Polyomavirus tumor induction in mice: effects of polymorphisms of VP1 and large T antigen.

By testing recombinants between "high tumor" (inducing a high incidence of tumors) and "low tumor" (inducing a low incidence of tumors) strains of polyomavirus, we have previously shown that the key determinant(s) for induction of a high tumor profile resides in coding regions of the high tumor strain (R. Freund, G. Mandel, G. G. Carmichael, J. P. Barncastle, C. J. Dawe, and T. L. Benjamin, J. Virol. 61:2232-2239, 1987). Three single-amino-acid differences between the PTA (high tumor) and RA (low tumor) virus strains have now been identified by DNA sequencing, one each in the large T antigen, in the region common to the middle and small T antigens, and in the major capsid protein VP1. Further tests of appropriate recombinants and oligonucleotide-induced mutants show that VP1 of PTA is the major determinant for induction of a high tumor profile, including all tumors of epithelial origin. The differential effect of the VP1s of PTA and RA on the tumor profile is discussed in terms of a likely contribution of the polymorphic region of VP1 to binding of receptors and infection of different cell types in the animal. The polymorphism in the large T antigen has a more restricted action, which is seen only when tested in virus carrying the VP1 type of PTA; the PTA large T antigen then promotes more rapid growth of tumors of salivary gland and thymus than the RA large T antigen.     

Mapping of attenuating sequences of an avirulent poliovirus type 2 strain.

A mouse model for poliomyelitis was used to identify genomic sequences that attenuate neurovirulence of poliovirus strain P2/P712. This type 2 strain is avirulent in primates and mice yet grows as well as virulent strains in cell culture. The approach used was to exchange portions of the genome of the mouse-virulent P2/Lansing strain with the corresponding region from P2/P712 to identify sequences that could attenuate Lansing neurovirulence in mice. A full-length infectious cDNA of P2/P712 was assembled and used to construct recombinants between P2/P712 and P2/Lansing. The results of neurovirulence testing of 11 recombinants indicated that strong attenuating determinants are located in the 5' noncoding region of P2/P712 and a region encoding capsid protein VP1 and 2Apro, 2B, and part of 2C. An attenuating determinant was further localized to between nucleotides 456 and 628 of P2/P712. A third sequence from P2/P712, nucleotides 752 to 2268, encoding VP4, VP2, and part of VP3, was weakly attenuating. The sequence from nucleotide 4454, approximately halfway through the 2C-coding region, to the end of the P2/P712 genome did not contain attenuating determinants. Nucleotide sequence analysis revealed that P2/P712 differs from the type 2 Sabin vaccine strain by only 22 nucleotides. Six differences lead to amino acid changes in the coding region, and four differences are in the 5' noncoding region. These studies show that, like the type 1 and type 3 Sabin vaccine strains, the attenuated type 2 strain P712 contains multiple attenuating sequences, including strongly attenuating sequences in the 5' noncoding region of the genome.     

Identification of two determinants that attenuate vaccine-related type 2 poliovirus.

The poliovirus P2/P712 strain is an attenuated virus that is closely related to the type 2 Sabin vaccine strain. By using a mouse model for poliomyelitis, sequences responsible for attenuation of the P2/P712 strain were previously mapped to the 5' noncoding region of the genome and a central region encoding VP1, 2Apro, 2B, and part of 2C. To identify specific determinants that attenuate the P2/P712 strain, recombinants between this virus and the mouse-adapted P2/Lansing were constructed and their neurovirulence in mice was determined. By using this approach, the attenuation determinant in the central region was mapped to capsid protein VP1. Candidate attenuating sequences in VP1 and the 5' noncoding region were identified by comparing the P2/P712 sequence with that of vaccine-associated isolate P2/P117, and the P2/117 sequences were introduced into the P2/Lansing-P2/P712 recombinants by site-directed mutagenesis. Results of neurovirulence assays in mice indicate that an A at nucleotide 481 in the 5' noncoding region and isoleucine (Ile) at position 143 of capsid protein VP1 are the major determinants of attenuation of P2/P712. These determinants also attenuated neurovirulence in transgenic mice expressing human poliovirus receptors, a new model for poliomyelitis in which virulent viruses are not host restricted. These results demonstrate that A-481 and Ile-143 are general determinants of attenuation.     

Duplication of noncoding sequences in polyomavirus specifically augments the development of thymic tumors in mice.

