Chimeric Clones Research Articles

Identification of virulence determinants of porcine reproductive and respiratory syndrome virus through construction of chimeric clones

In order to determine virulence associated genes in porcine reproductive and respiratory syndrome virus (PRRSV), a series of chimeric viruses were generated where specific genomic regions of a highly virulent PRRSV infectious clone (FL12) were replaced with their counterparts of an attenuated vaccine strain (Prime Pac). Initial genome-wide scanning using a sow reproductive failure model indicated that non-structural (ORF 1a and 1b) and structural (ORF2-7) genomic regions appear to be sites where virulence determinants of PRRSV may reside. These results thus confirm the multigenic character of PRRSV virulence. Additional chimeras containing each individual structural ORFs (2 through 7) of Prime Pac and ORF5 of Neb-1 (parental strain of Prime Pac) within the FL12 backbone were generated and tested individually for further mapping of virulence determinants. Our results allow to conclude that NSP3–8 and ORF5 are the location of major virulence determinants, while other virulence determinants may also be contained in NSP1–3, NSP10–12 and ORF2.

The methyltransferase domain of the 1a protein of cowpea chlorotic mottle virus controls local and systemic accumulation in cowpea

The type strain of cowpea chlorotic mottle virus (CCMV-T) induces a local and systemic infection in California Blackeye cowpea (Vigna unguiculata (L.) Walp. subs. unguiculata cv. California Blackeye), but accumulates to low levels in inoculated leaves and fails to accumulate systemically in the cowpea plant introduction (PI) 186465. CCMV-R, a mutant strain derived from CCMV-T, accumulates to higher levels than CCMV-T in inoculated leaves and systemically infects PI 186465 plants. The phenotypic determinant of CCMV-R was previously mapped to viral RNA1, but the location of the determinant within RNA1 was not identified. Pseudorecombinants generated from genomic cDNA clones of CCMV-T and CCMV-R indicated that the phenotypic differences on PI 186465 were independent of replication. Through the use of chimeric RNA1 cDNA clones containing portions of CCMV-T and CCMV-R and site-directed mutagenesis, two nucleotides, 299 (amino acid residue 77) and 951 (amino acid residue 294), were identified as being independently critical for the local and systemic accumulation patterns of CCMV-R in PI 186465 plants. A second independently derived CCMV-R-like mutant, identified nucleotide 216 (amino acid residue 49) as being critical for induction of the CCMV-R infection phenotype. Amino acid residues 49, 77, and 294 are within the methytransferase domain of the CCMV 1a protein, suggesting that the methytransferase domain has a role in cell-to-cell and systemic accumulation of the virus that is independent of replication.

Reducing cloning artifacts for recovery of allelic sequences by T7 endonuclease I cleavage and single re-extension of PCR products — A benchmark

Occurrence of chimeric sequences and related artifacts in PCR cloning procedures gives us risks of over-estimation of haplotypes or alleles. Recombination among haplotypes occurs through template switching during PCR cycles or through random repair of mismatch sites on heteroduplex DNA by the host cell. To eliminate the chimeric cloning artifacts, we tested two alternative protocols using T7 endonuclease I cleavage of mismatch sites and re-extension of nascent strands. Though T7 endonuclease I effectively eliminated chimeric clones in some cases, it produced many short fragments. Protocol with single re-extension of PCR products successfully recovered non-recombinant clones with fewer short fragments. In spite of the improvement of allelic recovery through these two protocols, there were still a few recombinants that remained in both reaction mixtures, and thus interpretation of the results for haplotype diversity in a PCR-amplified DNA population should be cautionary. Because re-extension in a diluted reaction mixture is quick, inexpensive and effective, it is advisable to use this procedure for recovery of chromosomal alleles with PCR cloning.

