Molecular characterization of the 3' terminus of the simian hemorrhagic fever virus genome.
ABSTRACT The 3' end of the simian hemorrhagic fever virus (SHFV) single-stranded RNA genome was cloned and sequenced. Adjacent to the 3' poly(A) tract, we identified a 76-nucleotide noncoding region preceded by two overlapping reading frames (ORFs). The ultimate 3' ORF of the viral genome encodes the capsid protein, and the penultimate ORF encodes the smallest SHFV envelope protein. These two ORFs overlap each other by 26 nucleotides. Northern (RNA) blot hybridization analyses of cytoplasmic RNA extracts from SHFV-infected MA-104 cells with gene-specific probes revealed the presence of full-length genomic RNA as well as six subgenomic SHFV-specific mRNA species. The subgenomic mRNAs are 3' coterminal. In its virion morphology and size, genome structure and length, and replication strategy, SHFV is most similar to lactate dehydrogenase-elevating virus, equine arteritis virus, and porcine reproductive and respiratory syndrome virus.
[show abstract] [hide abstract]
ABSTRACT: cDNA clones prepared from genomic RNA of coronavirus IBV have been sequenced. The nucleotide sequence for the complete 5' region of mRNA C, which is not present in mRNAs A and B, has been determined. A sequence of 1224 bases is presented which contains a long open reading frame predicting a polypeptide of molecular weight 25 443. This is in agreement with the molecular weight of 23 000 reported for the unglycosylated form of the membrane polypeptide.Virus Research 02/1984; 1(4):303-13. · 2.94 Impact Factor
Article: Structure and chemical-physical characteristics of lactate dehydrogenase-elevating virus and its RNA.[show abstract] [hide abstract]
ABSTRACT: Lactate dehydrogenase-elevating virus (LDV) was purified from culture fluid of infected primary cultures of various mouse tissues (peritoneal macrophage, bone marrow, spleen, and embryo) and from plasma of infected mice. Electron microscopy of negatively stained virus and positively stained sections of LDV revealed spherical particles of uniform size with a diameter of about 55 nm, containing an electron-dense core with a diameter of about 30 nm. During sample preparation the envelope had a tendency to slough off and disintegrate to form aggregates of various sizes and small hollow particles with a diameter of 8 to 14 nm. Two strains of LDV exhibited a density of 1.13 g/cm3 in isopycnic sucrose density gradient centrifugation whether propagated in primary cultures of the various mouse tissues or isolated from plasma of infected mice. A brief incubation of LDV in a solution containing 0.01% Nonidet P-40 or Triton X was sufficient to release the viral nucleocapsid, whereas a similar treatment had no effect on Sindbis virus. The nucleocapdis of LDV exhibited a density of 1.17 g/cm3, was devoid of phosphatidylcholine, and contained only the smallest of the viral proteins, VP-1, which had a molecular weight of about 15,000. The envelope contained two proteins. VP-2 with a molecular weight of 18,000 and a glycoprotein, VP-3, which migrated heterogenously (24,000 to 44,000 daltons) during polyacrylamide gel electrophoresis. When compared to the sedimentation rate of 29S rRNA, the RNAs of LDV and Sindbis virus sedimented at 48 and 45S, respectively, whether analyzed by zone sedimentation in sucrose density gradients containing low or high salt concentrations or denatured by treatment with formaldehyde. Our results indicate that LDV should be classified as a togavirus, but that LDV is sufficiently different from alpha and flaviviruses to be excluded from these groups.Journal of Virology 09/1975; 16(2):420-33. · 5.40 Impact Factor
Article: Sequences of 3' end of genome and of 5' end of open reading frame 1a of lactate dehydrogenase-elevating virus and common junction motifs between 5' leader and bodies of seven subgenomic mRNAs.[show abstract] [hide abstract]
