Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK1
Department of Virology, The WrightFleming Institute, Faculty of Medicine, Imperial College, St Marys Campus, Norfolk Place, London W2 1PG, UK2
Author for correspondence: Geoffrey Smith (at Imperial College). Fax +44 207 594 3973. e-mail glsmith{at}ic.ac.uk
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The terminal regions of the VV genome are variable as exemplified by the isolation of spontaneous mutants with rearrangements or deletions in these regions (Wittek et al., 1978 ; Panicali et al., 1981
). One such VV strain, Western Reserve (WR) mutant (6/2), contains a large deletion near the left terminus (Moss et al., 1981
); it exhibits normal growth properties in tissue culture, but is attenuated in vivo (Buller et al., 1985
). Nucleotide sequence analysis of this region of the parental virus genome (HindIII C and N fragments) and comparison to virus 6/2 revealed that the 6/2 genome had undergone a transposition event resulting in the loss of 17 open reading frames (ORFs) (Kotwal & Moss, 1988
). Among the ORFs missing from the 6/2 mutant is N1L, which was predicted to encode a 13·8 kDa protein (Kotwal & Moss, 1988
). Subsequently, this ORF was reported to encode one of several proteins of approximately 12 kDa in the supernatants of VV-infected cells (Kotwal et al., 1989
). The contribution of the N1L protein to the attenuated phenotype of the 6/2 mutant was analysed by construction of a VV-recombinant lacking a functional N1L gene. This virus was attenuated in intracranial and intraperitoneal mouse infection models (Kotwal et al., 1989
), although a revertant virus was not reported.
Here the VV N1L protein has been characterized more extensively and its role in the virus life-cycle has been studied by the construction of deletion and revertant viruses. The N1L protein synthesized by VV WR-infected cells is a non-glycosylated, non-covalent homodimer that is present predominately within infected cells. The deletion mutant was attenuated compared to wild-type and revertant controls in two murine models of infection.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Construction of VV N1L deletion and revertant viruses.
A virus lacking 88% of the N1L ORF (nucleotides -78 to +276 relative to the N1L initiation codon) was constructed by transient dominant selection (Falkner & Moss, 1990 ) using the E. coli guanine xanthine phosphoribosyltransferase (Ecogpt) gene as the selectable marker (Boyle & Coupar, 1988
). The entire N1L ORF and left and right flanking regions from VV strain WR were amplified by a PCR using VV WR genome DNA as template, and as primers oligonucleotides (5' ACAGAGCTCCGGATATTCTTCTAC and 5' TCGCTGCAGACCTAATCAACATCACACC) that contained SstI and PstI restriction sites (underlined), respectively. This DNA fragment was digested with these enzymes and cloned into plasmid pSJH7 (Hughes et al., 1991
) forming pN1L and the fidelity of the DNA sequence was confirmed by DNA sequencing. To generate p
N1L, plasmid pN1L was linearized with Tth3I and digested with Bal31 exonuclease before religation. The size of the deletion within individual clones was characterized initially by PCR using the above primers and then by sequencing. A plasmid with a deletion from nucleotide -78 to +276 relative to the 5' end of the ORF was called p
N1L. This was transfected into VV WR-infected CV-1 cells and a deletion mutant (v
N1L) and a wild-type control virus (vN1L) derived from the same intermediate virus were isolated as described for the A41L gene (Ng et al., 2001
). A revertant virus (vN1L-rev) was derived in a similar way by transfecting plasmid pN1L into cells infected with v
N1L.
Expression and purification of recombinant N1L protein.
The N1L protein was expressed from Autographa californica nuclear polyhedrosis virus (AcNPV). The N1L ORF with or without six histidine residues at the C terminus was amplified by PCR using as primers oligonucleotides (5' TAAGGTACCAAGCATGAGGACTC and 5' ATTGGATCCTTATTTTTCACCATATAG) that contained KpnI and BamHI sites (underlined), or (5' AAAGGTACCGAATTCTAAGCATGAGGACTCTAC and 5' ATTCTCGAGTTTTTCACCATATAG) that contained EcoRI and XhoI sites (underlined), and VV WR DNA as template. The resultant DNA fragments were digested with the appropriate enzymes and cloned into plasmids pAcCL29-1 and pBAC1, respectively. The resultant plasmids, pAcN1L and pAcN1L-his, were sequenced to confirm the fidelity of the PCR-derived material and were used to construct AcNPV recombinants AcN1L and AcN1L-his, respectively. Recombinant proteins made by these viruses were called AN1L and AN1L-his. AN1L-his was purified by nickel chelate and ion exchange chromatography as described for the A41L protein (Ng et al., 2001 ).
