were not detected
when RT was omitted from the cDNA synthesis
reaction (Fig.
3B).
Poly(A)
LRT was also detected at 1 day p.i. (Fig.
3A, lane 1), and the
PCR product appeared to be slightly
smaller than genomic viral
DNA. Furthermore, poly(A)
LRT
from latently infected animals (60 days p.i.) migrated as
a 455-bp
amplified product (Fig.
3C, lane 1), and the specificity
of the
amplified product was confirmed by Southern blot hybridization
(data
not shown). As expected, amplified products were not observed
when RT
was left out of the cDNA synthesis reaction (Fig.
3C,
lane 2). RNA from
TG of a second set of infected calves yielded
similar bands (data not
shown). In summary, these results demonstrated
that (i) alternative
splicing of poly(A)
+ LRT occurred in TG during acute
infection, (ii) splicing of poly(A)
LRT apparently
occurred at 1 day p.i., and (iii) splicing of
poly(A)
LRT
in TG at 2, 7, 15, or 60 days p.i. was not readily detected.
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FIG. 3.
Analysis of poly(A) LR RNA. (A) Southern
blot analysis of PCR-amplified products derived from the reaction
performed with RT. BHV-1 genomic DNA was used as a positive control
(lane V). Lane M, RNA from an uninfected calf. Numbers above the lanes
indicate RNA prepared from TG which were obtained at different days
p.i. The arrow indicates the position of LRT. (B) Southern blot
analysis of PCR products derived from the reaction performed without
RT. Water was used as a negative control (lane V). The remainder of the
lanes are labeled as described for panel A. 174 DNA digested with
HaeIII was used as a marker, and the positions of the bands
are designated in base pairs. (C) Amplification of
poly(A) LR RNA from latently infected calves (60 days
p.i.). Lane M, 174 DNA digested with HaeIII; lane U, RNA
prepared from TG of an uninfected calf; lane 1, reaction with RT and
LRT cDNA subsequently amplified; lane 2, reaction without RT followed
by PCR to amplify LRT cDNA.
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Analysis of LRT synthesized in transfected or infected cells.
A protein product was identified in COS-7 cells transfected with the LR
gene or after MDBK cells were infected (35, 65). In this
study, splicing of LRT was investigated after COS-7 cells were
transfected with a plasmid expressing LR gene products or after MDBK
cells were infected with BHV-1. Total RNA was extracted 48 h after
transfection or 24 h after infection. Following DNase I treatment,
poly(A)+ RNA was used as a template to synthesize
single-stranded cDNA with an oligo(dT) primer, and LRT cDNA was
amplified. Although poly(A)+ LRT was detected in
transfected or infected cells, spliced poly(A)+ LRT was not
detected, as judged by the size of the PCR product (Fig.
4A, lane 2 or 5). A previous study
detected small amounts of spliced poly(A)+ LRT in infected
MDBK cells (35). Although the results in Fig. 4A appear to
be at odds with that conclusion, the RNA used for this study was
purified by oligo(dT) chromatography. Thus, we hypothesize that either
the spliced poly(A)+ LRT was degraded during purification,
the poly(A) tail was too short to be stably bound on an oligo(dT)
column, or the use of a strand-specific primer in the previous study
(35) allowed detection of small amounts of spliced
poly(A)+ LRT in infected MDBK cells.
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FIG. 4.
Analysis of LRT expressed in transfected COS-7 or
infected MDBK cells. (A) PCR amplification of poly(A)+ LRT
from transfected or infected cells. Lane M, 174 DNA digested with
HaeIII; lane 1, RNA prepared from untransfected COS-7 cells;
lanes 2 and 3, reactions with (lane 2) and without (lane 3) RT, using
poly(A)+ LRT prepared from COS-7 cells transfected with
pcDNA1/LRT; lane 4, RNA prepared from uninfected MDBK cells; lanes 5 and 6, reactions with (lane 5) and without (lane 6) RT, using
poly(A)+ LRT prepared from MDBK cells which were infected
for 24 h. The arrow indicates the position of LRT. (B) PCR
amplification of poly(A) LRT from transfected or infected
cells. Lanes are as in panel A. The top arrow indicates the position of
unspliced LRT, and the bottom arrows indicate the positions of spliced
LRT. Sizes are indicated in base pairs.
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When cDNA synthesis of poly(A)
RNA was primed with a
random primer and LRT cDNA was amplified by nested PCR, bands smaller
than amplified BHV-1 DNA (455 bp) were detected (Fig.
4B, lanes
2 and
5). poly(A)
RNA which was prepared from productively
infected cells yielded
amplified products migrating as 280-, 240-, or
200-bp fragments
(Fig.
