Plum Island Animal Disease Center, Agricultural Research Service, US Department of Agriculture, Orient Point, Long Island, PO Box 848 Greenport, NY 11944-0848, USA1
Author for correspondence: Luis L. Rodriguez. Fax +1 631 323 2507. email lrodriguez{at}piadc.ars.usda.gov
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Abstract |
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Introduction |
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The viral genome consists of a linear, single-stranded, negative-sense RNA molecule of approximately 11 kb encoding five genes: the nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and polymerase (L). A 47 nucleotide leader (Le) RNA is transcribed from the 3' genomic terminus. Only 70 nucleotides two nucleotides at each of the four gene junctions, three nucleotides at the LeN junction and a 59 nucleotide trailer (Tr) sequence at the 5' terminus are not transcribed (Gallione et al., 1981 ). Two additional small proteins (C and C) are encoded in a second open reading frame within the P gene (Spiropoulou & Nichol, 1993
). Transcription of the viral genes occurs in sequential order from a single promoter at the 3' end of the genome resulting in decreasing amounts of each transcript in the order: 3' LeNPMGL 5' (Iverson & Rose, 1982
). Individual monocistronic mRNAs are transcribed by the viral transcriptase by a mechanism of pause and reinitiation controlled by a 23 nucleotide conserved sequence located at each gene junction, which contains the polyadenylation and transcription initiation signals.
Despite its widespread use as a laboratory model, only one full-length genomic sequence of VSV-IN1 is currently available in GenBank (the same sequence is found under two accession numbers: NC_001560 and J02428). This sequence is based on a composite of several laboratory strains of VSV-IN1 that have been extensively passaged in tissue culture for many years, mainly the San Juan strain (New Mexico, 1956) and the MuddSummers strain (Colorado, prior to 1956). Most studies on the molecular biology and functional analyses of the VSV genome are based on these sequences. However, it is not certain that they are representative of strains circulating under natural conditions.
This work describes the first complete genomic sequencing of three natural isolates of VSV-IN1, each representing a distinct genetic lineage of different geographical origin. We show detailed analyses of all genes and gene junctions for each of these viruses and compare them with those of the laboratory strain previously described in the literature. These full-length genomic sequences should be useful in the design and interpretation of future functional genomic analyses using reverse genetics.
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Methods |
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Total RNA was extracted directly from 1 g of macerated epithelium by the acid guanidine thiocyanate method using Trizol (Invitrogen), as previously described (Rodriguez et al., 1997 ). RNA pellets were resuspended in sterile water and kept at -70 °C until tested. In order to obtain enough RNA for sequencing the genomic termini, virion RNA was extracted similarly from sucrose-gradient-purified virions from passage 1 virus in BHK-21 cells, as described previously (Rodriguez et al., 1993
).
RTPCR and cloning.
Reverse transcription was carried out using random hexamers (Invitrogen) and SuperScript II RNase H Reverse Transcriptase (Invitrogen), following the manufacturer's instructions. Alternatively, RTPCR was carried out using the one-tube rTth RNA PCR System (GeneAmp EZ rTth RNA PCR kit, Perkin-Elmer), as previously described (Rodriguez et al., 1997 ). To determine the sequence of genomic termini, template RNA was extracted from sucrose-gradient-purified virions and ligated using T4 RNA ligase (Mandl et al., 1991
). Primers for PCR and sequencing reactions were designed based on the published sequence of VSV-IN1 (GenBank accession no. J02428) or based on newly obtained sequences (primer sequences are available from the corresponding author on request). PCR reactions were performed using Pfu DNA polymerase (Stratagene), according to the manufacturer's instructions. Nucleic acid amplifications were performed in a 9600 Perkin-Elmer thermocycler (PE Applied Biosystems) using the temperature profiles within the following ranges, depending on expected product size and primer sequence: 94 °C for 3 min, followed by 35 cycles of 94 °C for 3060 s, 5060 °C for 3060 s, 72 °C for 13 min, and a final elongation step at 72 °C for 715 min. Products were analysed by electrophoresis on agarose gels and visualized by ethidium bromide staining. To characterize the sequence of the insert at the 5' (genomic sense) non-coding region of the glycoprotein, we cloned an RTPCR product comprising the region from the G stop codon to the L start codon of virus 94GUB using the Zero Blunt TOPO PCR (Cloning Kit Version D, Invitrogen). Ten different colonies containing an insert were selected and plasmid DNA was purified for sequencing.
DNA sequencing, alignments and phylogenetic analysis.
