Assessment of Virally Vectored Autoimmunity as a Biocontrol Strategy for Cane Toads

BACKGROUND: The cane toad, Bufo (Chaunus) marinus, is one of the most notorious vertebrate pests introduced into Australia over the last 200 years and, so far, efforts to identify a naturally occurring B. marinus-specific pathogen for use as a biological control agent have been unsuccessful. We explored an alternative approach that entailed genetically modifying a pathogen with broad host specificity so that it no longer caused disease, but carried a gene to disrupt the cane toad life cycle in a species specific manner. METHODOLOGY/PRINCIPAL FINDINGS: The adult beta globin gene was selected as the model gene for proof of concept of autoimmunity as a biocontrol method for cane toads. A previous report showed injection of bullfrog tadpoles with adult beta globin resulted in an alteration in the form of beta globin expressed in metamorphs as well as reduced survival. In B. marinus we established for the first time that the switch from tadpole to adult globin exists. The effect of injecting B. marinus tadpoles with purified recombinant adult globin protein was then assessed using behavioural (swim speed in tadpoles and jump length in metamorphs), developmental (time to metamorphosis, weight and length at various developmental stages, protein profile of adult globin) and genetic (adult globin mRNA levels) measures. However, we were unable to detect any differences between treated and control animals. Further, globin delivery using Bohle iridovirus, an Australian ranavirus isolate belonging to the Iridovirus family, did not reduce the survival of metamorphs or alter the form of beta globin expressed in metamorphs. CONCLUSIONS/SIGNIFICANCE: While we were able to show for the first time that the switch from tadpole to adult globin does occur in B. marinus, we were not able to induce autoimmunity and disrupt metamorphosis. The short development time of B. marinus tadpoles may preclude this approach

High-Mobility-Group Protein I Can Modulate Binding of Transcription Factors to the U5 Region of the Human Immunodificiency Virus Type 1 Proviral Promoter

HMG I/Y appears to be a multifunctional protein that relies on in its ability to interact with DNA in a structure-specific manner anti with DNA, binding transcriptional activators via distinct protein-protein interaction surfaces. To investigate the hypothesis that HMG I/Y may have a role in human immunodeficiency virus type 1 (HIV-1) expression, we have analyzed whether HMG I/Y interacts with the 5' long terminal repeat anti whether this interaction can modulate transcription factor binding. Using purified recombinant HMG I, we have identified several high-affinity binding sites which overlap important transcription factor binding sites. One of these HMG I binding sites coincides with an important binding site for AP-1 located downstream of the transcriptional start site, in the 5' untranslated region at the boundary of a positioned nucleosome. HMG I binding to this composite site inhibits the binding of recombinant AP-1. Consistent with this observation, using nuclear extracts prepared from Jurkat T cells, we show that HMG I (but not HMG Y) is strongly induced upon phorbol myristate acetate stimulation and this induced HMG I appears to both selectively inhibit the binding of basal DNA-binding proteins and enhance the binding of an inducible AP-1 transcription factor to this AP-1 binding site. We also report the novel finding that a component present in this inducible AP-1 complex is ATF-3. Taken together, these results argue that HMG I may play a fundamental role in HIV-1 expression by determining the nature of transcription factor-promoter interactions

Distinct importin recognition properties of histones and chromatin assembly factors.

Synthesis of the protein components of nuclear chromatin occurs in the cytoplasm, necessitating specific import into the nucleus. Here, we report the binding affinities of the nuclear localisation sequence (NLS)-binding importin subunits for a range of histones and chromatin assembly factors. The results suggest that import of histones to the nucleus may be mediated predominantly by importin β1, whereas the import of the other components probably relies on the conventional α/β1 import pathway. Differences in recognition by importin β1 were observed between histone H2A and the variant H2AZ, as well as between histone H3/4 with or without acetylation. The results imply that different histone variants may possess distinct nuclear import properties, with acetylation possibly playing an inhibitory role through NLS masking

Time course detection of rHb within tadpoles after injection.