Regulation of Plasminogen Activator Inhibitor-1 Expression by Tumor Suppressor Protein p53

H1299 lung carcinoma cells lacking p53 (p53-/-) express minimal amounts of plasminogen activator inhibitor-1 (PAI-1) protein as well as mRNA. p53(-/-) cells express highly unstable PAI-1 mRNA. Transfection of p53 in p53(-/-) cells enhanced PAI-1 expression and stabilized PAI-1 mRNA. On the contrary, inhibition of p53 expression by RNA silencing in non-malignant human lung epithelial (Beas2B) cells decreased basal as well as urokinase-type plasminogen activator-induced PAI-1 expression because of accelerated degradation of PAI-1 mRNA. Purified p53 protein specifically binds to the PAI-1 mRNA 3'-un-translated region (UTR), and endogenous PAI-1 mRNA forms an immune complex with p53. Treatment of purified p53 protein with anti-p53 antibody abolished p53 binding to the 3'-UTR of PAI-1 mRNA. The p53 binding region maps to a 70-nucleotide PAI-1 mRNA 3'-UTR sequence, and insertion of the p53-binding sequence into beta-globin mRNA destabilized the chimeric transcript. Deletion experiments indicate that the carboxyl-terminal region (amino acid residues 296-393) of p53 protein interacts with PAI-1 mRNA. These observations demonstrate a novel role for p53 as an mRNA-binding protein that regulates increased PAI-1 expression and stabilization of PAI-1 mRNA in human lung epithelial and carcinoma cells.

Construction and Characterization of an Infectious Molecular Clone of HIV Type 1 CRF07_BC

HIV-1 CRF07_BC constitutes a large fraction of HIV-1 strains circulating in China and is responsible for the rapidly expanding epidemic across the country. Little is known about the biological characteristics of the CRF07_BC viruses, particularly the propensity by which this circulating recombinant form of HIV replicates within an infected individual. In this study, a near-full-length genome of a CRF07_BC virus from an injecting drug user (IDU) from the Xingjiang region in China was cloned and rescued by the functional 5' LTR (long terminal repeat region) and 3' LTR from pNL4-3 to produce a chimeric infectious molecular clone (NLXJDC6441X2). When cultured in interleukin-2-stimulated peripheral blood mononuclear cells (PBMCs) and the Ghost.R5 cell line, NLXJDC6441X2 produced a lower replication yield than expected. More study is needed to explore the key role that leads to the poor infectious activity of NLXJDC6441X2. This study has produced the first isolation of the HIV-1 CRF07_BC DNA clone and has provided a versatile molecular model for research focusing on the biological properties of this subtype.

Animal reproduction biotechnology in Poland

The key research areas of the Department are: in vitro production of embryos, embryo cryopreservation, animal transgenesis, cloning, cytometric semen sexing and evaluation. Research has been focused on the in vitro production of animal embryos, including the development of complex methods for oocyte maturation, fertilization and embryo culture. Moreover, experiments on long-term culturing of late preantral and early antral bovine ovarian follicles have been developed. Studies on the cloning of genetically modified pigs with "humanized" immunological systems have been undertaken. A cloned goat was produced from oocytes reconstructed with adult dermal fibroblast cells. The novel technique of rabbit chimeric cloning for the production of transgenic animals was applied; additionally, the recipient-donor-cell relationship in the preimplantation developmental competences of feline nuclear transfer embryos has been studied. Regarding transgenic animal projects, gene constructs containing growth hormone genes connected to the mMt promoter were used. Modifications of milk composition gene constructs with tissue-specific promoters were performed. Moreover, pigs for xenotransplantation and animal models of human vascular diseases have been produced. Over the last 15 years, our flow cytometry research group has focused its work on new methods for sperm quality assessment and sex regulation. In the 1970s, our team initiated studies on embryo cryopreservation. As a result of vitrification experiments, the world's first rabbits and sheep produced via the transfer of vitrified embryos were born.