4B, lane 5). In contrast, poly(A)
RNA in transiently transfected COS-7 cells contained bands migrating
as
455-, 300-, or 200-bp fragments (Fig.
4B, lane 2). All of the
amplified
products hybridized to the LR-specific probe described
in Fig.
1 (data
not shown). No PCR products were observed when
RT was left out of the
cDNA synthesis reaction (Fig.
4A and B,
lanes 3 and 6). Unspliced
poly(A)
LRT was also detected in transfected cells
because a 455-bp band
was amplified (Fig.
4B, lane 2). Plasmid
pcDNA1/LRT does not contain
the IE2.9/E2.6 gene, demonstrating that LRT
can be spliced in
the absence of any other known viral gene and a
subset of LRT
was not spliced. In summary, these results indicated that
poly(A)
LRT was spliced in MDBK cells after infection or
when COS-7 cells
were transfected with pcDNA1/LRT.
Sequencing of cloned fragments spanning LRT splice sites.
Although it was possible to sequence the PCR products directly, they
were cloned into the pCR-Script vector prior to DNA sequencing. This
approach was used in an attempt to identify minor splice site variants
and to enhance selection of full-length products. Purified PCR products
shown in Fig. 2A, 3A and C, and 4B were cloned into pCR-Script vector.
A fragment migrating as a 200-bp fragment was the only band less than
455 bp which was able to be cloned from transfected COS-7 cells or
infected MDBK cells (Fig. 4B, lanes 2 and 5). Prior to DNA sequencing,
plasmids were analyzed by restriction enzyme digestion to verify that
the inserts were similar in size to the amplified products. Less than
2% of the plasmids had different-size inserts, suggesting that most of
the PCR products were not deleted during cloning. DNA sequence of these
variants did not match the published LRT gene sequence, indicating they
were rearranged during cloning or were not bona fide LRT cDNAs. In
contrast, fragments migrating at the expected position of the amplified
product yielded DNA sequence which matched the LR gene sequence with an
interruption in the middle. The 455-bp PCR product matched the known
sequence of the LR gene but did not contain an interruption and thus
was not spliced. Figure 5 shows
representative examples of the DNA sequence spanning
splice junction sites at 1, 7, or 15 days p.i. from TG. At least 10 independent clones were sequenced for each time point, and they yielded
the same sequence (locations of splice sites are summarized in Fig. 6).
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FIG. 5.
Determination of DNA sequences of the LRT splice
junctions. The splice junction site was amplified with primers as
described in Fig. 1. The sequence presented on the right is the sense
strand. Double asterisks mark the splice sites of LRT and the sequence
which was spliced out (41). The 5' and 3' splice sites are
marked with asterisks. The nucleotides that differ from the published
LR gene sequence (41) are underlined. The nucleotides in
parentheses (T at position 1368 in panel A and C and T at positions
1173 and 1368, respectively, in panel B) were detected in this study
but not in the previous study (41). (A) 1 day p.i.; (B) 7 days p.i.; (C) 15 days p.i.
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FIG. 6.
Summary of spliced poly(A)+ or
poly(A) LRT. (A) Schematic of BHV-1 genome, positions of
repeats, and positions of map units (mu). Locations of immediate-early
transcripts with respect to LRT are shown (82). Introns of
immediate-early transcripts are presented as dashed lines. Exons are
listed as e1 and e2. The e1 of IE2.9 and that of IE4.2 are identical
but not protein coding. The E2.6 transcript does not contain e1, and
transcription initiates from a novel promoter at the 5' terminus of e2.
Consequently, the proteins encoded by IE2.9 and E2.6 are identical. (B)
Alternatively spliced variants of LRT. The numbers on the left indicate
that RNA was prepared from TG of calves infected for 1, 7, 15, or 60 days. Tr, spliced LRT which was synthesized in COS-7 cells after
transfection with pcDNA1/LRT; In, spliced LRT which was synthesized in
MDBK cells infected with BHV-1 for 24 h. Numbers above the lines
indicate the 5' and 3' boundaries of the intron. The schematic in panel
A is not drawn to scale with respect to panel B.
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Regardless of whether LRT was poly(A)
+ or
poly(A)
, splice sites did not match consensus 5' or 3'
splice sites (Table
1). The
5' splice
sites of poly(A)
+ LRT at 60 days p.i. were GC, and they
match the 5' splice site
of HSV-1 LAT (
71,
74), duck
-globin, or bovine aspartyl protease
(reviewed in references
36 and
47). The 5' GC splice site
was also identified at the second exon/intron border in transiently
transfected COS-7 cells. The remainder of the 5' splice sites
were CG,
and to date no transcript has been identified with this
splice donor.