For expression in E. coli the N1L ORF was amplified by a PCR using VV WR genomic DNA as template and as primers oligonucleotides (5' GGTTCCCATATGAGGACTCTACTTATTAGATATATTC and 5' CCTTGAATTCTTATTTTTCACCATATAGATC) that contained NdeI and EcoRI sites (underlined). Alternatively, the N1L ORF with a C-terminal (6)His tag was amplified by PCR using the same 5' primer and as the 3' primer an oligonucleotide (5' CCTTCTCGAGTTTTTCACCATATAGATC) containing a XhoI site (underlined) and lacking a stop codon. The resultant DNA fragments were digested with the appropriate enzymes and cloned into NdeI- and EcoRI- or NdeI- and XhoI-restricted pET24a (Novagen) to generate pETN1L and pETN1Lhis, respectively. Recombinant proteins were expressed from E. coli using the pET system (Novagen) according to the manufacturers specifications and were called EN1L and EN1L-his, respectively. EN1L-his was purified by Ni2+-affinty chromatography [HiTrap chelating HP column, AmershamPharmacia Biotech (APB)] and gel filtration using a calibrated Superdex 75 HR column (APB). EN1L protein was purified by ion-exchange chromatography using a ResourceQ column (APB), gel filtration (as described above) and a final polishing step using a MonoQ HR column (APB). Buffers all contained 1 mM dithiothreitol.
Production of polyclonal antiserum and purification of IgG.
A polyclonal antiserum (-N1L) was generated by immunizing New Zealand White rabbits with the purified AN1L-his protein according to standard protocols (Harlow & Lane, 1988
). The IgG fraction was isolated from the antiserum by precipitation with 65% (w/v) (NH4)2SO4 and affinity purification on a HiTrap protein ASepharose column (APB) as instructed by the manufacturer.
Immunoblotting.
BS-C-1 cells were mock-infected or were infected with the indicated viruses at 10 p.f.u. per cell in the presence, where specified, of 40 µg/ml -D-arabinofuranoside (AraC), 1 µg/ml tunicamycin or 1 µM monensin (all Sigma). At 24 h post-infection (p.i.) cell extracts were prepared as described previously (Ng et al., 2001
). Supernatants were centrifuged (3000 g, 10 min) to remove cellular debris before concentration in a centrifugal filter device (Amicon). Where indicated, supernatants were ultracentrifuged (14000 g for 2 h) to pellet extracellular virus. Cell extracts and supernatants were analysed by SDSPAGE (15% gel), transferred to nitrocellulose and probed with rabbit
-N1L IgG or mouse mAb AB1.1 against the VV D8L protein (Parkinson & Smith, 1994
) (each diluted 1:1000). Bound IgG was detected by incubation with the appropriate horseradish peroxidase-conjugated IgG (Sigma) (diluted 1:2000) followed by the enhanced chemiluminescence (ECL) detection system (APB).
Pulsechase and immunoprecipitation.
BS-C-1 cells were infected at 10 p.f.u. per cell with VV WR. At 4 h p.i. the culture medium was removed, the cells were washed in methionine- and cysteine-free medium and the cells were incubated in the same medium for 20 min. Cells were pulsed with 100 µCi [35S]methionine and [35S]cysteine (1000 Ci/mmol)/106 cells (APB) for 20 min, the cells were washed with DMEM containing 2 mM unlabelled methionine and cysteine and then incubated in the same. At the indicated times, the supernatants were collected and filtered (0·2 µm) to remove cellular debris. Cells were washed with ice-cold PBS before being scraped into lysis buffer (1·0 ml/106 cells) and harvested. For immunoprecipitations, cell extracts and supernatants were pre-adsorbed against protein Aagarose (Pierce) and then incubated with either -N1L or
-A41L. Immune complexes were captured with protein Aagarose, washed, eluted in Laemmli buffer, and proteins were resolved by SDSPAGE (15% gel) and visualized by fluorography.
