Cary Rue - Academia.edu (original) (raw)

Papers by Cary Rue

Journal of General Virology, Feb 1, 2002

Pseudorabies virus first attaches to cells through an interaction between the envelope glycoprote... more Pseudorabies virus first attaches to cells through an interaction between the envelope glycoprotein C (gC) and the cell surface heparan sulfate (HS) that is linked to proteoglycans (HSPGs). The HSbinding domain of gC is composed of three discrete heparin-binding domains (HBDs), designated HBD1,-2 and-3 for their proximity to the amino terminus of gC. Each HBD can independently mediate virus attachment to HS, yet each also exhibits a distinct binding preference for differentially sulfated derivatives of heparin. To demonstrate this, affinity columns composed of wild-type gC or mutant gC retaining a single HBD to capture several HSPGs from cultured pig and bovine kidney cells were used. The wild-type gC column bound all of the HSPGs well and, overall, bound more than 90 % of the total sample applied to the column. Columns composed of either HBD2 or-3 bound intermediate amounts (40 %) of the total sample applied, while the HBD1 column bound low amounts of HSPGs. HBD2 and-3 columns did not uniformly bind all of the HSPGs from bovine kidney cells, but the same HSPGs were bound with equal efficiency on each column. Thus, despite their different preferences for sulfation patterns on HS side-chains, HBD2 and-3 appear to bind the same proteoglycan cores. These results established a hierarchy of HBD2 l HBD3 HBD1 in importance for HSPG binding. These in vitro-binding results correlated with the attachment phenotype of virus strains expressing gC with a single HBD in their envelopes.

Virus Genes, Aug 1, 2008

Marek's disease (MD) is the leading cause of losses in chicken production in the world. Over the ... more Marek's disease (MD) is the leading cause of losses in chicken production in the world. Over the past 40 years significant progress has been made in the control of MD through the use of vaccines which reduce or delay tumor formation in vaccinated flocks. However, these vaccines fail to induce an immune response that protects against infection and virus shedding. Little is known about the genetic changes that lead to attenuation and are necessary for the generation of vaccine strains. Previous research has demonstrated that serial passage of virulent strains in cell culture results in the generation of attenuated progeny. Obtaining detailed knowledge of the changes which are needed for attenuation will be important for advancing our understanding of MD biology and should facilitate the development of more potent vaccines. We have determined the complete nucleotide sequence of a bacterial artificial chromosome (BAC) construct representing the 80th passage of a very virulent plus (vv+) MD virus strain termed 584A. Pathotyping studies have indicated that this strain (584Ap80) is indeed attenuated. Bioinformatic analysis of the sequencing data has identified numerous gross genetic changes clustering in the inverted repeat regions of the genome, as well as subtle changes (single nucleotide polymorphisms or SNPs) scattered throughout the genome. Relative to the parental strain (584Ap9), insertional mutations were identified in the MD-specific genes encoding RLORF1, RLORF3, RLORF6, 23 kDa, RLORF7 (Meq), vIL8, vLip, RSORF1, and five uncharacterized novel genes. Deletions were found in four locations within the 584Ap80 genome. A large deletion (297nt) was found in the diploid genes 85.6/98.6 and a 321 nt deletion within the intergenic region between the U L 3 and U L 3.5 genes is predicted to create a fusion polypeptide. A single nucleotide deletion was identified within the origin of replication. Both insertions and deletions were found in the dipoid genes MDV3.4/78.3 encoding the virulence factor RLORF4. The sequencing of the attenuated strain 584Ap80 and comparison to that of the virulent parent 584A passage 9 (584Ap9) has provided a wealth of information regarding genetic changes which have occurred during the attenuation process.

Virology, Mar 1, 2003

Glycoprotein C (gC) of pseudorabies virus, a swine herpesvirus, initiates virus attachment by bin... more Glycoprotein C (gC) of pseudorabies virus, a swine herpesvirus, initiates virus attachment by binding to heparan sulfate (HS) linked to proteoglycans (HSPGs) on the cell surface. This interaction facilitates a required step in virus entry, the binding to a non-HS coreceptor, likely by another viral glycoprotein, gD. We demonstrate that gC has an even more direct role in virus entry than simply promoting adhesion strengthening. A porcine cell line expressing gC trans-complemented the penetration, but not attachment, defect of gC null mutants. In addition, gC promoted the colocalization of cell surface HSPGs and the actin cytoskeleton, suggesting a role for filamentous actin in virus entry. This was supported by results showing that both the engagement of a non-HS coreceptor and entry events subsequent to coreceptor binding were impaired if cells were first treated with an actin depolymerizing agent, cytochalasin D. Our results suggest a model in which gC-HS interactions promote not only virus attachment but also virus entry by usurping the normal properties of HSPGs.