Except for the 3' TC splice site identified at 7
days p.i., the
remainder of the 3' splice sites have been described
for other mRNAs.
The 3' TG splice site identified at 1 day p.i.
or in transfected cells
(second 3' splice site) is present in
the 3' splice acceptor site of
human or
Drosophila melanogaster subunit of guanine
nucleotide-binding protein (
39,
53).
The 3' CC splice site
observed at 15 days p.i. in transfected
COS-7 cells (first 3' splice
site) or infected MDBK cells was
described for yeast HAC1 mRNA
(
67). HAC1 and
D. melanogaster subunit of
guanine nucleotide-binding protein RNAs are alternatively
spliced
(
39,
67). Finally, the 3' AG splice site present at
60 days
p.i. matches HSV-1 LAT (
71,
74). In summary, these
studies
indicated that (i) in TG of latently infected calves,
the 5' and 3'
splice sites of LRT match HSV-1 LAT; and (ii) most
of the nonconsensus
splice sites are utilized by other mRNAs.
Effects of splicing on LR ORFs.
The LR gene contains two open
reading frames (ORFs) and two reading frames without an initiating
methionine. One reading frame without an initiating methionine is
contained after the three in-frame stop codons of ORF 2, and the other
is in reading frame C. Each spliced LRT isoform was examined to
determine the effect of splicing on the ORFs. A fusion between ORF 2 and ORF 1 was generated by splicing at 7 days p.i. in infected MDBK
cells or transfected COS-7 cells (Fig.
7). However, these putative proteins would not be identical because the splice junction signals are different. At 15 days p.i., the stop codons at the 3' end of ORF 2 were
removed and fused to the reading frame which is in frame with ORF 2 (Fig. 7). At 1 day or 60 days p.i., the ORFs were organized in a
similar fashion (Fig. 7). Interestingly, at 1 or 60 days p.i., a new
ORF which is a fusion between reading frame C and ORF 1 is generated
(Fig. 7). When ORF 2 is fused to ORF 1 (7 days p.i., infected or
transfected cells) or to reading frame B (15 days p.i.), the predicted
molecular masses of these proteins were 35 to 45 kDa. This finding
agrees with previous conclusions that the P2 antibody directed against
the amino terminus of LR ORF 2 recognizes a 40-kDa protein (35,
65). Although we do not know if these proteins are expressed in
neurons, these studies suggest that alternative splicing of LRT has the
potential to generate novel proteins.
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FIG. 7.
Organization of ORFs in the LR gene or in alternatively
spliced LRT isoforms. The organization of ORFs in the LR gene was
described originally by Kutish et al. (41). The reading
frame in B (open box) that follows LR ORF 2 (stippled box) does not
contain a methionine at its amino terminus. The reading frame in C
(black box) does not contain a methionine at its amino terminus.
Asterisks indicate the positions of in-frame stop codons. The hatched
box denotes ORF 1. Numbers at the top indicate nucleotide positions,
and those on the left indicate that RNA was prepared from TG of calves
infected for 1, 7, 15, or 60 days. Tr, LRT synthesized in COS-7 cells
transfected with pcDNA1/LRT; In, LRT synthesized in MDBK cells infected
with BHV-1 for 24 h. The sequence of each LRT variant was analyzed
and translated by using the IBI MacVector sequence analysis software
(Kodak).
|
|
| |
DISCUSSION |
This study demonstrated that alternative splicing of LRT occurred
in bovine TG compared to nonneural cells. Several conclusions were
drawn from the sequencing data: (i) poly(A)+ LRT in bovine
TG at 7 days p.i. was spliced differently than at 15 or 60 days p.i.,
(ii) poly(A) LRT detected in bovine TG at 1 day p.i. was
not the same as LRT detected at 7, 15, or 60 days p.i., (iii)
poly(A)+ LRT in transfected or infected cells which
migrated with viral genomic DNA was not spliced, (iv)
poly(A) LRT was spliced in productively infected MDBK
cells, and (v) poly(A) LRT was apparently spliced at two
positions in transiently transfected COS-7 cells. Although DNA sequence
analysis of splice junction sites suggested that samples from different
times p.i. contained one spliced product, the procedures used for
amplifying and cloning splice junction sites would yield the major
spliced product. It is also possible that at other times p.i.,
different spliced versions of LRT exist or certain splice variants were
not stably cloned. The finding that nonconsensus splice sites were
utilized suggests that splicing was regulated by a combination of
cell-specific and viral or virus-induced factors.
Several LRT introns are smaller than the 80-nt minimum intron size
which has been proposed for eukaryotes (81). For example, we
detected a 35-nt intron at 60 days p.i. in TG, a 44-nt intron at 1 day
p.i. in TG, and a 44-nt intron in transiently transfected COS-7 cells.