Radio-iodination of the N1L protein.
N1L protein was purified from the supernatants of AcN1L-infected insect cells as described above. This protein was then radio-iodinated using the BoltonHunter reagent as described previously for human interferon-2 (Symons et al., 1995
).
Immunofluorescence.
BS-C-1 cells were grown on 13 mm glass coverslips and were infected with VV WR or vN1L at 1 p.f.u. per cell. For surface staining of live cells, the culture medium was removed at 18 h p.i. and was replaced with DMEM containing rabbit
-N1L IgG (diluted 1:100) or rat anti-B5R mAb 19C2 (Schmelz et al., 1994
) (hybridoma culture supernatant diluted 1:100) and the incubation was continued for 1 h at 4 °C. Cells were washed three times with ice-cold PBS and then fixed and permeabilized by incubation in methanol at -20 °C for 10 min. Cells were washed thoroughly with PBS, and then incubated in PBS containing 10% FBS for 20 min at room temperature followed by rhodamine-conjugated donkey anti-rabbit IgG, or Texas red b-isothiocyanate (TRITC)-conjugated donkey anti-rat IgG (both from Jackson Laboratories) in PBS containing 10% FBS for 30 min at 37 °C. Cells were washed three times in PBS containing 10% FBS and once in water before mounting in Mowiol as described previously (Sanderson et al., 1996
).
To stain fixed and permeabilized cells, the culture medium was removed at 18 h p.i. and cells were fixed and permeabilized in methanol as described above. Primary antibody staining was performed at 37 °C for 1 h. Addition of secondary conjugated antibodies and mounting were as described above. All samples were examined with a Zeiss LSM 510 laser scanning confocal microscope and images were captured and processed using Zeiss LSM Image Browser version 3.1.0.99.
Mouse infection models.
The virulence of the recombinant viruses was determined in female BALB/c mice 68 weeks old. Mice were anaesthetized and infected intranasally with VV in 25 µl of PBS (Gardner et al., 2001 ), or by intradermal injection of 104 p.f.u. of VV in 10 µl in the left ear pinna (Tscharke et al., 2002
). For intranasal infections, mice were weighed daily and signs of illness were scored as described previously (Alcamí & Smith, 1992
). Mice were euthanized if they lost more than 30% of their body weight. For intradermal infections, the sizes of lesions on infected ears were estimated daily with the aid of a micrometer. To determine virus titres, infected ears were ground with a tissue homogenizer, subjected to three cycles of freezing and thawing and two sonications and the resulting homogenate was assayed on BS-C-1 cells (Tscharke & Smith, 1999
).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Growth properties of N1L deletion and revertant viruses
Previous work demonstrated that the N1L gene is not essential for virus replication in tissue culture and that a mutant virus with the N1L gene disrupted was attenuated in vivo (Kotwal et al., 1989 ). However, in the absence of a revertant virus it was not proven that the loss of N1L contributed to this attenuated phenotype. Therefore, we constructed wild-type, deletion and revertant viruses (Methods) and studied their properties in cell culture and in animal models. The genomic structure of these viruses was analysed by PCR and Southern blotting (see supplementary data 1 at JGV Online: http://vir.sgmjournals.org). After DNA was extracted from purified virus and digested with HindIII, the HindIII N and C fragments (approximately 1·6 kb and >12·2 kb, respectively) were present with vN1L and vN1L-rev. In contrast, with v
N1L the HindIII N fragment was not detected and the C fragment increased in size slightly due to the loss of the HindIII site between the N and C fragments (Supplementary data Fig. 1a
). Similarly, after digestion with either EcoRI or BglII, bands of equivalent size were detected with vN1L and vN1L-rev but these were reduced in size by approximately 350 bp for v
N1L. These observations confirmed the absence of the N1L gene from v
N1L, consistent with the lack of N1L protein expression by this virus (Fig. 2c
).