ABSTRACT: The sequences of the 3'-terminal 3.7 kb of the genome and of a 1.7 kb 5' end cDNA clone of one isolate of lactate dehydrogenase-elevating virus (LDV) are reported. The 3' end sequence encodes six major independent open reading frames (ORFs 2 to 7), which are overlapping by between one and 130 nucleotides. Each ORF is expressed at the 5' end of one of six 3'-coterminal subgenomic mRNAs (mRNAs 2 to 7, respectively; 3.5 to 0.8 kb). The smallest mRNA, mRNA 7, encodes the nucleocapsid protein, VP1; mRNA 6 probably encodes the non-glycosylated envelope protein, VP2; and mRNAs 2 to 5 encode proteins of 26.0K, 21.5K, 19.2K and 22.4K, respectively, each possessing several potential N-glycosylation sites and membrane-spanning segments. About 72% of the LDV genome segment carrying ORFs 2 to 7 exhibits about 50% or higher nucleotide identity with the corresponding genome segment of swine infertility and respiratory syndrome (Lelystad) virus (LV), whereas only limited similarity is observed in discontinuous regions of the same corresponding genome segments of LDV and equine arteritis virus (EAV). EAV and LV belong to the same new group of positive-strand RNA viruses as LDV. One additional subgenomic mRNA of about 4 kb is produced in LDV- but not in EAV- or LV-infected cells. The 5' end of this mRNA (1-1) carries a continuous coding sequence. The N-terminal 80 amino acids of the predicted product exhibit about 50% identity with segments in the ORF 1b proteins of both EAV and LV. These segments are located 117 to 150 amino acids upstream of the C termini of the ORF 1b proteins of these viruses. The 5' end cDNA clone contains part of a 5' leader associated with all seven subgenomic mRNAs and the 5' end of ORF 1a. The junctions between the 5' leader and the bodies of all seven subgenomic mRNAs have been determined. Only a single junction sequence was detected for each mRNA. Linkage occurs between a 5' UAUAACC 3' sequence at the 3' end of the leader and only partially identical segments specified downstream in the genome preceding ORFs 2 to 7. The generated junctions differ for different subgenomic mRNAs but possess the consensus sequence 5' U(A/G)(U/A)AACC 3'. In mRNA 7, the UA in positions 1 and 2 are derived from the leader, but a G in position 2 in mRNAs 1-1, 3 and 4 and an A in position 3 in mRNA 6 seem to be specified by the 3' genomic sequences.(ABSTRACT TRUNCATED AT 400 WORDS)Journal of General Virology 05/1993; 74 ( Pt 4):643-59. · 3.36 Impact Factor
JOURNAL OF VIROLOGY, Apr. 1995, p. 2679–2683
Copyright ? 1995, American Society for Microbiology
Vol. 69, No. 4
Molecular Characterization of the 3? Terminus of the
Simian Hemorrhagic Fever Virus Genome
E. K. GODENY,1* L. ZENG,2S. L. SMITH,1AND M. A. BRINTON2
Department of Veterinary Microbiology and Parasitology, Louisiana State University School of
Veterinary Medicine, Baton Rouge, Louisiana 70803,1and Department of Biology,
Georgia State University, Atlanta, Georgia 303032
Received 23 September 1994/Accepted 10 January 1995
The 3? end of the simian hemorrhagic fever virus (SHFV) single-stranded RNA genome was cloned and
sequenced. Adjacent to the 3? poly(A) tract, we identified a 76-nucleotide noncoding region preceded by two
overlapping reading frames (ORFs). The ultimate 3? ORF of the viral genome encodes the capsid protein, and
the penultimate ORF encodes the smallest SHFV envelope protein. These two ORFs overlap each other by 26
nucleotides. Northern (RNA) blot hybridization analyses of cytoplasmic RNA extracts from SHFV-infected
MA-104 cells with gene-specific probes revealed the presence of full-length genomic RNA as well as six
subgenomic SHFV-specific mRNA species. The subgenomic mRNAs are 3? coterminal. In its virion morphology
and size, genome structure and length, and replication strategy, SHFV is most similar to lactate dehydroge-
nase-elevating virus, equine arteritis virus, and porcine reproductive and respiratory syndrome virus.