A 40-base-pair duplication of noncoding sequences in polyomavirus specifically augmented the development of thymic epitheliomas following inoculation of virus into newborn mice. Virus strains carrying only one copy of this sequence induced a full spectrum of tumors except for overt thymic tumors. This 40-base-pair repeat, on the early side of the replication origin, constituted a tissue-specific regulatory determinant for tumor induction.     

Tandemization of a Subregion of the Enhancer Sequences from SRS 19-6 Murine Leukemia Virus Associated with T-Lymphoid but Not Other Leukemias

Most simple retroviruses induce tumors of a single cell type when infected into susceptible hosts. The SRS 19-6 murine leukemia virus (MuLV), which originated in mainland China, induces leukemias of multiple cellular origins. Indeed, infected mice often harbor more than one tumor type. Since the enhancers of many MuLVs are major determinants of tumor specificity, we tested the role of the SRS 19-6 MuLV enhancers in its broad disease specificity. The enhancer elements of the Moloney MuLV (M-MuLV) were replaced by the 170-bp enhancers of SRS 19-6 MuLV, yielding the recombinants DeltaMo+SRS(+) and DeltaMo+SRS(-) M-MuLV. M-MuLV normally induces T-lymphoid tumors in all infected mice. Surprisingly, when neonatal mice were inoculated with DeltaMo+SRS(+) or DeltaMo+SRS(-) M-MuLV, all tumors were of T-lymphoid origin, typical of M-MuLV rather than SRS 19-6 MuLV. Thus, the SRS 19-6 MuLV enhancers did not confer the broad disease specificity of SRS 19-6 MuLV to M-MuLV. However, all tumors contained DeltaMo+SRS M-MuLV proviruses with common enhancer alterations. These alterations consisted of tandem multimerization of a subregion of the SRS 19-6 enhancers, encompassing the conserved LVb and core sites and adjacent sequences. Moreover, when tumors induced by the parental SRS 19-6 MuLV were analyzed, most of the T-lymphoid tumors had similar enhancer alterations in the same region whereas tumors of other lineages retained the parental SRS 19-6 MuLV enhancers. These results emphasize the importance of a subregion of the SRS 19-6 MuLV enhancer in induction of T-cell lymphoma. The relevant sequences were consistent with crucial sequences for T-cell lymphomagenesis identified for other MuLVs such as M-MuLV and SL3-3 MuLV. These results also suggest that other regions of the SRS 19-6 MuLV genome contribute to its broad leukemogenic spectrum.     

Spontaneous emergence of an Hfr strain with a cit plasmid from natural isolates of citrate-positive Escherichia coli bovine origin.

From citrate-utilizing (Cit+) Escherichia coli strain C53 of bovine origin, strains C53A and C53B were obtained. Upon mating with recA+ but not with recA mutant recipients of K-12, C53A produced chromosomal recombinants at quite high frequencies, leading to the following conclusions: (i) C53A is an Hfr strain; (ii) the site of integration of the Cit plasmid (IncH1) is between metA (89 min) and ara (1 min); (iii) the direction of chromosome transfer is clockwise; and (iv) the plasmid-associated determinants are transferred as the terminal markers. A transductant of a dnaA(Ts) strain, CRT46, which acquired Cit determinants from a recombinant, SG13, was also an Hfr strain similar to SG13, and thermoresistant due to suppressive integration. On the other hand, unstable C53B did not produce recombinants, but the frequency of RecA-independent transfer of the Cit plasmid was high, indicating that the Cit plasmid (IncH1) exists autonomously in C53B. Attempts to isolate an Hfr strain from C53B failed.     

Receptor-binding and oncogenic properties of polyoma viruses isolated from feral mice

Laboratory strains of the mouse polyoma virus differ markedly in their abilities to replicate and induce tumors in newborn mice. Major determinants of pathogenicity lie in the sialic binding pocket of the major capsid protein Vp1 and dictate receptor-binding properties of the virus. Substitutions at two sites in Vp1 define three prototype strains, which vary greatly in pathogenicity. These strains replicate in a limited fashion and induce few or no tumors, cause a disseminated infection leading to the development of multiple solid tumors, or replicate and spread acutely causing early death. This investigation was undertaken to determine the Vp1 type(s) of new virus isolates from naturally infected mice. Compared with laboratory strains, truly wild-type viruses are constrained with respect to their selectivity and avidity of binding to cell receptors. Fifteen of 15 new isolates carried the Vp1 type identical to that of highly tumorigenic laboratory strains. Upon injection into newborn laboratory mice, the new isolates induced a broad spectrum of tumors, including ones of epithelial as well as mesenchymal origin. Though invariant in their Vp1 coding sequences, these isolates showed considerable variation in their regulatory sequences. The common Vp1 type has two essential features: 1) failure to recognize "pseudoreceptors" with branched chain sialic acids binding to which would attenuate virus spread, and 2) maintenance of a hydrophobic contact with true receptors bearing a single sialic acid, which retards virus spread and avoids acute and potentially lethal infection of the host. Conservation of these receptor-binding properties under natural selection preserves the oncogenic potential of the virus. These findings emphasize the importance of immune protection of neonates under conditions of natural transmission.     