Construction, Characterization, and Chromosomal Mapping of a Fosmid Library of the White-Cheeked Gibbon ( Nomascus leucogenys)

Gibbons have experienced extensive karyotype rearrangements during evolution and represent an ideal model for studying the underlying molecular mechanism of evolutionary chromosomal rearrangements. It is anticipated that the cloning and sequence characterization of evolutionary chromosomal breakpoints will provide vital insights into the molecular force that has driven such a radical karyotype reshuffle in gibbons. We constructed and characterized a high-quality fosmid library of the white-cheeked gibbon (Nomascus leucogenys) containing 192,000 non-redundant clones with an average insert size of 38 kb and 2.5-fold genome coverage. By end sequencing of 100 randomly selected fosmid clones, we generated 196 sequence tags for the library. These end-sequenced fosmid clones were then mapped onto the chromosomes of the white-cheeked gibbon by fluorescence in situ hybridization, and no spurious chimeric clone was detected. BLAST search against the human genome showed a good correlation between the number of hit clones and the number of chromosomes, an indication of unbiased chromosomal distribution of the fosmid library. The chromosomal distribution of the mapped clones is also consistent with the BLAST search result against human and white-cheeked gibbon genomes. The fosmid library and the mapped clones will serve as a valuable resource for further studying gibbons’ chromosomal rearrangements and the underlying molecular mechanism as well as for comparative genomic study in the lesser apes.

Attenuation of porcine reproductive and respiratory syndrome virus strain MN184 using chimeric construction with vaccine sequence

Two genetically distinct infectious recombinant virus clones (pMLV, constructed from Ingelvac® PRRS MLV and pMN184, constructed from virulent strain MN184) were developed to study attenuation of contemporary porcine reproductive and respiratory syndrome virus (PRRSV) strain MN184. Two reciprocal chimeric clones (pMLVORF1/MN184 and pMN184ORF1/MLV) were then constructed, such that the 5′UTR/ORF1 of one genotype was linked to ORF2–7/3′UTR from the other genotype. In vitro studies demonstrated that the rescued chimeric viruses possessed intermediate growth properties compared to recombinant rMLV and rMN184. Swine inoculation with rMN184 and rMLV verified that these viruses fully mimicked the respective parent virus. In addition, earlier and higher antibody responses were detected in animals infected with rMN184 in contrast to those infected with rMLV. Chimeric virus treatment groups showed similar antibody responses as seen with these parent viruses, but much less severe pathogenesis when compared to the rMN184 group. These data suggested that genetic aspects of Ingelvac® PRRS MLV 5′UTR/ORF1 replicase region and/or the structural proteins/3′UTR can serve to attenuate virulent strain MN184. The data also indicated that designed PRRSV vaccines could be developed, keeping the known 5′UTR/replicase region of an early vaccine strain such as Ingelvac® PRRS MLV intact, but replacing the structural protein/3′UTR domain with that of an emerging virulent virus.

Significance of arginine 20 in the 2A protease for swine vesicular disease virus pathogenicity

Pathogenic and attenuated strains of swine vesicular disease virus (SVDV), an enterovirus, have been characterized previously and, by using chimeric infectious cDNA clones, the key determinants of pathogenicity in pigs have been mapped to the coding region for 1D-2A. Within this region, residue 20 of the 2A protease is particularly significant. Inoculation of pigs with mutant viruses containing single amino acid substitutions at this residue leads to the appearance of revertants, often containing an arginine at this position encoded by an AGA codon, one of six codons for this residue. The properties in pigs of two chimeric viruses, each with an arginine residue at this position but encoded by different codons, have been investigated in parallel with the parental pathogenic and attenuated strains. Presence of the arginine residue, but not of the AGA codon, is essential for induction of high viraemia and appearance of significant disease.