Simian hemorrhagic fever virus (SHFV) causes a persistent
infection in patas monkeys (Erythrocebus patas) with no overt
disease symptoms (15, 21). In contrast, this virus induces a fatal
hemorrhagic fever in monkeys within the genus Macaca (26).
Death occurs 1 to 2 weeks after the onset of symptoms in
macaque monkeys and is usually due to hypovolemic shock
(26). Within an infected primate center colony, mortality rates
of up to 100% in susceptible monkeys have been observed (21).
The SHF virion is enveloped and 45 to 50 nm in diameter.
The nucleocapsid possesses icosahedral symmetry (35). The
positive-sense, single-stranded RNA genome, which was esti-
mated to be 15,000 nucleotides (nt) in length (28), contains a
poly(A) tract at its 3? terminus (29) and a type I cap at its 5?
On the basis of virion morphology, SHFV was initially
placed in the family Togaviridae (35). However, because this
virus was shown to bud through the endoplasmic reticular
membrane (35), as do the flaviviruses, and not through the
plasma membrane of the cell, as do the togaviruses, SHFV was
subsequently reclassified into the family Flaviviridae (39). This
virus was later further classified into the hepatitis C virus group
within the family Flaviviridae (38). In 1993, on the basis of our
preliminary results (14), SHFV was reclassified as a member of
the genus Arterivirus by the International Committee on the
Taxonomy of Viruses at the International Congress of Virol-
ogy in Glasgow, Scotland.
The genus Arterivirus, originally classified within the family
Togaviridae but now free-standing, was created for equine ar-
teritis virus (EAV) when it was discovered that EAV utilized
six subgenomic mRNAs during its replication cycle (36, 40). In
contrast, other togaviruses produce only a single subgenomic
message during their replication cycle. When the EAV genome
was sequenced, its genome organization was found to be most
similar to that of the coronaviruses and the toroviruses (7).
However, EAV differs from the coronaviruses and toroviruses
in virion size and morphology as well as in genome length.
Recently, two viruses with RNA genomes closely related to
that of EAV have been sequenced: Lelystad virus (LV), a
European isolate of porcine reproductive and respiratory syn-
drome virus (PRRSV) (6, 22), and lactate dehydrogenase-
elevating virus (LDV) (12), which causes persistent infections
in mice. Both of these viruses are also morphologically similar
to EAV and have been included in the genus Arterivirus. A
study group has been formed by the International Committee
on the Taxonomy of Viruses to debate the taxonomical classi-
fication of this new group of viruses.
As a first step in determining the molecular characteristics of
SHFV, we cloned and sequenced the 3? end of the SHFV RNA
genome and examined the replication strategy of this virus.
The evidence reported here demonstrates that SHFV is most
similar to EAV, LDV, and LV/PRRSV in its genome organi-
zation and replication strategy.
To obtain purified virus as a source of genome RNA, MA-
104 cells were infected at a multiplicity of infection of 0.2 with
the prototype strain of SHFV, LVR 42-0/M6941. Tissue cul-
ture fluid was collected 24 h postinfection, clarified, and lay-
ered atop a 10 and 20% discontinuous glycerol gradient made
in TNE (10 mM Tris [pH 8.0], 0.1 M NaCl, 1 mM EDTA).
Virus was pelleted through the gradient at 24,500 rpm for 16 h.
SHFV genomic RNA was purified by sedimentation through a
15 to 40% sodium dodecyl sulfate (SDS)-sucrose gradient after
treatment of the virion pellet with SDS, pronase, and vanadyl
ribonucleoside complex as previously described (2). Peak RNA
fractions were pooled, ethanol precipitated, aliquoted, repre-
cipitated with ethanol, and stored at ?70?C until use.