<p>Western blot using rabbit antibody to adult globin to detect persistence of rHb emulsion. n = 3 animals pooled per time point. Actin indicates loading per protein sample (mAb mouse anti-actin used at 1∶5000).</p

No effect on globin protein profile following infection of tadpoles with virus carrying rAdglob.

<p>200 ng protein per well from lysed red blood cell samples taken from metamorphs then stained with silver stain. a: Lanes 1 and 12, See Blue Plus2 Pre Stained Standard (Invitrogen); Lanes 2–4, blood from 3 control animals bathed in 10² TCID₅₀/ml of rBIV/neo^r; Lanes 5–10, blood from 6 test animals bathed in 10² TCID₅₀/ml of rBIV/neo^r/adglo. Lane 11, positive control, globin from a 2 month old toadlet. b: Panel shows silver stain and Western blot antibody detection of adult globin; lane i See Blue Plus2 Pre Stained Standard (Invitrogen); lane ii, positive control, globin from a 2 month old toadlet; lanes iii, iv and v, 500, 1000 and 5000 ng of protein from fluid of untreated stage 42 tadpoles.</p

Tadpole to adult globin switch detected in normal cane toad development.

<p>a: mRNA data expressed as number of copies of adult or tadpole globin mRNA detected by real time PCR across various tadpole and metamorphic stages. Mean copy numbers were normalised using a toad actin housekeeping gene. Animals were staged according to Limbaugh and Volpe, 1957. Toadlet (*) development was approximately one month post-metamorphosis. b: Detection of globin proteins as determined by western blot analysis using specific antibodies to tadpole and adult globins. Coomassie staining indicates the loading level for each lane. Recombinant proteins for adult and tadpole globin (rAdglob and rTadglob, respectively), as well as native adult globin (Adult) were included as positive controls.</p

IgG antibody response to globin antigen not detected in metamorphs by ELISA.

<p>IgG levels as measured by ELISA in metamorphs (stage 46) injected with adult globin (rAdglob treated) compared with untreated metamorphs (FCA control). Positive controls show IgG levels in metamorphs and an adult injected with ovalbumin. Sera from rAdglob treated and FCA control animals used at 1∶80; ovalbumin treated metamorph sera used at 1∶320; ovalbumin treated adult sera used at 1∶1000.</p

Physical measures of <i>B. marinus</i> development across metamorphosis unchanged by recombinant adult globin injection.

<p>a: Wet weights (mg), lengths (mm) and speed of development (days) for animals at six different developmental stages injected with rAdglob (□) or FCA control (▴, dotted line), as well as normal uninjected animals (◊) were recorded. n = 10 animals for each stage and error bars show standard deviation. b: Swimming performance of rAdglob treated and FCA control tadpoles at approximately stage 36 (or 10 days post injection) is shown. Burst swim speed represents the absolute swim speed normalised to body length, and the full data range (vertical line), standard deviation (box) and mean (horizontal line) are indicated. c: Jumping performance of rAdglob treated and FCA control amimals at stage 46 is shown. Longest jump distance was normalised to body lengths.</p

Adult globin mRNA and protein expression levels in treated and untreated <i>Bufo marinus</i>.

<p>a: The profile of adult globin mRNA expression across metamorphosis for treated rAdglob injected (grey shading) and control FCA only injected (white) animals was determined by real time-PCR analysis. Each stage point indicates the overall expression level for five pooled (∧) animals, with treated and control animals showing similar copy numbers. The last stage point represents an average of 6 individuals. Adult globin copy number was normalised to a toad actin housekeeping gene. b: Western blot analysis of whole animal extracts (upper panel) provides a general indication of the adult globin protein levels in normal developing animals (stages 28, 36 and 46), as well as FCA control and rAdglob treated animals. The lower panel shows the Coomassie brilliant blue staining for the same blot and provides an indication of total protein loading. rAdglob (∼) is an additional 3.3 kDa larger than native adult globin due to His_x6 tag and linker. Adult (*) control was generated using purified globin from the RBCs of adult <i>B. marinus</i>. The blot is representative of rAdglob treatments (n = 10) and FCA controls (n = 9).</p