The NS3 Helicase and NS5B-to-3′X Regions Are Important for Efficient Hepatitis C Virus Strain JFH-1 Replication in Huh7 Cells

The JFH-1 strain of hepatitis C virus (HCV) is a genotype 2a strain that can replicate autonomously in Huh7 cells. The J6 strain is also a genotype 2a strain, but its full genomic RNA does not replicate in Huh7 cells. However, chimeric J6/JFH-1 RNA that has J6 structural-protein-coding regions and JFH-1 nonstructural-protein-coding regions can replicate autonomously and produce infectious HCV particles. In order to determine the mechanisms underlying JFH-1 RNA replication, we constructed various J6/JFH-1 chimeras and tested their RNA replication and virus particle production abilities in Huh7 cells. Via subgenomic-RNA-replication assays, we found that both the JFH-1 NS5B-to-3'X (N5BX) and the NS3 helicase (N3H) regions are important for the replication of the J6CF replicon. We applied these results to full-length genomic RNA replication and analyzed replication using Northern blotting. We found that a chimeric J6 clone with JFH-1 N3H and N5BX could replicate autonomously but that a chimeric J6 clone with only JFH-1 N5BX had no replication ability. Finally, we tested the virus production abilities of these clones and found that a chimeric J6 clone with JFH-1 N3H and N5BX could produce infectious HCV particles. In conclusion, the JFH-1 NS3 helicase and NS5B-to-3'X regions are important for efficient replication and virus particle formation of HCV genotype 2a strains.

Long terminal repeat sequences from virulent and attenuated equine infectious anemia virus demonstrate distinct promoter activities

In the early 1970s, the Chinese Equine Infectious Anemia Virus (EIAV) vaccine, EIAV DLA, was developed through successive passages of a wild-type virulent virus (EIAV L) in donkeys in vivo and then in donkey macrophages in vitro. EIAV attenuation and cell tropism adaptation are associated with changes in both envelope and long terminal repeat (LTR). However, specific LTR changes during Chinese EIAV attenuation have not been demonstrated. In this study, we compared LTR sequences from both virulent and attenuated EIAV strains and documented the diversities of LTR sequence from in vivo and in vitro infections. We found that EIAV LTRs of virulent strains were homologous, while EIAV vaccine have variable LTRs. Interestingly, experimental inoculation of EIAV DLA into a horse resulted in a restriction of the LTR variation. Furthermore, LTRs from EIAV DLA showed higher Tat transactivated activity than LTRs from virulent strains. By using chimeric clones of wild-type LTR and vaccine LTR, the main difference of activity was mapped to the changes of R region, rather than U3 region.

α7 Nicotinic Receptor Gene Promoter Polymorphisms in Inbred Mice Affect Expression in a Cell Type-specific Fashion

Inbred mouse strains display significant differences in their levels of brain alpha7 nicotinic acetylcholine receptor (alpha7 nAChR) expression, as measured by binding of the alpha7-selective antagonist alpha-bungarotoxin. Variations in alpha-bungarotoxin binding have been shown to correlate with an animal's sensitivity to nicotine-induced seizures and sensory gating. In two inbred mouse strains, C3H/2Ibg (C3H) and DBA/2Ibg (DBA/2), the inter-strain binding differences are linked to a restriction length polymorphism in the alpha7 nAChR gene, Chrna7. Despite this finding, the molecular mechanism(s) through which genetic variability in Chrna7 may contribute to alpha7 nAChR expression differences remains unknown. However, studies of the human alpha7 nAChR gene (CHRNA7) previously have demonstrated that CHRNA7 promoter polymorphisms are associated with differences in promoter activity as well as differences in sensory processing. In the present study, a 947-base pair region of the Chrna7 promoter was cloned from both the C3H and DBA/2 inbred mouse strains in an attempt to identify polymorphisms that may underlie alpha7 nAChR differential expression. Sequence analysis of these fragments identified 14 single nucleotide polymorphisms (SNPs). A combination of two of these SNPs affects promoter activity in an in vitro luciferase reporter assay. These results suggest a mechanism through which the Chrna7 promoter genotype may influence interstrain variations in alpha7 nAChR expression.