Because it had previously been demonstrated that SHFV
contains a poly(A) tract at the 3? terminus of its genome (29),
oligodeoxythymidine was used to prime the viral genome for
reverse transcription. The resulting cDNA was made double
stranded with DNA polymerase by the method of Okayama
and Berg (25) and inserted into the pUC18 plasmid vector
(Pharmacia Biotec, Inc., Piscataway, N.J.), using EcoRI link-
ers. The plasmid DNA was amplified in Escherichia coli DH5?
and purified by using a Miniprep Kit Plus (Pharmacia).
Northern (RNA) blot hybridization analysis (31) using pu-
rified, full-length SHFV genome RNA and radiolabeled cDNA
probes synthesized from the cDNA clones revealed SHFV
specificity of the clones (data not shown). Two clones were
chosen and then sequenced by the dideoxy-chain termination
* Corresponding author.
method (32), using the Sequenase enzyme (United States Bio-
chemical Corp., Cleveland, Ohio). The sequences obtained
from these two clones were identical except that one clone
contained an additional adenosine residue in the poly(A) tract
relative to the other clone (12 versus 11 nt). Preceding the
poly(A) tract, 806 unique 3?-terminal SHFV nucleotides were
identified (Fig. 1). An additional 148 nt were obtained by direct
sequencing of the SHFV genomic RNA, as previously de-
scribed (12), using a primer complementary to nt 175 through
197 (Fig. 1). The amino acid sequences of the potential open
reading frames (ORFs) encoded by the SHFV 3? sequence
were derived by using the University of Wisconsin Genetics
Computer Group (GCG) software (9). No other reasonably
sized ORFs were found in this sequence.
As shown in Fig. 1, there is a 76-nt noncoding region (NCR)
adjacent to the poly(A) tract of the SHFV genome. The length
of the SHFV 3? NCR is similar to the lengths of the 3? NCRs
of EAV, LDV, and LV/PRRSV, which are 59 (7), 80 (12), and
114 (6, 22) nt, respectively. Using the GCG software GAP
program (24) with a gap weight of 5.0 and a gap length weight
of 0.3, we determined that the nucleotide identities between
the SHFV 3? NCR and the 3? NCRs of EAV, LDV, and
LV/PRRSV were only 37, 32, and 34%, respectively. The iden-
tity between the EAV and LV/PRRSV 3? NCRs was 44%,
while the identity in this region between EAV and LDV was
37%. The 3? NCRs of LDV and LV/PRRSV were most similar,
with a nucleotide identity of 47%.
It had previously been shown that the last 8 nt at the 3?
termini of the 3? NCRs of SHFV, LDV, and LV/PRRSV were
conserved, with a consensus sequence of 5? CC(A/G)(T/G/
A)AATT 3? (12). The 3?-terminal nucleotides of the EAV
genome (5? CCAGGAACC 3?) were more divergent but did
resemble the consensus sequence present in these other vi-
ruses. It has been proposed that this conserved 3? sequence
may function as a cis-acting signal for viral RNA replication
Adjacent to the SHFV 3? NCR is an ORF of 336 nt (Fig. 1)
which encodes a very basic protein of 111 amino acids with a
calculated molecular mass of 12.3 kDa and an estimated pI of
11.7 at neutral pH. Since the capsid protein genes of EAV,
LDV, and LV/PRRSV each map to the ultimate 3? ORFs of
their respective genomes and since the capsid proteins of most
RNA viruses are basic proteins, it seemed likely that the 3?
ORF of SHFV encodes the capsid protein.