The ciliate Paramecium tetraurelia has introns which are 20 to 33 nt long, and these introns have consensus eukaryotic splice
signals, G(T/A)G (21, 60). Most fungi or insects, including Drosophila, have introns which range in size from 31 to 70 nt (51, 61, 73; reviewed in references 31 and 48)). More importantly, the polyomavirus small tumor antigen transcript contains a
48-nt-long intron which is excised by a novel mechanism
(27), demonstrating that small introns can be excised in
mammalian systems. We hypothesize that cis-acting sequences
within LRT regulate alternative splicing and mediate excision of short
introns. The three in-frame stop codons at the C terminus of ORF 2 (reference 41 and Fig. 7) may be important for
alternative splicing because it is known that multiple in-frame stop
codons influence cell-specific splicing (2). Although
splicing of LRT has unusual features (intron length and nonconsensus
splicing signals, for example), there is precedence for unusual introns
in a variety of organisms.
Splicing is regulated by a complex array of trans-acting
factors, some of which are cell or tissue specific (reviewed in
references 12 and 42). Although
5' splice signals are usually recognized by small ribonucleoprotein
complexes (snRNPs) which contain the U1 small nuclear RNA (reviewed in
references 5 and 8), introns containing nonconsensus splice sites are frequently spliced by less
abundant snRNPs (30, 75, reviewed
in reference 69). Serine/arginine (SR) proteins are
also important for selection of 5' and 3' splice sites (reviewed in
references 13, 25, 45, 49, and
78). Adenovirus (33, 38), bovine
papillomavirus (84), and HSV-1 (46, 63) alter the
distribution or activity of SR proteins. Neuron-specific or
brain-specific alternative splicing of specific mRNAs has frequently
been observed (1, 6, 7, 44, 72, 77). A neuron-specific
splicing regulator (KSRP) is crucial for neuron-specific splicing of
c-src (43; reviewed in reference
29). Finally, alternative splicing of HSV-1 LAT
occurs in neural cells (44) or murine TG (3),
suggesting that neuron-specific splicing has functional significance.
Latency has conveniently been divided into three distinct steps: (i)
establishment, (ii) maintenance, and (iii) reactivation. The finding
that LRT is alternatively spliced during establishment (1 to 15 days
p.i.) relative to maintenance (60 days p.i.) suggests that LR gene
products have specialized functions which are necessary for the various
stages of latency. During establishment of latency, it is reasonable to
hypothesize that a viral function represses viral gene expression and
enhances neuronal survival. BHV-1 gene expression in TG, early or
immediate-early, is detected as early as 2 days p.i. and peaks at 7 days p.i. (66). Spliced LRT was detected at 1 day p.i. in
TG, suggesting that it accumulates prior to productive viral gene
expression and thus participates in establishment of latency. HSV-1 LAT
promotes establishment of latency (64, 76) in mice by
repressing productive viral gene expression (16, 26), adding
support to the hypothesis that the LR gene plays a role in
establishment. During maintenance of latency, promoting neuronal
survival would still be important but repression of viral gene
expression does not appear to be as important. A viral function which
promotes viral gene expression or DNA replication but prevents neuronal
death would be advantageous during reactivation from latency. A number
of studies have concluded that HSV-1 LAT mutants do not reactivate from
latency efficiently in vivo (reviewed in references
57 and 58), but the mechanism by
which LAT functions in this capacity is unknown. Although it is
unlikely that LRT regulates every aspect of latency, we hypothesize
that alternative splicing of LRT yields novel proteins with specialized
functions and that these protein isoforms are important for certain
steps of latency. Cloning and characterizing the various LRT cDNAs
should allow a better understanding of how LR gene products regulate latency.
| |
ACKNOWLEDGMENTS |
We are grateful to Luis Schang and Maria Teresa Winkler for
assisting with the animal studies and preparing RNA from TG. We appreciate suggestions made by Harikrishna Nakshatri (IU Medical Center) and Stephen Mount (University of Maryland) for advice on
nonconsensus splice sites and on intron lengths. Finally, we thank
Ruben Donis for critically reading the manuscript.
This work was supported by grants 9402117, 9502236, and 9702394 from
the USDA.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Center for
Biotechnology, Dept. of Veterinary and Biomedical Sciences, University
of NebraskaLincoln, Fair St. at East Campus Loop, Lincoln, NE
68583-0905. Phone: (402) 472-1890. Fax: (402) 472-9690. E-mail:
cj{at}unlinfo.unl.edu.
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Journal of Virology, September 1998, p. 7294-7301, Vol. 72, No. 9
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