The growth properties of vN1L, vN1L and vN1L-rev were examined in cell culture and found to be indistinguishable. The rate of increase in virus infectivity after infection and the final titre obtained were the same for each virus (supplementary data 2a). Moreover, there were no differences in the amount of intracellular or extracellular virus produced and the plaques formed by each virus were of equal size (supplementary data 2b). These data confirm that the N1L gene is dispensable for virus growth in cell culture.
The N1L protein is predominantly intracellular
To examine the kinetics of N1L protein expression, cells were infected with VV WR in the presence or absence of AraC, an inhibitor of DNA replication and late gene expression. Immunoblotting detected the 14 kDa N1L protein in cell extracts in the presence of AraC indicating early expression (Fig. 3a). This was consistent with transcriptional data that identified early and late RNA start sites upstream of the N1L initiation codon (Kotwal & Moss, 1988
). Addition of monensin or tunicamycin did not affect the size of the N1L protein indicating this protein was not post-translationally modified by attachment of O- or N-linked carbohydrate (Fig. 3a
).
|
To examine the release of the N1L protein further, the kinetics of N1L protein release from cells were investigated by pulsechase analysis and were compared with the secretion of the VV A41L protein, an immunomodulatory glycoprotein secreted into the culture supernatant (Ng et al., 2001 ). VV-infected cells were pulsed with [35S]methionine and [35S]cysteine and chased for the indicated periods of time. Cells and supernatants were harvested and proteins were immunoprecipitated with either
-N1L IgG or anti-A41L polyclonal serum, resolved by SDSPAGE and visualized by fluorography (Fig. 4
). The A41L protein was exported efficiently from infected cells and the majority of radioactive protein was in the supernatant within 60 min of pulse. In contrast, the N1L protein appeared to be released from cells slowly and at all times examined the great majority remained in the cell extract. Therefore, it is unlikely that N1L is a true secreted protein, but rather its presence in supernatants is most likely a result of leakage from infected cells.
|
The location of the N1L protein was also examined by immunofluorescence (Fig. 5). BS-C-1 cells were infected with vN1L or v
N1L for 16 h and were stained with
-N1L IgG and mAb 19C2 directed against the B5R protein that is present on the cell surface and cell-associated enveloped virus and extracellular enveloped virus particles. Bound Abs were detected with appropriate secondary Abs and visualized by confocal microscopy (Methods). The NIL protein was detected predominantly in the cytoplasm of WR-infected cells that had been permeabilized prior to addition of Ab (Fig. 5a
), but only background fluorescence was observed if the cells were stained prior to permeabilization (Fig. 5d
), or if the cells were infected with v
N1L (Fig. 5b
, g
). Phase contrast images confirmed the presence of cells in the fields shown in panels (d) and (g) (Fig. 5e
, h
, respectively). In contrast, the B5R protein was detected in cells infected with either virus and was detected in cells that had or had not been permeabilized (Fig. 5c
, f
, i
). These data show that N1L is an intracellular protein and is not present on the cell surface.
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although the N1L protein was described originally as a secreted protein, analysis of the distribution of the N1L protein showed that it was present predominantly within infected cells, and only a small fraction (<10 %) was released into the cell culture medium (Fig. 3b). How this protein is released is unclear, but the N terminus of N1L lacks a conventional signal peptide and amino acid sequencing showed that the N1L protein released from insect cells infected with AcN1L is uncleaved at the N terminus. Moreover, pulsechase analyses established that in comparison with glycoprotein A41L, which is expressed by VV during the same phase of infection, has a signal peptide and is secreted from infected cells, the N1L protein leaks out of cells very slowly and the majority of the protein is always present within cells (Fig. 4
). The N1L protein also has a distribution comparable with the VV D8L protein that is described as intracellular and a component of released virions. In addition, immunofluorescence showed the protein was predominantly cytoplasmic and was not present on the cell surface (Fig. 5
). Collectively, these observations suggest that the small fraction of the total N1L protein released into the cell supernatant might be due to lysis of some cells following virus infection; alternatively, some N1L protein might be released by an unidentified and unconventional pathway.