Genome Sequence of Herpes Simplex Virus 1 Strain McKrae

The herpes simplex virus 1 (HSV-1) strain McKrae is highly virulent compared to other wild-type strains of HSV-1. To help us better understand the genetic determinants that lead to differences in the pathogenicity of McKrae and other HSV-1 strains, we sequenced its genome. Comparing the sequence of McKrae's genome to that of strain 17 revealed that the genomes differ by at least 752 single nucleotide polymorphisms (SNPs) and 86 insertion/deletion events (indels). Although the majority of these polymorphisms reside in noncoding regions, 241 SNPs and 10 indels alter the protein-coding sequences of 58 open reading frames. Some of these variations are expected to contribute to the pathogenic phenotype of McKrae.     

Influence of the viral regulatory region on tumor induction by simian virus 40 in hamsters

Most of the simian virus 40 (SV40) genome is conserved among isolates, but the noncoding regulatory region and the genomic region encoding the large T-antigen C terminus (T-ag-C) may exhibit considerable variation. We demonstrate here that SV40 isolates differ in their oncogenic potentials in Syrian golden hamsters. Experimental animals were inoculated intraperitoneally with 10⁷ PFU of parental or recombinant SV40 viruses and were observed for 12 months to identify genetic determinants of oncogenicity. The viral regulatory region was found to exert a statistically significant influence on tumor incidence, whereas the T-ag-C played a minor role. Viruses with a single enhancer (1E) were more oncogenic than those with a two-enhancer (2E) structure. Rearrangements in the 1E viral regulatory region were detected in 4 of 60 (6.7%) tumors. Viral loads in tumors varied, with a median of 5.4 SV40 genome copies per cell. Infectious SV40 was rescued from 15 of 37 (40%) cell lines established from tumors. Most hamsters with tumors and many without tumors produced antibodies to T antigen. All viruses displayed similar transforming frequencies in vitro, suggesting that differences in oncogenic potential in vivo were due to host responses to viral infection. This study shows that SV40 strains differ in their biological properties, suggests that SV40 replicates to some level in hamsters, and indicates that the outcome of an SV40 infection may depend on the viral strain present.     

Comparison of the DNA sequence of the Crawford small-plaque variant of polyomavirus with those of polyomaviruses A2 and strain 3.

The DNA sequence of two wild-type strains of polyomavirus (A2 and strain 3) are known. We have determined the majority of the DNA sequence of a third strain, the Crawford small-plaque virus. This virus has been noted for its capacity to induce readily detected tumor-specific transplantation antigen in hamster cells, a property that is most likely attributable to an altered middle T-antigen. A comparison of its DNA sequence with those of the A2 and strain 3 viruses reveals numerous nucleotide substitutions, insertions, and deletions throughout the genome. Most sequence changes in coding regions are silent mutations; however, variability in proteins can be predicted from these sequence data at 5 locations in middle T-antigen, 10 in large T-antigen, and 10 in VP1. The Crawford small-plaque virus noncoding regulatory region contains, in addition to nucleotide substitutions, a 44-base-pair tandem repeat of sequences on the late side of the origin of DNA replication.     

Properties of intracellular bovine papillomavirus chromatin.

Episomal nucleoprotein complexes of bovine papillomavirus type 1 (BPV-1) in transformed cells were exposed to DNase I treatment to localize hypersensitive regions. Such regions, which are indicative for gene expression, were found within the noncoding part of the genome, coinciding with the origin of replication and the 5' ends of most of the early mRNAs. However, there were also regions of hypersensitivity within the structural genes. These intragenic perturbations of the chromatin structure coincide with regulatory sequences at the DNA level. One of these regions maps in close proximity to a Z-DNA antibody-binding site which is located near the putative BPV-1 enhancer sequence.     