To determine the map position of the capsid protein gene on
the SHFV genome, we first identified the SHFV capsid protein
among the viral structural proteins and then determined its
N-terminal amino acid sequence. Extracellular SHFV that had
been metabolically radiolabeled with a14C-amino acid mixture
(DuPont-NEN Research Products, Boston, Mass.) was har-
vested 48 h after infection at a multiplicity of infection of 0.1
and partially purified by pelleting through a discontinuous glyc-
erol gradient as described above. Pelleted virus was resus-
pended in Laemmli sample buffer (19), and the viral proteins
were separated by SDS-polyacrylamide gel electrophoresis
(PAGE) analysis. The gel was fixed, stained, and autoradio-
graphed to visualize the viral protein bands. Four prominent
SHFV protein bands were observed in the SHFV-containing
lane of the gel by this procedure (Fig. 2). These proteins
migrated with apparent molecular masses of 15, 20, 38 to 46,
and 48 to 60 kDa and were designated p15, p20, p42, and p54,
respectively (Fig. 2, lane A). Two additional protein bands with
apparent molecular masses of 28 and 35 kDa were observed;
however, preliminary Western blot (immunoblot) analyses sug-
FIG. 1. Nucleotide sequence of the 3? terminus of the SHFV genome. The amino acids encoded by the ORFs are shown below the nucleotide sequence. An asterisk
indicates a termination codon. Amino acids which were also obtained by N-terminal amino acid sequencing of the SHFV p15 (single underline) and p20 (double
underline) proteins are shown. Potential N-linked glycosylation sites are indicated in boldface.
2680NOTES J. VIROL.
gest that these two proteins may be cellular contaminants (data
not shown and reference 11). The SDS-PAGE pattern for the
SHFV proteins is similar to that previously reported for the
LDV structural proteins, which have estimated molecular
masses of 14, 18, and 24 to 44 kDa (3, 23), and the structural
proteins of EAV, with estimated molecular masses of 14, 16,
25, and 30 to 42 kDa (10).
SHFV particles were next incubated with 1% Nonidet P-40,
to remove the viral envelope, and the nucleocapsids were then
pelleted. SDS-PAGE analysis of the nucleocapsids revealed a
single protein, p15 (Fig. 2, lane C). These data indicated that
p15 is the SHFV capsid protein and that p20, p42, and p54 are
associated with the viral envelope.
To determine the N-terminal amino acid sequence of p15,
the SHFV structural proteins were separated by SDS-PAGE,
electrophoretically transferred to a polyvinylidene difluoride
membrane (Millipore Corp., Bedford, Mass.), and stained as
previously described (13). The capsid protein, p15, was excised
from the membrane, and its amino-terminal sequence was
determined by David W. Speicher of the Protein Microchem-
istry Facility at the Wistar Institute (Philadelphia, Pa.), using
an Applied Biosystems model 475A protein sequencer as de-
scribed previously (34). A 26-amino-acid sequence was ob-
tained for the N terminus of the SHFV capsid protein; this
amino acid sequence mapped to the 5? end of the ultimate 3?
ORF of the SHFV genome (Fig. 1).
Analyzed with the GCG software GAP program with a gap
weight of 3.0 and a gap length weight of 0.1, the SHFV capsid
protein showed about the same degree of amino acid similarity
to the capsid proteins of EAV (39%), LDV (39%), and LV/
PRRSV (41%). Amino acid similarity between the capsid pro-
teins of EAV and LDV was 47%, and that between EAV and
LV was 42%. The capsid proteins of LDV and LV/PRRSV
showed the greatest degree of amino acid similarity (57%).
The amino acid sequence of the SHFV capsid protein con-
tains two potential N-linked glycosylation sites: one site is
located in the middle of the capsid protein sequence, while the
other site is located toward the C terminus of the protein (Fig.
1). The capsid protein sequence of LV/PRRSV contains one
potential N-linked glycosylation site near the N terminus of the
protein (6, 22), and the EAV capsid protein sequence contains
no potential N-linked glycosylation sites (7). Although the cap-
sid protein sequence of LDV contains three putative N-linked
glycosylation sites located near the C terminus of the protein
(12, 17), it has been reported that the mature capsid protein of
LDV is not glycosylated (3). Recent attempts to metabolically
radiolabel the sugar residues on SHF virion particles indicate
that the capsid protein in mature virus particles is also not
glycosylated (data not shown and reference 11).