Cloning and Characterization of Functional Subtype A HIV-1 Envelope Variants Transmitted Through Breastfeeding

Previous studies of HIV-1 variants transmitted from mother-to-infant have focused primarily on computational analyses of partial envelope gene sequences, rather than analyses of functional envelope variants. There are very few examples of well-characterized functional envelope clones from mother-infant pairs, especially from envelope variants representing the most prevalent subtypes worldwide. To address this, we amplified the envelope variants present in 4 mother-infant transmission pairs, all of whom were infected with subtype A and three of whom presumably transmitted HIV-1 during the breastfeeding period. Functional envelope clones were constructed, either encoding full-length envelope sequences from the mother and baby or by making chimeric envelope clones in a common backbone sequence. The infant envelope sequences were genetically homogeneous compared to the maternal viruses, and pseudoviruses bearing these envelopes all used CCR5 as a coreceptor. The infant viruses were generally resistant to neutralization by maternal antibodies present near the time of transmission. There were no notable differences in sensitivity of the mother and infant envelope variants to neutralization by heterologous plasma or monoclonal antibodies 2G12 and b12, or to inhibition by sCD4, PSC-RANTES or TAK779. This collection of viral envelopes, which can be used for making pseudotyped viruses, may be useful for examining the efficacy of interventions to block mother-infant transmission, including sera from vaccine candidates, purified antibodies under consideration for passive immunization and viral entry inhibitors.

The double-resistance-breaking Tomato mosaic virus strain ToMV1-2 contains two independent single resistance-breaking domains

Tomato mosaic virus (ToMV) causes a serious loss of yield and fruit quality in tomato crops. To control ToMV, three resistance genes, Tm-1, Tm-2, and Tm-2(2) from wild tomato species were introduced into commercial tomato cultivars. Soon after, however, single and, rarely, double-resistance-breaking virus strains for Tm-1 and Tm-2 emerged. Sequence analysis of a Tm-1/Tm-2 double-resistance-breaking virus, designated ToMV1-2, revealed 30 nucleotide substitutions compared with wild-type ToMV. Of these, six substitutions result in amino acid exchanges. Two exchanges are in the methyl transferase/helicase domain of the ToMV1-2 130/180-kDa proteins (D-1097 to V, R-1100 to Q), two exchanges are found in the 30-kDa movement protein (MP; E-52 to K, E-133 to K), and two exchanges are in the coat protein (I-22 to V, A-87 to S). Construction of chimeric full-length viral cDNA clones containing the base substitutions resulting in altered amino acids, either in the 130/180 kDa proteins or the MP, revealed that the amino acid exchanges in the 130/180-kDa proteins enable ToMV1-2 to break the Tm-1 resistance, while those in the MP enable ToMV1-2 to overcome the Tm-2 resistance. Furthermore, a novel Tm-1/Tm-2 double-resistance-breaking virus was generated by combining the Tm-1-breaking domain of ToMV1-2 and the MP of a new virus, ToMV2, containing the amino acid exchanges E-133 to K and N-135 to S. Together, the data suggest that double-resistance-breaking ToMV1-2 may have resulted from recombination between Tm-1 and Tm-2 single resistance-breaking virus strains.

The usefulness of the gfp reporter gene for monitoring Agrobacterium-mediated transformation of potato dihaploid and tetraploid genotypes