Construction of a piggyBac-based enhancer trap system for the analysis of gene function in silkworm Bombyx mori

Enhancer trapping and insertional mutagenesis are powerful tools for analyzing genetic function. To construct an enhancer trap system in the silkworm Bombyx mori, we developed efficient jumpstarter strains by inserting the piggyBac transposase gene under the control of Bombyx cytoplasmic actin gene (BmA3) promoter into the genome. To stabilize the inserted transgene, the jumpstarter strains were constructed using the Minos transposon as a vector. The ability of each of the 13 jumpstarter strains to remobilize their respective transposons was tested by crossing the jumpstarters with a mutator strain carrying a GAL4 construct containing the BmA3 promoter. Four strains with high remobilization activity were then selected and used to produce enhancer trap lines by crossing with the mutator strains and hybridizing the F1 progeny with a UAS-EGFP strain. Several enhancer trap lines showing characteristic expression patterns at the embryonic, larval, pupal, and adult stages were detected in the subsequent generation. Approximately 10-40% of the silkworms from each cross in the hybridized brood had a remobilized mutator. An analysis of the insertion positions in 105 lines by inverse PCR using a silkworm genome database revealed that remobilization occurred randomly in each chromosome. The frequency of insertion of the remobilized mutator into putative exons, introns, intergenic regions, and repetitive sequences was 12, 9, 36, and 40%, respectively. We concluded that the piggyBac-based GAL4 enhancer trap system developed in this study is applicable for large-scale enhancer trapping in the silkworm.     

A recombinant virus between the Sabin 1 and Sabin 3 vaccine strains of poliovirus as a possible candidate for a new type 3 poliovirus live vaccine strain.

Biological tests including the monkey neurovirulence test performed on recombinants between the virulent Mahoney and attenuated Sabin 1 strains of type 1 poliovirus indicated that the genome region encoding mainly the viral capsid proteins had little correlation with the neurovirulence or attenuation phenotype of the virus. The results suggested that new vaccine strains of type 2 and type 3 polioviruses may be constructed in vitro by replacing the sequence encoding the antigenic determinants in viral capsid proteins of the Sabin 1 genome by the corresponding sequences of the type 2 and type 3 genome, respectively. Accordingly, we constructed recombinants between the Sabin 1 and Sabin 3 strains of poliovirus in which genome sequences of the Sabin 1 strain encoding most or all capsid proteins were replaced by the corresponding genome sequences of the Sabin 3 strain. One of the recombinant viruses thus constructed was fully viable and showed antigenicity and immunogenicity identical to those of type 3 poliovirus. The monkey neurovirulence tests and in vitro phenotypic marker tests (temperature sensitivity of growth, sodium bicarbonate concentration dependency of growth under agar overlay, and size of plaque) were performed on the recombinant virus. The stability of the virus in regard to the temperature sensitivity phenotype was also tested. The results suggested that the recombinant virus is a possible candidate for a new type 3 poliovirus vaccine strain.     

Distinct segments within the enhancer region collaborate to specify the type of leukemia induced by nondefective Friend and Moloney viruses.

The nondefective Moloney and Friend murine leukemia viruses induce T-cell lymphomas and erythroleukemias, respectively, after being injected into newborn NFS mice. In previous studies, we showed that the distinct disease specificities of the two viruses could be switched by exchanging a small segment, about 200 nucleotides in length, encompassing their enhancer regions. This segment included the direct repeat sequence and an adjacent GC-rich region of about 20 nucleotides defined in studies of Moloney murine sarcoma virus enhancer-promoter function (L. A. Laimins, P. Gruss, R. Pozzatti, and G. Khoury, J. Virol. 49:183-189, 1984). The direct repeats of Friend and Moloney viruses are identical in a central core sequence of 32 nucleotides but have sequence differences on either side of this core as well as in their GC-rich segments. To determine whether disease specificity resides in part or in all of the direct repeat and GC-rich region, we constructed recombinants between Friend and Moloney viruses within this segment and tested them for their disease-inducing phenotypes. We found that disease specificity, in particular the ability of Friend virus sequence to confer erythroleukemogenicity on Moloney virus, is encoded throughout the region in at least three separable segments: the 5' and 3' halves of the direct repeat and the GC-rich segment. When just one of these segments (either both 5' halves of the direct repeat, both 3' halves, or just the GC-rich segment) from Friend virus was substituted into a Moloney virus genome, it conferred only a negligible or low incidence of erythroleukemia (less than or equal to 5% to between 10 and 15%). Any two segments together were considerably more potent (35 to 95% erythroleukemia), with the most effective pair being the two halves of the direct repeat. Individual segments and pairs of segments were considerably more potent determinants when they were matched with a genome of the same origin. Thus, although sequences outside the enhancer region are minor determinants of disease specificity when the enhancer is derived entirely from either Friend or Moloney virus, they can play a significant role when the enhancer is of mixed origin. Some recombinant enhancers conferred a long latent period of disease induction. This was particularly striking when the 5' halves of each copy of the direct repeat sequence were derived from Moloney virus and the 3' halves were derived from Friend virus.(ABSTRACT TRUNCATED AT 400 WORDS)     

Assembly of Theiler's virus recombinants used in mapping determinants of neurovirulence.