Adjacent to the SHFV p15 ORF is an ORF of 489 nt, which
overlaps the capsid protein ORF by 26 nt (Fig. 1). Overlapping
ORFs are characteristic of the EAV, LDV, and LV/PRRSV
genomes but are not found in the coronavirus, torovirus, to-
gavirus, or flavivirus genomes. The second ORF encodes a very
hydrophobic protein of 162 amino acids with a calculated mass
of 17.8 kDa. A hydropathicity plot of this peptide, by the
method of Kyte and Doolittle (18), and an estimation of the
secondary structure, by the method of Chou and Fasman (5),
suggest that three hydrophobic, potential membrane-spanning
regions are present within the first 90 N-terminal amino acids
of this sequence (data not shown). Proteins with similar triple
membrane-spanning structures have been shown to be en-
coded by the 3? penultimate ORFs in the genomes of LDV
(12), EAV (7, 10), and LV/PRRSV (22). A similar protein
structure has also been described for the M proteins of the
coronaviruses (1, 8, 27).
To confirm that the smallest of the SHFV envelope proteins,
p20, mapped to the penultimate 3? ORF of the SHFV genome,
the N-terminal sequence of this viral protein was determined
by the method described above for the SHFV capsid protein. A
30-amino-acid sequence was obtained, and as shown in Fig. 1,
this amino acid sequence mapped to the 5? end of the penul-
timate 3? SHFV ORF.
The amino acid similarities between the SHFV p20 se-
quence and the sequences of those proteins encoded by the
penultimate ORFs of EAV, LDV, and LV/PRRSV were de-
termined to be 48, 54, and 57%, respectively, using the GAP
program with the constraints described above for the capsid
protein comparisons. The amino acid sequence similarity be-
tween the EAV and LDV proteins was 44%, and that between
the EAV and LV/PRRSV proteins was 47%. Surprisingly, a
70% similarity was observed between the LDV and LV/
The protein sequence encoded by this second ORF contains
two potential N-linked glycosylation sites (Fig. 1). Similarly,
the peptide sequence encoded by this ORF in LV/PRRSV (6,
22) contains two putative N-linked glycosylation sites, whereas
the corresponding peptide sequences encoded by EAV (7) and
LDV (12) contain no potential N-linked glycosylation sites.
Preliminary evidence indicates that the mature SHFV p20 pro-
tein is not glycosylated (data not shown and reference 11).
EAV, LDV, and LV/PRRSV express their 3? genes from a
3?-coterminal set of subgenomic mRNAs during replication
(17, 22, 36). To investigate the possibility that SHFV generates
a similar set of subgenomic mRNAs, Northern blot hybridiza-
tion analyses were performed on cytoplasmic RNA extracts
obtained from SHFV-infected MA-104 cells. Cells were in-
fected with SHFV at a multiplicity of infection of 10. After 7 h,
cytoplasmic RNA was extracted as described by Sawicki et al.
(33), denatured with glyoxal, separated on a 1% agarose gel,
and transferred to a Magna nylon transfer membrane (Micron
Separations, Inc., Westborough, Mass.) by standard techniques
(31). The nylon membrane was baked at 80?C for 2 h and
prehybridized in 5? SSC (1? SSC is 0.15 M NaCl plus 0.015 M
FIG. 2. Autoradiograph of
teins. Proteins from extracellular SHFV particles (lane A), media obtained from
mock-infected MA-104 cells (lane B), and viral nucleocapsids (lane C) were
pelleted through a glycerol gradient and separated by SDS-PAGE. Lines indicate
the positions (kilodaltons) of standard proteins, and arrows indicate possible
SHFV structural protein bands.
