Sarah K. Wootton

Homo-Oligomerization of the Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein and the Role of Disulfide Linkages

ABSTRACT As a step toward understanding the assembly pathway of the porcine reproductive and respiratory syndrome virus (PRRSV), the oligomeric properties of the nucleocapsid (N) protein were investigated. In this study, we have demonstrated that under nonreducing conditions the N protein forms disulfide-linked homodimers. However, inclusion of an alkylating agent (N-ethylmaleimide [NEM]) prevented disulfide bond formation, suggesting that these intermolecular disulfide linkages were formed as a result of spurious oxidation during cell lysis. In contrast, N protein homodimers isolated from extracellular virions were shown to have formed NEM-resistant intermolecular disulfide linkages, the function of which is probably to impart stability to the virion. Pulse-chase analysis revealed that N protein homodimers become specifically disulfide linked within the virus-infected cell, albeit at the later stages of infection, conceivably when the virus particle buds into the oxidizing environment of the endoplasmic reticulum. Moreover, NEM-resistant disulfide linkages were shown to occur only during productive PRRSV infection, since expression of recombinant N protein did not result in the formation of NEM-resistant disulfide-linked homodimers. Mutational analysis indicated that of the three conserved cysteine residues in the N protein, only the cysteine at position 23 was involved in the formation of disulfide linkages. The N protein dimer was shown to be stable both in the presence and absence of intermolecular disulfide linkages, indicating that noncovalent interactions also play a role in dimerization. Non-disulfide-mediated N protein interactions were subsequently demonstrated both in vitro by the glutathione S-transferase (GST) pull-down assay and in vivo by the mammalian two-hybrid assay. Using a series of N protein deletion mutants fused to GST, amino acids 30 to 37 were shown to be essential for N-N interactions. Furthermore, since RNase A treatment markedly decreased N protein-binding affinity, it appears that at least in vitro, RNA may be involved in bridging N-N interactions. In cross-linking experiments, the N protein was shown to assemble into higher-order structures, including dimers, trimers, tetramers, and pentamers. Together, these findings demonstrate that the N protein possesses self-associative properties, and these likely provide the basis for PRRSV nucleocapsid assembly.

Phosphorylation of the porcine reproductive and respiratory syndrome virus nucleocapsid protein.

ABSTRACT Porcine reproductive and respiratory syndrome virus (PRRSV) is a cytoplasmic RNA virus with the unique or unusual feature of having a nucleocapsid (N) protein that is specifically transported to the nucleolus of virus-infected cells. In this communication, we show that the N protein is a phosphoprotein. Phosphoamino acid analysis of authentic and recombinant N proteins demonstrated that serine residues were exclusively phosphorylated. The pattern of phosphorylated N protein cellular distribution in comparison with that of [35S]methionine-labeled N protein suggested that phosphorylation does not influence subcellular localization of the protein. Time course studies showed that phosphorylation occurred during, or shortly after, synthesis of the N protein and that the protein remained stably phosphorylated throughout the life cycle of the virus to the extent that phosphorylated N protein was found in the mature virion. Two-dimensional electrophoresis and acid-urea gel electrophoresis showed that one species of the N protein is predominant in virus-infected cells, suggesting that multiple phosphorylated isoforms of N do not exist.

Peptide domains involved in the localization of the porcine reproductive and respiratory syndrome virus nucleocapsid protein to the nucleolus.

Abstract The nucleocapsid (N) protein of porcine reproductive and respiratory syndrome virus (PRRSV) is the principal component of the viral nucleocapsid and localizes to the nucleolus. Peptide sequence analysis of the N protein of several North American isolates identified two potential nuclear localization signal (NLS) sequences located at amino acids 10–13 and 41–42, which were labeled NLS-1 and NLS-2, respectively. Peptides containing NLS-1 or NLS-2 were sufficient to accumulate enhanced green fluorescent protein (EGFP) in the nucleus. The inactivation of NLS-1 by site-directed mutagenesis or the deletion of the first 14 amino acids did not affect N protein localization to the nucleolus. The substitution of key lysine residues with uncharged amino acids in NLS-2 blocked nuclear/nucleolar localization. Site-directed mutagenesis within NLS-2 identified the sequence, KKNKK, as forming the core localization domain within NLS-2. Using an in vitro pull-down assay, the N protein was able to bind importin-α, importin-β nuclear transport proteins. The localization pattern of N-EGFP fusion peptides represented by a series of deletions from the C- and N-terminal ends of the N protein identified a region covering amino acids 41–72, which contained a nucleolar localization signal (NoLS) sequence. The 41–72 N peptide when fused to EGFP mimicked the nucleolar–cytoplasmic distribution of native N. These results identify a single NLS involved in the transport of N from the cytoplasm and into nucleus. An additional peptide sequence, overlapping NLS-2, is involved in the further targeting of N to the nucleolus.