Virus Genes, Jun 1, 2008

The complete DNA sequence of the mildly virulent Gallid herpesvirus type 2 strain CU-2 was determ... more The complete DNA sequence of the mildly virulent Gallid herpesvirus type 2 strain CU-2 was determined and consists of 176,922 bp with an overall gene organization typical of class E herpesviruses. Phylogenetically, this strain partitions in its own branch between the virulent strains RB-1B, Md11, and Md5, and the vaccine strain CVI988. Overall, the genome of CU-2 is more similar to that of CVI988, with identically sized unique short regions of 11,651 bp. As in CVI988, an insertion of 177 bp was identified in the overlapping genes encoding the Meq, RLORF6, and 23 kDa proteins within the repeat long region of the genome. A total of 15 single nucleotide polymorphisms (SNPs) common to both CU-2 and CVI988, and not occurring in virulent strains, were identified in the genes encoding UL29, UL45, UL50, UL52, LORF10, RLORF14a, RLORF12, Meq(RLORF7), 23kDa, ICP4, US3, and two hypothetical proteins MDV071.4 and MDV076.4. Each gene encoding UL29 and Meq contained two SNPs. Only one major open reading frame (ORF) encoding UL41, the virus host shutoff (VHS) ribonuclease, was disrupted in the CU-2 genome. An additional cytosine after the 25 codon is predicted to produce a truncated protein of 97 aa. Since GaHV-2 mutants lacking UL41 have been reported to retain their virulence, other factors are likely responsible for the low virulence of CU-2. It is largely suspected that SNPs in common with CVI988 along with the insertions in the Meq loci are responsible for its phenotype. Conversely, we identified 43 nonsynonymous mutations (within 23 genes) that may contribute to the virulence of CU-2. These SNPs are shared exclusively with all sequenced virulent strains (Md5, Md11, and RB-1B) and not present within the CVI988 genome. Although most occur in proteins of unknown function, a significant percentage is in proteins involved in virion assembly.

Journal of Virological Methods, Jul 1, 2008

Pseudorabies virus (PRV) glycoprotein C (gC) initiates virus attachment to cells by binding to he... more Pseudorabies virus (PRV) glycoprotein C (gC) initiates virus attachment to cells by binding to heparan sulfate (HS) proteoglycans. The gC:HS interaction is not essential since gC null mutants still infect; however, they are more easily removed from cells during the initial stages of infection. The expendability of gC has facilitated a genetic mapping of the HS-binding domain, which is composed of three independent heparin-binding domains (HBDs) of six to eight amino acids each. Previous results suggested that at least one of the HBDs (HBD 1) functioned in a context-dependent manner. To define the context better, a reversion analysis was performed in which a defective gC containing a nonfunctional but intact HBD 1 regained HS-binding ability. To increase the reversion frequency, an efficient method for targeted, yet random mutagenesis of the gC gene was developed. The method involves random mutagenesis of a plasmid-borne copy of gC, and highly efficient recombination of the plasmid-borne genes into the virus genome at the site of a double-strand break in the viral gC locus. Revertants were recovered readily, and their gC alleles suggested that HS-binding could be restored by several different amino acid substitutions. This approach should be applicable to targeted mutagenesis of other herpesvirus genes.

Journal of Virology, Nov 15, 2004

Cyclooxygenase-2 (COX-2) is a cellular enzyme in the eicosanoid synthetic pathway that mediates t... more Cyclooxygenase-2 (COX-2) is a cellular enzyme in the eicosanoid synthetic pathway that mediates the synthesis of prostaglandins from arachidonic acid. The eicosanoids function as critical regulators of a number of cellular processes, including the acute and chronic inflammatory response, hemostasis, and the innate immune response. Human cytomegalovirus (HCMV), which does not encode a viral COX-2 isoform, has been shown to induce cellular COX-2 expression. Importantly, although the precise role of COX-2 in CMV replication is unknown, COX-2 induction was shown to be critical for normal HCMV replication. In an earlier study, we identified an open reading frame (Rh10) within the rhesus cytomegalovirus (RhCMV) genome that encoded a putative protein (designated vCOX-2) with high homology to cellular COX-2. In the current study, we show that vCOX-2 is expressed with early-gene kinetics during RhCMV infection, resulting in production of a 70-kDa protein. Consistent with the expression of a viral COX-2 isoform, cellular COX-2 expression was not induced during RhCMV infection. Finally, analysis of growth of recombinant RhCMV with vCOX-2 deleted identified vCOX-2 as a critical determinant for replication in endothelial cells.

Journal of Clinical Microbiology, Mar 17, 2010

Eight Newcastle disease virus isolates from Pakistan were sequenced and characterized. A PCR matr... more Eight Newcastle disease virus isolates from Pakistan were sequenced and characterized. A PCR matrix gene assay, designed to detect all avian paramyxovirus 1, did not detect four of the isolates. A new matrix gene test that detected all isolates was developed. Phylogenetic analysis and pathotyping confirmed that virulent viruses of different genotypes are circulating in Pakistan.

Virus Genes, Mar 20, 2008

Journal of General Virology, 2002

Virology

Journal of General Virology, 2011

Newcastle disease virus (NDV) is an avian paramyxovirus that causes significant economic losses t... more Newcastle disease virus (NDV) is an avian paramyxovirus that causes significant economic losses to the poultry industry worldwide. There is limited knowledge about the avian immune response to infection with virulent NDVs, and how this response may contribute to disease. In this study, pathogenesis and the transcriptional host response of chickens to a virulent NDV strain that rapidly causes 100 % mortality was characterized. Using microarrays, a strong transcriptional host response was observed in spleens at early times after infection with the induction of groups of genes involved in innate antiviral and pro-inflammatory responses. There were multiple genes induced at 48 h post-infection including: type I and II interferons (IFNs), several cytokines and chemokines, IFN effectors and inducible nitric oxide synthase (iNOS). The increased transcription of nitric oxide synthase was confirmed by immunohistochemistry for iNOS in spleens and measured levels of nitric oxide in serum. In vitro experiments showed strong induction of the key host response genes, alpha IFN, beta interferon, and interleukin 1b and interleukin 6, in splenic leukocytes at 6 h post-infection in comparison to a non-virulent NDV. The robust host response to virulent NDV, in conjunction with severe pathological damage observed, is somewhat surprising considering that all NDV encode a gene, V, which functions as a suppressor of class I IFNs. Taken together, these results suggest that the host response itself may contribute to the pathogenesis of this highly virulent strain in chickens.