14C-amino acid-labeled SHFV structural pro-
VOL. 69, 1995 NOTES2681
sodium citrate)–2 mM EDTA–0.01% SDS–200 ?g of salmon
sperm DNA per ml–50% formamide for 2 h. The RNA probe
was added, and incubation continued at 65?C for 16 h. The
membrane was washed three times with 0.1? SSC–0.1% SDS
for 20 min at 65?C and then autoradiographed at ?70?C.
The RNA probe was produced from a cloned PCR fragment
template. A forward antisense primer complementary to two
residues of the poly(A) tract and the last 18 nt in the 3? NCR
of the SHFV genome and a reverse genomic sense primer
starting 47 nt from the 5? end of the p15 ORF (Fig. 1) were
used to generate a 367-nt PCR product from the purified virion
RNA template. The PCR product was cloned into the pCRII
vector (Invitrogen Corp., San Diego, Calif.) and transfected
into E. coli DH5?. The recombinant 3?-pCRII plasmid DNA
template was linearized with BamHI, and RNA was tran-
scribed in vitro with T7 RNA polymerase in the presence of 50
?Ci of [?-32P]UTP (3,000 Ci/mmol; Amersham Corp., Arling-
ton Heights, Ill.). The DNA template was digested with DNase
after transcription, and the RNA was precipitated with etha-
nol. This antisense 3? probe detected full-length viral RNA (15
kb) as well as six subgenomic mRNAs with estimated lengths
of 4.7, 3.3, 2.7, 2.0, 1.2, and 0.65 kb in cytoplasmic extracts from
SHFV-infected MA-104 cells (Fig. 3, lane 5). No SHFV-spe-
cific RNA was detected in uninfected cell extracts (Fig. 3, lane
4). The lengths of the SHFV subgenomic mRNAs are very
similar to those of EAV (7), LDV (4), and LV/PRRSV (6, 22),
except that the largest SHFV subgenomic mRNA is approxi-
mately 1.4 kb longer than the comparable subgenomic mRNAs
produced by the other viruses. The detection of six SHFV
subgenomic RNAs with the 3? probe indicates that these
mRNAs are 3? coterminal.
Northern blot hybridization analysis was also performed
with a probe specific for the p20 gene. The cloning strategy
used to construct the DNA template for this probe and the
method used for in vitro synthesis of this RNA probe were
similar to those described above for the 3? probe. The p20
RNA probe was 200 nt in length and started with the first 5?
nucleotide of the p20 ORF (Fig. 1). As shown in Fig. 3, lane 3,
this probe hybridized to full-length SHFV genome RNA and
to the five largest subgenomic mRNAs; the smallest sub-
genomic mRNA was not detected with this probe. The p20
probe also did not detect RNA in uninfected cell extracts (Fig.
3, lane 2). It has previously been shown that the smallest EAV
subgenomic mRNA contains only the capsid protein ORF and
that the EAV capsid protein is translated from that mRNA
species (37). The evidence reported here indicates that the
smallest SHFV subgenomic RNA (0.65 kb) also contains only
the capsid protein ORF; therefore, p15 is likely to be trans-
lated from this subgenomic mRNA. It has been proposed for
EAV (37) and the coronaviruses (20) that only the 5? ORF on
each subgenomic mRNA is translated. Accordingly, it is ex-
pected that p20 is translated from the 1.2-kb SHFV sub-
We report here molecular evidence which supports the re-
cent reclassification of SHFV with EAV, LDV, and LV/
PRRSV. First, the genome organization of SHFV is similar to
that of EAV, LDV, and LV/PRRSV; the ultimate 3?-terminal
ORF of the SHFV genome encodes the capsid protein, and the
smallest envelope protein maps to the penultimate 3? ORF.
Second, the SHFV genome consists of multiple overlapping
ORFs which, among the positive-sense RNA viruses with ico-
sahedral nucleocapsids, is a characteristic unique to EAV,
LDV, and LV/PRRSV. Third, SHFV produces genome-length
RNA as well as six subgenomic mRNAs during replication.