Antigenic Importance of the Carboxy-Terminal Beta-Strand of the Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein

ABSTRACT Five domains of antigenic importance were previously mapped on the nucleocapsid protein (N) of the porcine reproductive and respiratory syndrome virus (PRRSV), and a domain comprised of the 11 C-terminal-most amino acids (residues 112 to 123) was shown to be essential for binding of N-specific conformation-dependent monoclonal antibodies (MAbs). In the present study, the importance of individual residues within this C-terminal domain for antigenicity was investigated using eight different mutant constructs of N expressed in HeLa cells. Single amino acid substitutions were introduced into the C-terminal domain of the N protein, and the significance of individual amino acids for MAb reactivity was determined by immunoprecipitation. None of the MAbs tested recognized the mutant with a leucine-to-proline substitution at residue 114 (L114P), while V112P, R113P, R113D, I115P, and R116P reduced MAb binding significantly. Conversely, substitution of amino acids at positions 118 (T118S) and 121 (P121A) had little effect on MAb binding. Secondary-structure predictions indicate that amino acids 111 to 117 form a beta-strand. In view of the fact that replacement of beta-strand-forming amino acids with proline elicited the greatest effect on MAb binding, it appears that secondary structure in the C terminus of the N protein is an important determinant of conformational epitope formation. While the crystal structure of the PRRSV N protein remains to be determined, results from these studies broaden our understanding of the secondary structures that make up the PRRSV N protein and shed some light on how they may relate to function.

Opposing functions of Akt isoforms in lung tumor initiation and progression.

Background The phosphatidylinositol 3-kinase–regulated protein kinase, Akt, plays an important role in the initiation and progression of human cancer. Mammalian cells express three Akt isoforms (Akt1–3), which are encoded by distinct genes. Despite sharing a high degree of amino acid identity, phenotypes observed in knockout mice suggest that Akt isoforms are not functionally redundant. The relative contributions of the different Akt isoforms to oncogenesis, and the effect of their deficiencies on tumor development, are not well understood. Methods Here we demonstrate that Akt isoforms have non-overlapping and sometimes opposing functions in tumor initiation and progression using a viral oncogene-induced mouse model of lung cancer and Akt isoform-specific knockout mice. Results Akt1 ablation significantly delays initiation of lung tumor growth, whereas Akt2 deficiency dramatically accelerates tumorigenesis in this mouse model. Ablation of Akt3 had a small, not statistically significant, stimulatory effect on tumor induction and growth by the viral oncogene. Terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling and Ki67 immunostaining of lung tissue sections revealed that the delayed tumor induction in Akt1−/− mice was due to the inhibitory effects of Akt1 ablation on cell growth and survival. Conversely, the accelerated growth rate of lung tumors in Akt2−/− and Akt3−/− mice was due to increased cell proliferation and reduced tumor cell apoptosis. Investigation of Akt signaling in tumors from Akt knockout mice revealed that the lack of Akt1 interrupted the propagation of signaling in tumors to the critical downstream targets, GSK-3α/β and mTOR. Conclusions These results demonstrate that the degree of functional redundancy between Akt isoforms in the context of lung tumor initiation is minimal. Given that this mouse model exhibits considerable similarities to human lung cancer, these findings have important implications for the design and use of Akt inhibitors for the treatment of lung cancer.

Full-length genome sequence analysis of enzootic nasal tumor virus reveals an unusually high degree of genetic stability.