Single-band products were purified directly from the RTPCR reaction using QIAquick PCR purification kits (Qiagen). Multiple-band products were separated in agarose gels and extracted using the QIAquick gel extraction kit (Qiagen). PCR products were sequenced by dideoxy-sequencing using a BigDye Terminator Sequencing kit on 373XL or 3700 automated sequencing instruments (PE Applied Biosystems), as previously described (Rodriguez et al., 2000 ). The Sequencher software (GeneCodes) was used to analyse nucleotide sequence fragments and assemble contigs. Consensus sequences were derived from at least two independent forward and reverse sequences, but in most cases there was extensive sequence overlap and at least four sequences in each direction were available. Protein alignments were performed using MEGALIGN (DNAStar) or ClustalX (Thompson et al., 1997
). Sliding window analysis was performed with similarity scores obtained from ClustalX using the Gonnet PAM250 protein weight matrix (Thompson et al., 1994
). Divergence scores (100-similarity score) for each 30-amino acid window were plotted using Excel (Microsoft). Phylogenetic analysis was performed by maximum parsimony using PAUP 4.0 beta version (available from D. L. Swofford) in a Power Macintosh G3. Maximum parsimony settings included a character weighting of 2:1 transition/transversion (ts/tv) ratio, and the branch-swapping algorithm was tree-bisection-reconnection (TBR). Bootstrap analysis was carried out by performing 1000 replicates.
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Results and Discussion |
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The (U)7 polyadenylation signal was followed by two non-transcribed nucleotides. These non-transcribed intergenic nucleotides are an important part of the cis-acting signals involved in termination and reinitiation of transcription in VSV (Barr et al., 1997a ; Stillman & Whitt, 1997
). The non-transcribed intergenic nucleotides 3' GA 5' and 3' CA 5' between N and P and between P and M, respectively, were conserved among all viruses sequenced (Table 1
). In contrast, the MG and GL intergenic nucleotides were more permissive to changes in the natural isolates from Central and South America, with each virus lineage having different intergenic nucleotides at the MG junction, including the trinucleotide 3' GGA 5' in the South American lineage and either 3' GG 5'or 3' GA 5' in the Central American lineage (Table 1
). We determined the sequence of the intergenic nucleotides between M and G and between G and L for five additional viruses from each genetic lineage and found them to be identical to the representative full-length sequence within the North American or South American lineages. However, the GL junction was variable in the Central American lineage with three different intergenic nucleotides found among the five viruses sequenced (Table 1
).
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Nucleocapsid
N is the most abundant viral protein in infected cells. It binds both genomic and replicative intermediate viral RNA and, together with the L and P proteins, is an indispensable component of the transcriptionreplication complex. We found that N, which is 422 amino acids long, was the most conserved protein among all the viruses (Fig. 3). Only five amino acid substitutions (positions 33, 259 and 360 in 94GUB and positions 11 and 110 in 85CLB) were found among all three viruses sequenced. None of these changes were located within the last 60 amino acids of the C-terminal end of N, where sequences required for interaction with the phosphoprotein and also for encapsidation are believed to be located (Pattnaik et al., 1995
). Its high conservation, coupled with the fact that N is the most abundant transcript in infected cells, makes it an ideal target for diagnostic probes.
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C proteins
Two small basic proteins, C' and C (67 and 55 amino acids, respectively), are encoded in a second open reading frame (ORF) within the P gene of VSV-IN1 (Spiropoulou & Nichol, 1993 ; Kretzschmar et al., 1996
). The role of these proteins in the VSV life cycle remains unknown, since viruses in which C protein expression has been abrogated grow in vitro similarly to the wild-type strain (Kretzschmar et al., 1996
). All viruses had the second ORF with conserved start codons for both C and C. In all cases, the AUG of the smaller C protein was in better context for translation, indicating that it is preferentially translated (Spiropoulou & Nichol, 1993
; Kozak, 1986
). The C protein was the most divergent among all the VSV proteins (followed by P, which was the most divergent among the structural proteins) (Fig. 3
). However, the predicted isoelectric point, dictated by the number of arginines in each protein, remained between 10·86 and 11·28 in all viruses. Interestingly, C was the only VSV gene product with a substantially higher number of non-synonymous versus synonymous substitutions (Fig. 3
). This indicates that C is under different selective pressures than P, despite the fact that both proteins are encoded by the same mRNA.
Matrix protein
M is an important component in the virus structure, playing a major role in virus morphology, assembly and budding. It is also involved in inducing host-cell cytopathic effect, inhibition of host gene expression, nuclear transport and apoptosis (Kopecky et al., 2001 ; Desforges et al., 2001
; Petersen et al., 2000
; Wagner & Rose, 1996
). M, which is 229 amino acids in length, was very conserved at the amino acid level, with only seven differences among all the viruses (positions 54, 133 and 225 in virus J02428; 70 and 221 in 98COE; and 126 and 171 in 94GUB) (Fig. 3
). At position 2427, all viruses had a PPPY motif similar to the late domains in retroviral Gag proteins, which seems to be required for the efficient release of viral particles (Jayakar et al., 2000
).