Potato is one of the main targets for genetic improvement by gene transfer. The aim of the present study was to establish a robust protocol for the genetic transformation of three dihaploid and four economically important cultivars of potato using Agrobacterium tumefaciens carrying the in vivo screenable reporter gene for green fluorescent protein (gfp) and the marker gene for neomycin phosphotransferase (nptII). Stem and leaf explants were used for transformation by Agrobacterium tumefaciens strain LBA4404 carrying the binary vector pHB2892. Kanamycin selection, visual screening of GFP by epifluorescent microscopy, PCR amplification of nptII and gfp genes, as well as RT-PCR and Southern blotting of gfp and Northern blotting of nptII, were used for transgenic plant selection, identification and analysis. Genetic transformation was optimized for the best performing genotypes with a mean number of shoots expressing gfp per explant of 13 and 2 (dihaploid line 178/10 and cv. 'Baltica', respectively). The nptII marker and gfp reporter genes permitted selection and excellent visual screening of transgenic tissues and plants. They also revealed the effects of antibiotic selection on organogenesis and transformation frequency, and the identification of escapes and chimeras in all potato genotypes. Silencing of the gfp transgene that may represent site-specific inactivation during cell differentiation, occurred in some transgenic shoots of tetraploid cultivars and in specific chimeric clones of the dihaploid line 178/10. The regeneration of escapes could be attributed to either the protection of non-transformed cells by neighbouring transgenic cells, or the persistence of Agrobacterium cells in plant tissues after co-cultivation.

A second RGD motif in the 1D capsid protein of a SAT1 type foot-and-mouth disease virus field isolate is not essential for attachment to target cells

The amino acid sequence motif Arg-Gly-Asp (RGD), located in the surface-exposed βG-βH loop of the 1D protein of different serotypes and subtypes of foot-and-mouth disease virus (FMDV), is highly conserved and participates in binding of FMDV to susceptible cells. Previous sequence analyses of the 1D-encoding region of a FMDV serotype SAT1 field isolate from Namibia (NAM/307/98) indicated the presence of a second RGD motif upstream of the conserved βG-βH loop RGD. The role of these RGD sequences in virus infection was investigated by mutating the βG-βH loop RGD to a KGE tripeptide, using a genome-length infectious chimeric cDNA clone. Although the infectivity of the derived mutant viruses for baby hamster kidney cells (BHK-21) was lost, subsequent replacement of the KGE sequence with RGD in the mutant cDNA clone led to recovery of infectious viruses. Furthermore, viral RNA replication could be demonstrated with the genetically engineered mutant and non-mutant viruses. The presence of virus particles in the transfected cells could be also demonstrated by electron microscopy. These results demonstrate that, in contrast to the βG-βH loop RGD motif, the second RGD sequence in the capsid protein 1D of NAM/307/98 does not function as a ligand for receptor binding in BHK-21 cells.

RETRACTED ARTICLE: Small interfering RNA targeted to stem-loop II of the 5' untranslated region effectively inhibits expression of six HCV genotypes

BackgroundThe antiviral action of interferon alpha targets the 5' untranslated region (UTR) used by hepatitis C virus (HCV) to translate protein by an internal ribosome entry site (IRES) mechanism. Although this sequence is highly conserved among different clinical strains, approximately half of chronically infected hepatitis C patients do not respond to interferon therapy. Therefore, development of small interfering RNA (siRNA) targeted to the 5'UTR to inhibit IRES mediated translation may represent an alternative approach that could circumvent the problem of interferon resistance.ResultsFour different plasmid constructs were prepared for intracellular delivery of siRNAs targeting the stem loop II-III of HCV 5' UTR. The effect of siRNA production on IRES mediated translation was investigated using chimeric clones between the gene for green fluorescence protein (GFP) and IRES sequences of six different HCV genotypes. The siRNA targeted to stem loop II effectively mediated degradation of HCV IRES mRNA and inhibited GFP expression in the case of six different HCV genotypes, where as siRNAs targeted to stem loop III did not. Furthermore, intracytoplasmic expression of siRNA into transfected Huh-7 cells efficiently degraded HCV genomic RNA and inhibited core protein expression from infectious full-length infectious clones HCV 1a and HCV 1b strains.ConclusionThese in vitro studies suggest that siRNA targeted to stem-loop II is highly effective inhibiting IRES mediated translation of the major genotypes of HCV. Stem-loop II siRNA may be a good target for developing an intracellular immunization strategy based antiviral therapy to inhibit hepatitis C virus strains that are not inhibited by interferon.