Enzootic nasal tumor virus (ENTV) is a betaretrovirus of sheep (ENTV-1) and goats (ENTV-2) associated with neoplastic transformation of epithelial cells of the ethmoid turbinate. Confirmation of the role of ENTV in the pathogenesis of enzootic nasal adenocarcinoma (ENA) has yet to be resolved due to the lack of an infectious molecular clone and the inability to culture the virus. Very little is known about the prevalence of this disease, particularly in North America, and only one full-length sequence is available for each of ENTV-1 and ENTV-2. In order to understand the molecular evolution of ENTV-1, the full-length genome sequence of ten ENTV-1 proviruses derived from clinical samples of ENA isolated from conventionally reared sheep in Canada and the United States was determined. The North American ENTV-1 (ENTV-1(NA)) genomes shared greater than 96% sequence identity with the European ENTV-1 sequence (ENTV-1(EU)). Most of the amino acid differences were found in Orf-x, which in the corresponding ENTV-1(EU) genome is truncated by 44 amino acids. Apart from Orf-x, the long terminal repeat (LTR) is where the majority of differences between ENTV-1(NA) and ENTV-1(EU) reside. Overall, there was an unusually high degree of amino acid conservation among the isolates suggesting that ENTV-1 is under stabilizing selection and K(a)/K(s) ratios calculated for each of the viral genes support this hypothesis. The unusually high degree of genetic stability of the ENTV-1 genome enabled us to develop a hemi-nested PCR assay for detection of ENTV-1 in clinical samples. Additionally, multiple nasal tumor cell clones were established and while most had lost the provirus by passage 5; one polyclonal line retained the provirus and attempts are being made to culture these cells. These tumor cells, the first of their kind, may provide a system for studying ENTV-1 in vitro. This work represents an important step in the study of ENTV and sets the foundation for the construction of an infectious molecular clone of ENTV-1.

Genetic characterization of small ruminant lentiviruses circulating in naturally infected sheep and goats in Ontario, Canada.

Maedi-visna virus (MVV) and caprine arthritis encephalitis virus (CAEV) are related members of a group of small ruminant lentiviruses (SRLVs) that infect sheep and goats. SRLVs are endemic in many countries, including Canada. However, very little is known about the genetic characteristics of Canadian SRLVs, particularly in the province of Ontario. Given the importance of surveillance and eradication programs for the control of SRLVs, it is imperative that the diagnostic tests used to identify infected animals are sensitive to local strains of SRLVs. The aim of this work was to characterize SRLV strains circulating in Ontario and to evaluate the variability of the immunodominant regions of the Gag protein. In this study, the nearly complete gag sequence of 164 SRLVs, from 130 naturally infected sheep and 32 naturally infected goats from Ontario, was sequenced. Animals belonged to distantly located single and mixed species (sheep and goats) farms. Ovine lentiviruses from the same farm tended to cluster more closely together than did caprine lentiviruses from the same farm. Sequence analysis revealed a higher degree of heterogeneity among the caprine lentivirus sequences with an average inter-farm pairwise DNA distance of 10% and only 5% in the ovine lentivirus group. Interestingly, amplification of SRLVs from ELISA positive sheep was successful in 81% of cases, whereas amplification of SRLV proviral DNA was only possible in 55% of the ELISA positive goat samples; suggesting that a significant portion of caprine lentiviruses circulating in Ontario possess heterogeneity at the primer binding sites used in this study. Sequences of sheep and goat SRLVs from Ontario were assembled into phylogenetic trees with other known SRLVs and were found to belong to sequence groups A2 and B1, respectively, as defined by Shah et al. (2004a). A novel caprine lentivirus with a pairwise genetic difference of 15.6-25.4% relative to other group B subtypes was identified. Thus we suggest the designation of a novel subtype, B4, within the caprine lentivirus-like cluster. Lastly, we demonstrate evidence of recombination between ovine lentiviruses. These results emphasize the broad genetic diversity of SRLV strains circulating in the province of Ontario and show that the gag region is suitable for phylogenetic studies and may be applied to monitor SRLV eradication programs.

Jaagsiekte Sheep Retrovirus and Enzootic Nasal Tumor Virus Promoters Drive Gene Expression in All Airway Epithelial Cells of Mice but Only Induce Tumors in the Alveolar Region of the Lungs