Biochemical analyses of N1L protein made by VV showed that the protein is a homodimer, and that this dimer can be disrupted by treatment with SDS but not reducing agents. So non-covalent bonds hold the dimer together. Recombinant N1L protein, synthesized in E. coli or in insect cells infected with recombinant baculoviruses, forms additional oligomeric structures that contain disulphide bonds. As there is only a single cysteine residue in N1L, these bonds must be intermolecular. Purification of recombinant N1L was therefore performed in the presence of reducing agent.
VV expresses many proteins that are non-essential for virus replication in cell culture but which contribute to virus virulence in vivo and several proteins of this group function as immunomodulators to affect the host response to infection. VV immunomodulators may be grouped according to their site of action. Those proteins secreted from the infected cell usually bind to and inhibit the function of host proteins such as interferons, cytokines, chemokines or complement, whereas intracellular proteins may interfere with interferon-induced antiviral proteins, cell signalling pathways or apoptosis. The correct location of the protein is essential to mediate these different functions. In several cases the function of VV immunomodulatory proteins has been inferred from computational analyses of amino acid sequences that revealed similarities between a VV protein and a host protein of known function. In other cases, the function of a virus protein has been deduced by functional studies. In the case of N1L, although bioinformatic analyses established that the protein is conserved in several orthopoxviruses and in other chordopoxvirus genera, no amino acid similarity with host proteins was detected. Predictions of the secondary structure of N1L showed a largely alpha-helical protein that has a similar overall topology to other small alpha-helical proteins such as IL-10 and interferon-. This similarity, taken together with the proposed secreted nature of N1L, led this protein to be described by some as a virokine (Chang et al., 1992
) and suggested a possible mechanism by which N1L contributed to virulence. However, data presented here cause the mechanism of action of N1L to be reconsidered. Computational analyses suggest that N1L is also related structurally to intracellular alpha-helical proteins such as the N-terminal actin-cross-linking domain of fimbrin (pdb 1aoa), histone acetyltransferase bromodomain (pdb 1b91) and the N-terminal domain of transcription elongation factor TFIIS (pdb 1eo0). It remains possible that the small fraction of N1L that is released from cells mediates an important extracellular function; however, it is more likely that the site of action of N1L is intracellular where the majority of the protein is located. Determination of the mechanism of action of N1L requires additional study.
Data presented here confirm that N1L promotes VV virulence and does so in both the intradermal and intranasal models of infection in mice. This is in contrast to a variety of other VV proteins that are associated with virulence only after inoculation by one of these routes (Tscharke et al., 2002 ). Furthermore in each model, the attenuation caused by deletion of N1L is amongst the strongest seen for genes that do not affect growth in culture. The attenuation seen in the intradermal model was accompanied by a reduction in virus titre in the infected skin, indicating that N1L expression may affect either virus growth in vivo or influence the rate at which virus is cleared by the host response to infection.
In conclusion, the N1L protein is demonstrated to be a non-covalent homodimer that is largely intracellular and contributes to virulence in intradermal and intranasal models of infection by an unknown mechanism.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
b Present address: Roche Bioscience, 3401 Hillview Avenue, Palo Alto, CA 94304, USA.
c Present address: Cell Biology and Viral Immunology Sections, Laboratory of Viral Diseases, NIAID, NIH, Bethesda, MD 20892, USA.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alcamí, A. & Smith, G. L. (1992). A soluble receptor for interleukin-1 beta encoded by vaccinia virus: a novel mechanism of virus modulation of the host response to infection. Cell 71, 153-167.[Medline]
Alcamí, A. & Smith, G. L. (1995). Vaccinia, cowpox, and camelpox viruses encode soluble gamma interferon receptors with novel broad species specificity. Journal of Virology 69, 4633-4639.[Abstract]
Alcamí, A., Symons, J. A., Collins, P. D., Williams, T. J. & Smith, G. L. (1998). Blockade of chemokine activity by a soluble chemokine binding protein from vaccinia virus. Journal of Immunology 160, 624-633.