BAC clones generated from sheared DNA

BAC libraries generated from restriction-digested genomic DNA display representational bias and lack some sequences. To facilitate completion of genome projects, procedures have been developed to create BACs from DNA physically sheared to create fragments extending up to 200 kb. The DNA fragments were repaired to create blunt ends and ligated to a new BAC vector. This approach has been tested by generating BAC libraries from Drosophila DNA with insert lengths between 50 and 150 kb. The libraries lack chimeric clone problems as determined by mapping paired BAC-end sequences to the assembled fly genome sequence. The utility of “sheared” libraries was demonstrated by closure of a previous clone gap and by isolation of clones from telomeric regions, which were notably absent from previous Drosophila BAC libraries.

Genetic diversity and geographical dispersal in grapevine clones revealed by microsatellite markers

Intravarietal genetic diversification associated with geographical dispersal of a vegetatively propagated species was studied using grapevine Vitis vinifera L. 'Cabernet Sauvignon' as a model. Fifty-nine clonal samples obtained from 7 countries (France, Chile, Spain, Australia, Hungary, USA, and Italy) were analyzed using 84 microsatellite markers. Eighteen polymorphic microsatellite loci (21.4%) were detected, finding 22 different genotypes in the population analyzed with a genetic similarity of over 97%. The presence of chimeric clones was evidenced at locus VMC5g7 by means of a segregation analysis of descendants by self-pollination of a triallelic Chilean clone and by somatic embryogenesis analysis, showing a mutation in L2 cell layer. Only 2 clones (obtained from France and Australia) presented the ancestral genotype, and the most divergent genotype was exhibited by another French clone, which had accumulated 5 somatic mutations. The 2 largest populations considered (from France and Chile) showed a clear divergency in the polymorphisms detected. These antecedents enabled the tracing of geographical dispersal with a phylogenetic hypothesis supporting France as the center of origin of diversification of Cabernet Sauvignon. The results obtained could help to explain diversification processes in other grapevine cultivars. The possibility that this kind of genetic variability occurs in other vegetatively propagated species is discussed, focusing on possible fingerprinting applications.

Virulence attenuation of Dengue virus due to augmented glycosaminoglycan-binding affinity and restriction in extraneural dissemination

To gain insight into the role of cell surface glycosaminoglycans (GAG) in dengue virus (DEN) cell tropism and virulence, DEN-2 mouse brain-adapted vaccine candidate, neurovirulent prototype strain (NGC) and low-passage strain, PUO-218, were passaged in BHK-21 and SW13 cells to isolate variants with high affinity for GAG. Sequence comparisons of parent and passage variants revealed five GAG-binding determinants, which all cluster in a surface-exposed region in domain II of the three-dimensional structure of the DEN envelope protein. Using an infectious cDNA clone of NGC and an NGC/PUO-218 prM-E chimeric clone, it was demonstrated that the GAG-binding determinants augment the specific infectivity for BHK-21 and/or SW13 cells by 10- to 170-fold and in some cases marginally reduce that for Vero cells. This altered cell tropism was due to a greater dependence of the variants on cell surface GAG for attachment/entry, given their increased susceptibility to heparin inhibition. The effect of the GAG-binding determinants on virulence was examined in mice deficient in alpha/beta/gamma interferon responses. High GAG affinity strongly correlated with low neuroinvasiveness due to rapid virus clearance from the blood. It was speculated that this mechanism accounts for the attenuation in primates of some DEN vaccine candidates. Interestingly, the GAG-binding variants did not display marked attenuation of neurovirulence and the opposing effect of enhanced neurovirulence was associated with one determinant (Lys126) already present in mouse brain-adapted NGC. This discrepancy of attenuated neuroinvasiveness and augmented neurovirulence may be reconciled by the existence of different mechanisms of virus dissemination in the brain and in extraneural tissues.