ABSTRACT Jaagsiekte sheep retrovirus (JSRV) induces tumors in the distal airways of sheep and goats, while the closely related enzootic nasal tumor virus type 1 (ENTV-1) and ENTV-2 induce tumors in the nasal epithelium of sheep and goats, respectively. When expressed using a strong Rous sarcoma virus promoter, the envelope proteins of these viruses induce tumors in the respiratory tract of mice, but only in the distal airway. To examine the role of the retroviral long terminal repeat (LTR) promoters in determining tissue tropism, adeno-associated virus (AAV) vectors expressing alkaline phosphatase under the control of the JSRV, ENTV-1, or ENTV-2 LTRs were generated and administered to mice. The JSRV LTR was active in all airway epithelial cells, while the ENTV LTRs were active in the nasal epithelium and alveolar type II cells but poorly active in tracheal and bronchial epithelial cells. When vectors were administered systemically, the ENTV-1 and -2 LTRs were inactive in major organs examined, whereas the JSRV showed high-level activity in the liver. When a putative transcriptional enhancer from the 3′ end of the env gene was inserted upstream of the JSRV and ENTV-1 LTRs in the AAV vectors, a dramatic increase in transgene expression was observed. However, intranasal administration of AAV vectors containing any combination of ENTV or JSRV LTRs and Env proteins induced tumors only in the lower airway. Our results indicate that mice do not provide an adequate model for nasal tumor induction by ENTV despite our ability to express genes in the nasal epithelium.

Spatial and temporal patterns of porcine reproductive and respiratory syndrome virus (PRRSV) genotypes in Ontario, Canada, 2004–2007

BackgroundThe spread of PRRSV among pig herds has been investigated experimentally, but few observational studies have investigated this subject. Because PRRSV is endemic and live modified vaccines are used in Ontario, the spatial and temporal distributions of 6 PRRSV genotypes were investigated in the province during the period from 2004–2007. The purpose was to find evidence of spread of PRRSV genotypes and determine if spread could be attributed to supplier or ownership connections between herds. Sequence information from PRRSV ORF5 and related source-herd demographic information were obtained from diagnostic submissions to the Animal Health Laboratory, University of Guelph.ResultsA spatial cluster that could not be attributed to supplier or ownership connections among herds in the cluster was detected for RFLP type 1-3-4. Because of genetic dissimilarity among members of the cluster, it was considered to be a result of past spread of the RFLP type. A spatio-temporal cluster detected for RFLP type 1-18-4 was attributed to a shared gilt supplier among the herds in the cluster. Significant spatio-temporal patterns detected for RFLP type 2-5-2, which is considered to be a vaccine-type virus were most likely due to grouping of herds in an ownership that used the corresponding vaccine. Clustering within herd-ownership was a risk factor for presence of five of the six genotypes investigated in the present study.ConclusionsAlthough the literature indicates that PRRSV can spread via aerosol between pig herds, the present study found no strong evidence of this occurring in Ontario. The evidence pointed toward transmission of PRRSV occurring in this population by common sources of animals or similarity of herd ownership, which is a proxy measure for other connections between herds. It is also apparent that the recognition and testing of these connections between herds is a necessary part of interpreting spatio-temporal patterns of PRRSV genotypes.

Adeno-Associated Virus Vector Mediated Expression of an Oncogenic Retroviral Envelope Protein Induces Lung Adenocarcinomas in Immunocompetent Mice

Lung cancer is the most common cause of cancer-related death worldwide. A poor overall survival rate of 16% necessitates the need for novel treatment strategies. Mouse models of lung cancer are important tools for analyzing the significance of somatic mutations in the initiation and progression of lung cancer. Of additional importance, however, are animal models of virally induced cancers. JSRV is a simple betaretrovirus that causes contagious lung cancer in sheep known as ovine pulmonary adenocarcinoma and closely resembles human lung adenocarcinoma. Previously we showed that expression of the JSRV envelope (Env) from an AAV vector induced lung tumors in immunodeficient mice, but not in immunocompetent mice. Because of the importance of studying lung cancer in the context of an intact immune system we sought to improve our mouse model. In this report, we employed the use of a strong JSRV enhancer-promoter combination to express Env at high levels and demonstrate for the first time, lung tumor induction in immunocompetent mice. This occurred despite a robust Env-specific antibody-mediated immune response. The PI3K/Akt and MAPK pathways were activated in both immunocompetent and immunodeficient mice, however, differential activation of PTEN, GSKα, p70S6K, p38MAPK, ATF2 and STAT5 was observed. A JSRV Env lung tumor-derived cell line was shown to have a similar signal transduction activation profile as Env-induced lung tumors in C57BL/6 mice. Given the similarities between our model and pulmonary adenocarcinomas in humans, and the ease with which tumors can be induced in any transgenic mouse, this system can be used to uncover novel mechanisms involved lung tumorigenesis.