Maximiliano Wilda

Tacaribe virus Z protein interacts with the L polymerase protein to inhibit viral RNA synthesis

ABSTRACT Tacaribe virus (TV) is the prototype of the New World group of arenaviruses. The TV genome encodes four proteins, the nucleoprotein (N), the glycoprotein precursor, the polymerase (L), and a small RING finger protein (Z). Using a reverse genetic system, we recently demonstrated that TV N and L are sufficient to drive transcription and full-cycle RNA replication mediated by TV-like RNAs and that Z is a powerful inhibitor of these processes (N. López, R. Jácamo, and M. T. Franze-Fernández, J. Virol. 65:12241-12251, 2001). In the present study we investigated whether Z might interact with either of the proteins, N and L, required for RNA synthesis. To that end, we used coimmunoprecipitation with monospecific antibodies against the viral proteins and coimmunoprecipitation with serum against glutathione S-transferase (GST) and binding to glutathione-Sepharose beads when Z was expressed as a fusion protein with GST. We demonstrated that Z interacted with L but not with N and that Z inhibitory activity was dependent on its ability to bind to L. We also evaluated the contribution of different Z regions to its binding ability and functional activity. We found that integrity of the RING structure is essential for Z binding to L and for Z inhibitory activity. Mutants with deletions at the N and C termini of Z showed that amino acids within the C-terminal region and immediately adjacent to the RING domain N terminus contribute to efficient Z-L interaction and are required for inhibitory activity. The data presented here provide the first evidence of an interaction between Z and L, suggesting that Z interferes with viral RNA synthesis by direct interaction with L. In addition, coimmunoprecipitation studies revealed a previously unreported interaction between N and L.

Mapping of the Tacaribe Arenavirus Z-Protein Binding Sites on the L Protein Identified both Amino Acids within the Putative Polymerase Domain and a Region at the N Terminus of L That Are Critically Involved in Binding

ABSTRACT Tacaribe virus (TacV) is the prototype of the New World group of arenaviruses. The TacV genome encodes four proteins: the nucleoprotein (N), the glycoprotein precursor, the polymerase (L), and a RING finger protein (Z). Using a reverse genetics system, we demonstrated that TacV N and L are sufficient to drive transcription and replication mediated by TacV-like RNAs and that Z is a powerful inhibitor of these processes (Lopez et al., J. Virol. 65:12241-12251, 2001). More recently, we provided the first evidence of an interaction between Z and L and showed that Zs inhibitory activity was dependent on its ability to bind to L (Jácamo et al., J. Virol. 77:10383-10393, 2003). In the present study, we mapped the TacV Z-binding sites on the 2,210-amino-acid L polymerase. To that end, we performed deletion analysis and point mutations of L and studied the Z-L interaction by coimmunoprecipitation with specific sera. We found that the C-terminal region of L was not essential for the interaction and identified two noncontiguous regions that were critical for binding: one at the N-terminus of L between residues 156 and 292 and a second one in the polymerase domain (domain III). The importance of domain III in binding was revealed by substitutions in D1188 and H1189 within motif A and in each residue of the conserved SDD sequence (residues 1328, 1329, and 1330) within motif C. Our results showed that of the substituted residues, only H1189 and D1329 appeared to be critically involved in binding Z.

Resurgence of canine parvovirus 2a strain in the domestic dog population from Argentina

Ninety-three rectal swab samples were taken, from dogs suspected of canine parvovirus (CPV) infection and analyzed by PCR. A fragment of the VP2 gene, was amplified in 41 (44%) of them, resulting CPV positive samples. Sequencing analysis of these PCR products showed that 37 samples (90.2%) belonged to the CPV2c type, whereas four samples (9.8%) were identified as CPV2a, which has not been found since 2008. It was also found that 24 out of 37 CPV2c samples (65%), carried the mutation Thr440Ala, whereas this mutation was absent in the four CPV2a strains reported herein. Using phylogenetic analysis of the full length VP2 gene, which was amplified by PCR in six local samples, it was seen that CPV2a Argentine strains reported in this study, were genetically closer to a previous local CPV2a isolate (year 2003) and to a South African CPV2a strain, than to any of the recently reported Uruguayan CPV2a strains. The results obtained in this work, together with those reported previously in Uruguay strongly suggest that, in spite of the geographical proximity, wild type CPV strains undergo different evolutive pathways in each country, resulting in the prevalence of different strains in related dog populations. Further extensive epidemiological studies are needed in order to improve the understanding of CPV evolution.