Glycoprotein
G has several functions, including fusion with the cell membrane during virus entry and membrane budding during exit from the cell. It is also the target of neutralizing antibodies and cell-mediated immune responses to VSV (Wagner & Rose, 1996 ). Trimers of G form the spikes protruding from the viral envelope, which interact with cellular receptors. Functional domains identified in G include glycosylation and palmitylation sites, a membrane anchor, a cytoplasmic domain and membrane fusion domains (Coll, 1995
).
After P, G was the second least conserved of the major viral proteins, with 95·597·5 amino acid conservation (Fig. 3). There were a total of 41 amino acid differences among the four viruses; 16 of these occurred within 30 amino acids of either the N terminus in the ectodomain, or the C terminus in the cytoplasmic domain. No changes occurred at or near glycosylation or palmitylation sites. Two amino acid changes (position 258 in 98COE and 259 in 94GUB) were observed at or near epitopes where neutralization-resistant mutations have been selected in vitro (Luo et al., 1988
). This could suggest that there is immune selection among these viruses under natural conditions. However, no accumulation of changes in these areas of the glycoprotein was observed among viruses from Central and North America previously sequenced (Rodriguez et al., 2000
). Two domains involved in fusion, one at amino acid position 118139 (Shokralla et al., 1998
) and the other at position 395462 (Fredericksen & Whitt, 1998
) were completely conserved among the four viruses compared.
Insertion in the genomic 5' non-coding region of the G gene
The 5' (genomic sense) non-coding region of the glycoprotein gene is highly variable both in length and sequence composition, among the genetic lineages of VSV. A reiterative 175 nucleotide insertion of 3' UUAAAAA 5' was found between the stop codon of G and the GL gene junction of 94GUB. We sequenced the G gene 5' non-coding regions of nine other VSV-IN1 isolates from throughout Central America, and found inserts of variable length only in viruses within one genetic lineage from northern Central America (Guatemala, Honduras, El Salvador) and not in viruses from southern Central America, North or South America (Fig. 1). Insertions had been previously noticed in the VSV-IN1 G mRNA by Bilsel & Nichol (1990)
. Until now, it was not clear whether this insert was added by the viral polymerase by stuttering during transcription of the mRNA, or if it was present in the viral genome. In order to clarify this, we used gradient-purified virion RNA as template for RTPCR of this region and confirmed the presence of the insert in the genome. How this insert arose and was maintained in this lineage is not certain. Our data supports the idea that it was created by stuttering of the viral polymerase on the sequence 5' AAUUUUU 3' near the U7 track in the GL junction of the positive-sense RNA template during virus replication, as first proposed by Bilsel & Nichol (1990)
. The polymerase stuttering event might have been favoured by the presence of the polyadenylation signal 3' AUAC 5', which occurs prior to the 5' AAUUUUU 3' only in the northern Central American lineage. Once this insert arose in an ancestral virus, it could have become fixed due to the lack of a recombination mechanism in Mononegavirales (Pringle, 1981).
Since the length of the 5' non-coding sequence in the G gene was variable among the viruses studied, we wanted to determine its diversity within the quasispecies of a single virus strain. In order to test this, we cloned the resulting RTPCR product of the GL region of 94GUB, from the stop codon of G to the start codon of L, into the TOPO vector and selected ten individual colonies for sequencing. Four of the colonies had a 1 nucleotide deletion at position 14, four had a 1 nucleotide deletion at position 55 and one colony had a 30 nucleotide deletion at position 142171 (Fig. 4). Despite this variability, we found the same consensus 175 nucleotide sequence in at least three independent 94GUB RNA preparations when RTPCR products were directly sequenced. Interestingly, the GL region is also the site where other rhabdoviruses such as snakehead rhabdovirus and rabies virus (Kurath et al., 1997
; Johnson et al., 2000
; Ravkov et al., 1995
) have additional genes. It seems that the GL junction of rhabdoviruses in general is very permissive to insertions and deletions and perhaps is the site of extinct genes in VSV.
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The exact domains within L associated with each of its functions are not clear but conserved motifs have been described in at least six areas of conservation among the Mononegavirales and these have been proposed as putative active sites (Poch et al., 1990 ). A sliding window analysis of the L amino acid alignment showed that, for the most part, these areas were of high amino acid conservation among the four virus sequences (Fig. 5
). The polymerase motif QGDNQ was found at position 712716 in all viruses in an area of low divergence within domain III (Fig. 5
). The putative template recognition sequences within domain II at amino acids 530660 were also completely conserved. Since these L sequences are the first from field strains without passage in tissue culture, they could be useful in determining the functional domains of this protein.
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Acknowledgments |
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Footnotes |
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a Present address: Advanced Life Science Products, Corning Inc., Corning, NY 14831, USA.
b Present address: Animal Health and Biomedical Sciences, University of WisconsinMadison, WI 53706, USA.
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References |
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Received 31 January 2002;
accepted 20 May 2002.