These subgenomic SHFV mRNAs are nested at the 3? end of
the genome. EAV (36) and LV/PRRSV (6, 22) each produce
six subgenomic mRNAs during replication, while LDV pro-
duces seven subgenomic mRNAs (4).
EAV, LDV, and LV/PRRSV produce 5? leader sequences
which are derived from the 5? termini of their respective ge-
nome RNAs. These leader sequences are joined to the mRNA
bodies at conserved junction sequences, but the mechanism
utilized to accomplish this is not well understood. The consen-
sus junction sequences of LDV and LV/PRRSV are similar: 5?
U(A/G)(U/A)AACC 3? for LDV (12, 16) and 5? GNUNAACC
3? for LV/PRRSV (6, 22). The consensus junction sequence of
EAV, 5? UCAAC 3? (7), shows only some similarity to those of
LDV and LV/PRRSV. In the smallest subgenomic mRNA
species, the junction sequences of LDV, LV/PRRSV, and
EAV are located 14 (12, 16), 17 (6, 22), and 55 (7) nt, respec-
tively, upstream of the capsid protein AUG initiation codon. A
sequence, 5? UUAACC 3?, similar to the consensus junction
sequences of LDV and LV/PRRSV is located 15 nt upstream
of the p15 ORF of SHFV and functions as the junction se-
quence for the smallest SHFV subgenomic mRNA (41). Al-
though the junction sequences for the penultimate 3? ORFs of
LDV, LV/PRRSV, and EAV are located 21 (12, 16), 32 (6, 22),
and 31 (7) nt, respectively, upstream of the AUG initiation
codon for these ORFs, the junction sequence, 5? UCAACC 3?,
preceding the SHFV p20 ORF is located 133 nt upstream of
the initiation codon (41). This indicates that the 5? NCR of the
second-smallest SHFV subgenomic mRNA is longer than
those of the corresponding subgenomic mRNAs produced by
the other three viruses.
Phylogenetic analysis of conserved amino acid domains
within the EAV, LDV, and LV/PRRSV RNA-dependent
RNA polymerases and helicases, encoded within ORF 1b lo-
cated in the central portion of their respective genomes, sug-
gest that LV/PRRSV and LDV are more closely related to
each other than either is to EAV (12). As presented in this
study, amino acid sequence comparisons of both the capsid
proteins and the smallest envelope proteins among these three
viruses support these relationships. Although the percent
amino acid similarities between the capsid protein of SHFV
and that of either LV/PRRSV, LDV, and EAV do not differ
significantly, amino acid sequence comparisons of the SHFV
p20 with the corresponding protein sequences of these three
viruses indicated a greater degree of similarity between SHFV
FIG. 3. Northern blot hybridization analyses of total cytoplasmic RNA from
mock-infected (lanes 2 and 4) and SHFV-infected (lanes 3 and 5) MA-104 cells.
The nylon membrane was incubated with a p20 probe (lanes 2 and 3) or with a
3?-terminus probe (lanes 4 and 5). Lane 1 shows the migration pattern of the
RNA standards (Gibco-BRL, Gaithersburg, Md.), which were stained with
2682NOTES J. VIROL.
and LV/PRRSV or LDV than between SHFV and EAV. How-
ever, because p15 and p20 are structural proteins whose evo-
lution may be influenced by immunologic selection as well as
host species variation, the sequences of the conserved domains
in the ORF 1b region of the SHFV genome must be deter-
mined before a meaningful phylogenetic analysis can be car-
Nucleotide sequence accession number. The sequence shown
in Fig. 1 has been assigned GenBank accession number
We thank David Speicher and Kay Speicher, Wistar Institute, Phil-
adelphia, Pa., for protein sequencing and Matthew Philpott, Louisiana
State University, Baton Rouge, for critical review of the manuscript.
This work was supported by Public Health Service grants RR06841
from NCRR and NS19013 from NINDS.
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