The immune enhancement of a novel soy lecithin/β-glucans based adjuvant on native Neospora caninum tachyzoite extract vaccine in mice

Efficient, cost-effective and safe Th1-immunity-inducing vaccine formulations are paramount for achieving protection against Neospora caninum. In this study, a new adjuvant (Providean-AVEC) was used in the development of a N. caninum vaccine and evaluated in a mouse model. Soluble N. caninum tachyzoite native protein extract (sNcAg) was selected as vaccine antigen based on its capacity to activate production of pro-inflammatory cytokines on dendritic cells. Vaccines containing 4 and 0.4 μg of sNcAg, and Providean-AVEC, ISCOM-Matrix or aluminum hydroxide (Alum) were tested in BALB/c mice. While mice vaccinated with 4μg of sNcAg + Providean-AVEC developed specific antibodies shortly after the first dose, the rest of the high antigen payload formulations only induced seroconversion after the booster. Mice immunized with the high payload ISCOM vaccine (4 μg sNcAg) or with either low or high payload Providean-AVEC formulations (0.4 μg and 4 μg sNcAg, respectively) elicited higher IgG2a than IgG1 serum levels, and IFN-γ anamnestic responses with a Th1-cytokine biased profile. These animals had no histological signs of cerebral lesions and parasite burden assessed by quantitative real-time PCR was not detected. Vaccine preparations including Providean-AVEC as adjuvant limited N. canimum multiplication even with only a tenth of antigen payload compared to vaccines containing other adjuvants. Using adjuvants to specifically activate dendritic cells, combined with a careful antigen selection can enhance cellular responses to inert N. caninum vaccines.

Immunization with Neospora caninum profilin induces limited protection and a regulatory T-cell response in mice.

Profilins are actin-binding proteins that regulate the polymerization of actin filaments. In apicomplexan parasites, they are essential for invasion. Profilins also trigger the immune response of the host by activating TLRs on dendritic cells (DCs), inducing the production of pro-inflammatory cytokines. In this study we characterized for the first time the immune response and protection elicited by a vaccine based on Neospora caninum profilin in mice. Groups of eight BALB/c mice received either two doses of a recombinant N. caninum profilin expressed in Escherichia coli. (rNcPRO) or PBS, both formulated with an aqueous soy-based adjuvant enriched in TLR-agonists. Specific anti-profilin antibodies were detected in rNcPRO-vaccinated animals, mainly IgM and IgG3, which were consumed after infection. Splenocytes from rNcPRO-immunized animals proliferated after an in vitro stimulation with rNcPRO before and after challenge. An impairment of the cellular response was observed in NcPRO vaccinated and infected mice following an in vitro stimulation with native antigens of N. caninum, related to an increase in the percentage of CD4+CD25+FoxP3+. Two out of five rNcPRO-vaccinated challenged mice were protected; they were negative for parasite DNA in the brain and showed no histopathological lesions, which were found in all PBS-vaccinated animals. As a whole, our results provide evidence of a regulatory response elicited by immunization with rNcPRO, and suggest a role of profilin in the modulation and/or evasion of immune responses against N. caninum.

Recombinant foot-and-mouth disease virus (FMDV) non-structural protein 3A fused to enhanced green fluorescent protein (EGFP) as a candidate probe to identify FMDV-infected cattle in serosurveys

Recombinant protein 3A-EGFP, a fusion construct between foot-and-mouth disease virus (FMDV) non-structural protein 3A and the enhanced green fluorescent protein (EGFP) was expressed in BL21-DE3 cells. The identity of the partially purified protein 3A-EGFP was confirmed by its reactivity with sera from cattle infected with FMDV and with a monoclonal antibody specific for FMDV-3ABC (MAb3H7) in Western blot assays. No reactivity was observed with sera from uninfected vaccinated animals. The performance of 3A-EGFP as an antigen in an indirect enzyme-linked immunosorbent assay (ELISA) was assessed and compared with that of a previously developed and validated capture ELISA that uses a 3ABC recombinant antigen (3ABC ELISA) and has been widely applied for serological surveys in Argentina. Parallel analysis of strongly and weakly positive reference sera from infected animals and 329 serum samples from uninfected vaccinated cattle showed that the 3A-EGFP antigen unequivocally identifies sera from FMDV-infected cattle with similar performance to its 3ABC counterpart. The 3A-EGFP ELISA is simpler and faster to perform than the 3ABC ELISA, since it does not require a capture step with a specific antibody. Moreover, the expression and storage of the recombinant 3A-EGFP is simplified by the absence of residual autoproteolytic activity associated to the 3C sequence. We conclude that the 3A-EGFP ELISA constitutes a promising screening method in serosurveys to determine whether or not animals are infected with FMDV.

Relevance of the N-terminal and major hydrophobic domains of non-structural protein 3A in the replicative process of a DNA-launched foot-and-mouth disease virus replicon

A foot-and-mouth disease virus (FMDV) DNA-launched reporter replicon containing a luciferase gene was used to assess the impact of non-structural (NS) protein 3A on viral replication. Independent deletions within the N-terminal region (amino acid [aa] residues 6 to 24) and the central hydrophobic region (HR, aa 59 to 76) of FMDV NS protein 3A were engineered, and luciferase activity in lysates of control and mutated replicon-transfected cells was measured. Triple alanine replacements of the N-terminal triplet Arg 18- His 19 -Glu 20 and a single alanine substitution of the highly charged Glu 20 residue both resulted in a 70-80% reduction in luciferase activity when compared with wild-type controls. Alanine substitution of the 17 aa present in the central HR, on the other hand, resulted in complete inhibition of luciferase activity and in the accumulation of the mutated 3A within the cell nucleus according to immunofluorescence analysis. Our results suggest that both the aa sequence around the putatively exposed hydrophilic E20 residue at the N-terminus of the protein and the hydrophobic tract located between aa 59 and 76 are of major relevance for maintaining the functionality of the 3A protein and preventing its mislocalization into the cell nucleus.