A major determinant of neurovirulence for the GDVII strain of Theiler's virus, a murine picornavirus, was mapped to the P1 capsid protein region. Chimeric viruses were constructed by using sequences from the 5' noncoding and P1 regions of the virulent GDVII strain to replace equivalent regions of the less virulent BeAn strain. Neurovirulence in mice progressively increased as larger regions of BeAn capsid protein-encoding sequences were replaced. The in vitro growth characteristics of the chimeras showed that some chimeras were growth delayed in BHK-21 cells even though the viral constructs exhibited larger plaque sizes, were less temperature sensitive, and were more thermally stable than BeAn. Examination of assembly intermediates revealed an altered pentamer conformation and delayed empty capsid formation for the growth-compromised viruses. For these constructs, their chimeric nature inadvertently resulted in virion assembly defects that complicated finer-scale mapping of the determinants of virulence within the capsid region. These results demonstrate the importance of determining in vitro growth characteristics of chimeras to correctly decipher the significance of their phenotypes. VP1 does not contain a complete determinate for virulence because a chimera with VP1-encoding sequences from GDVII in an otherwise BeAn virus has an attenuated phenotype but is not growth compromised in vitro. The source of sequences, BeAn or GDVII, in the 5' noncoding region had only slight effects on the virulence of recombinant constructs.     

Genomic sequence comparison of the human and mouse adenosine deaminase gene regions

A challenge for mammalian genetics is the recognition of critical regulatory regions in primary gene sequence. One approach to this problem is to compare sequences from genes exhibiting highly conserved expression patterns in disparate organisms. Previous transgenic and transfection analyses defined conserved regulatory domains in the mouse and human adenosine deaminase (ADA) genes. We have thus attempted to identify regions with comparable similarity levels potentially indicative of critical ADA regulatory regions. On the basis of aligned regions of the mouse and human ADA gene, using a 24-bp window, we find that similarity overall (67.7%) and throughout the noncoding sequences (67.1%) is markedly lower than that of the coding regions (81%). This low overall similarity facilitated recognition of more highly conserved regions. In addition to the highly conserved exons, ten noncoding regions >100 bp in length displayed >70% sequence similarity. Most of these contained numerous 24-bp windows with much higher levels of similarity. A number of these regions, including the promoter and the thymic enhancer, were more similar than several exons. A third block, located near the thymic enhancer but just outside of a minimally defined locus control region, exhibited stronger similarity than the promoter or thymic enhancer. In contrast, only fragmentary similarity was exhibited in a region that harbors a strong duodenal enhancer in the human gene. These studies show that comparative sequence analysis can be a powerful tool for identifying conserved regulatory domains, but that some conserved sequences may not be detected by certain functional analyses as transgenic mice. Received: 27 March 1998 / Accepted: 22 September 1998     

A single base deletion in the 5'' noncoding region of Theiler''s virus attenuates neurovirulence.

Viral chimeras have been constructed through in vitro manipulations of the infectious cDNA clones of two prototypes of Theiler's murine encephalomyelitis virus: (i) the virulent GDVII strain and (ii) the less virulent BeAn and VL strains. Previous studies have suggested that the phenotypic differences in virulence between the BeAn and GDVII strains map to both the 5' noncoding and the coat protein regions of these viral genomes. It is shown here that attenuation mapped to the 5' noncoding region is due, at least in part, to an inadvertent deletion resulting from a cloning artifact of one C nucleotide out of four between positions 876 and 879 in the BeAn sequences. The in vitro growth characteristics in BHK-21 cells, however, do not reflect the large differences in neurovirulence between chimeras that are identical except for the deleted C. Another chimera with a mutation at position 877 and a deletion at 976 is also attenuated. The wild-type sequences from the less virulent strains BeAn and VL between nucleotides 1 and 933, in an otherwise GDVII chimera, do not attenuate virulence. Sequences of the 500 nucleotides of the 5' noncoding region proximal to the translation initiation codon were obtained for nine additional Theiler's virus strains. The attenuating deletions are discussed in the context of these sequences and the proposed secondary structures for the 5' noncoding region.