Antoine, G., Scheiflinger, F., Dorner, F. & Falkner, F. G. (1998). The complete genomic sequence of the modified vaccinia Ankara strain: comparison with other orthopoxviruses. Virology 244, 365-396.[Medline]
Boyle, D. B. & Coupar, B. E. H. (1988). A dominant selectable marker for the construction of recombinant poxviruses. Gene 65, 123-128.[Medline]
Buller, R. M., Smith, G. L., Cremer, K., Notkins, A. L. & Moss, B. (1985). Decreased virulence of recombinant vaccinia virus expression vectors is associated with a thymidine kinase-negative phenotype. Nature 317, 813-815.[Medline]
Cameron, C., Hota-Mitchell, S., Chen, L., Barrett, J., Cao, J. X., Macaulay, C., Willer, D., Evans, D. & McFadden, G. (1999). The complete DNA sequence of myxoma virus. Virology 264, 298-318.[Medline]
Chang, P. Y., Lai, A. C. & Pogo, B. G. (1992). Attenuated deletion mutant of vaccinia virus IHD-W recovered virulence by reinsertion of a terminal restriction fragment. Microbial Pathogenesis 13, 49-59.[Medline]
Falkner, F. G. & Moss, B. (1990). Transient dominant selection of recombinant vaccinia viruses. Journal of Virology 64, 3108-3111.[Medline]
Gardner, J. D., Tscharke, D. C., Reading, P. C. & Smith, G. L. (2001). Vaccinia virus semaphorin A39R is a 5055 kDa secreted glycoprotein that affects the outcome of infection in a murine intradermal model. Journal of General Virology 82, 2083-2093.
Goebel, S. J., Johnson, G. P., Perkus, M. E., Davis, S. W., Winslow, J. P. & Paoletti, E. (1990). The complete DNA sequence of vaccinia virus. Virology 179, 247-266.[Medline]
Harlow, E. & Lane, D. (1988). Antibodies: a Laboratory Manual, pp. 1726. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Hughes, S. J., Johnston, L. H., de Carlos, A. & Smith, G. L. (1991). Vaccinia virus encodes an active thymidylate kinase that complements a cdc8 mutant of Saccharomyces cerevisiae. Journal of Biological Chemistry 266, 20103-20109.
Kelley, L. A., MacCallum, R. & Sternberg, M. J. E. (1999). Recognition of remote routeing homologies using three-dimensional information to generate a position specific scoring matrix in the program 3D-PSSM. In Third Annual Conference on Computational Molecular Biology , pp. 218-225. Edited by S. Istrail, P. Pevzner & W. Waterman. New York:The Association for Computational Machinery.
Kelley, L. A., MacCallum, R. M. & Sternberg, M. J. (2000). Enhanced genome annotation using structural profiles in the program 3D-PSSM. Journal of Molecular Biology 299, 499-520.[Medline]
Kotwal, G. J. & Moss, B. (1988). Analysis of a large cluster of nonessential genes deleted from a vaccinia virus terminal transposition mutant. Virology 167, 524-537.[Medline]
Kotwal, G. J., Hugin, A. W. & Moss, B. (1989). Mapping and insertional mutagenesis of a vaccinia virus gene encoding a 13,800-Da secreted protein. Virology 171, 579-587.[Medline]
Kyte, J. & Doolittle, R. F. (1982). A simple method for displaying the hydropathic character of a protein. Journal of Molecular Biology 157, 105-132.[Medline]
Mackett, M., Smith, G. L. & Moss, B. (1985). The construction and characterization of vaccinia virus recombinants expressing foreign genes. In DNA Cloning: a Practical Approach , pp. 191-211. Edited by D. M. Glover. Oxford:IRL Press.
Massung, R. F., Liu, L. I., Qi, J., Knight, J. C., Yuran, T. E., Kerlavage, A. R., Parsons, J. M., Venter, J. C. & Esposito, J. J. (1994). Analysis of the complete genome of smallpox variola major virus strain Bangladesh-1975. Virology 201, 215-240.[Medline]
Moss, B. (2001). Poxviridae: the viruses and their replication. In Virology , pp. 2849-2883. Edited by D. M. Knipe & P. M. Howley. Philadelphia:Lippincott Williams & Wilkins.
Moss, B. & Shisler, J. L. (2001). Immunology 101 at poxvirus U: immune evasion genes. Seminars in Immunology 13, 59-66.[Medline]
Moss, B., Winters, E. & Cooper, J. A. (1981). Deletion of a 9,000-base-pair segment of the vaccinia virus genome that encodes nonessential polypeptides. Journal of Virology 40, 387-395.[Medline]
Ng, A., Tscharke, D. C., Reading, P. C. & Smith, G. L. (2001). The vaccinia virus A41L protein is a soluble 30 kDa glycoprotein that affects virus virulence. Journal of General Virology 82, 2095-2105.
Nicholas, K. B. & Nicholas, H. B. (1997). GeneDoc: a tool for editing and annotating multiple sequence alignments, 2.5.000. Distributed by the author.
Panicali, D., Davis, S. W., Mercer, S. R. & Paoletti, E. (1981). Two major variants present in serially propagated stocks of the WR strain of vaccinia virus. Journal of Virology 37, 1000-1010.[Medline]
Parkinson, J. E. & Smith, G. L. (1994). Vaccinia virus gene A36R encodes a Mr 4350 K protein on the surface of extracellular enveloped virus. Virology 204, 376-390.[Medline]
Perkus, M. E., Goebel, S. J., Davis, S. W., Johnson, G. P., Norton, E. K. & Paoletti, E. (1991). Deletion of 55 open reading frames from the termini of vaccinia virus. Virology 180, 406-410.[Medline]
Sanderson, C. M., Parkinson, J. E., Hollinshead, M. & Smith, G. L. (1996). Overexpression of the vaccinia virus A38L integral membrane protein promotes Ca2+ influx into infected cells. Journal of Virology 70, 905-914.[Abstract]
Schmelz, M., Sodeik, B., Ericsson, M., Wolffe, E., Shida, H., Hiller, G. & Griffiths, G. (1994). Assembly of vaccinia virus: the second wrapping cisterna is derived from the trans Golgi network. Journal of Virology 68, 130-147.[Abstract]
Shchelkunov, S. N., Safronov, P. F., Totmenin, A. V., Petrov, N. A., Ryazankina, O. I., Gutorov, V. V. & Kotwal, G. J. (1998). The genomic sequence analysis of the left and right species-specific terminal region of a cowpox virus strain reveals unique sequences and a cluster of intact ORFs for immunomodulatory and host range proteins. Virology 243, 432-460.[Medline]
Smith, G. L., Symons, J. A., Khanna, A., Vanderplasschen, A. & Alcamí, A. (1997). Vaccinia virus immune evasion. Immunological Reviews 159, 137-154.[Medline]
Symons, J. A., Alcami, A. & Smith, G. L. (1995). Vaccinia virus encodes a soluble type I interferon receptor of novel structure and broad species specificity. Cell 81, 551-560.[Medline]
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research 22, 4673-4680.[Abstract]
Tscharke, D. C. & Smith, G. L. (1999). A model for vaccinia virus pathogenesis and immunity based on intradermal injection of mouse ear pinnae. Journal of General Virology 80, 2751-2755.
Tscharke, D. C., Reading, P. C. & Smith, G. L. (2002). Dermal infection with vaccinia virus reveals roles for virus proteins not seen using other inoculation routes. Journal of General Virology 83, 1977-1986.
Tulman, E. R., Afonso, C. L., Lu, Z., Zsak, L., Kutish, G. F. & Rock, D. L. (2001). Genome of lumpy skin disease virus. Journal of Virology 75, 7122-7130.
Willer, D. O., McFadden, G. & Evans, D. H. (1999). The complete genome sequence of Shope (rabbit) fibroma virus. Virology 264, 319-343.[Medline]
Wittek, R., Muller, K., Menna, A. & Wyler, R. (1978). Length heterogeneity in the DNA of vaccinia virus is eliminated by cloning the virus. FEBS Letters 90, 41-46.[Medline]
Received 7 February 2002;
accepted 22 March 2002.