Papaya mosaic potexvirus (PapMV) is a member of the potexvirus family. The virion is a flexible rod 500 nm in length and 15 nm in diameter composed of 1400 subunits of viral coat protein (CP) [1] assembled around a plus-strand genomic RNA of 6656 nucleotides (nt) [2]. In vitro reconstitution of PapMV nucleocapsid-like particles (NLPs) was previously studied using CP prepared from the acetic acid degradation of purified virus [3]. Using this method, PapMV CP can be isolated from the genomic RNA and used for in vitro assembly assays [3]. Extracted CP has been found as a variety of aggregates ranging from 14S to 25S [4] that include a disk-like structure (14 S) made of 18-20 subunits. These disks are helical structures (two turns of the helix) similar in architecture to the native virus particle [3,5]. The addition of RNA to the isolated disks triggers the assembly of long rod-shaped particles that are very similar to the WT virus [3]. The in vitro assembly of PapMV was shown to be specific and triggered by 47 nucleotides of the 5¢ noncoding region of the virus [6]. This region is free of any discernable secondary structure, an important feature for initiation of the assembly process [6]. In vitro assembly is specific when performed at a pH of 8.0-8.5 in a buffer of low ionic strength [4] and elongation proceeds in the 5¢fi3¢ direction [7]. One atom of Ca 2+ is attached to each subunit, which is probably important for the structure of the protein [8]. Alignment of PapMV CP with the CP of other potexviruses reveals that these proteins share 35% identity [9]. The N terminus of the proteins is the most divergent and their length is also variable. It can reach more than 50 amino acids in the case of potato aucuba mosaic virus. Phosphorylation of the N terminus of potato virus X CP by host Papaya mosaic potexvirus (PapMV) coat protein (CP) was expressed (CPDN5) in Escherichia coli and showed to self assemble into nucleocapsid like particles (NLPs). Twenty per cent of the purified protein was found as NLPs of 50 nm in length and 80% was found as a multimer of 450 kDa (20 subunits) arranged in a disk. Two mutants in the RNA binding domain of the PapMV CP, K97A and E128A showed interesting properties. The proteins of both mutants could be easily purified and CD spectra of these proteins showed secondary and tertiary structures similar to the WT protein. The mutant K97A was unable to self assemble and bind RNA. On the contrary, the mutant E128A showed an improved affinity for RNA and self assembled more efficiently in NLPs. E128A NLPs were longer (150 nm) than the recombinant CPDN5 and 100% percent of the protein was found as NLPs in bacteria. E128A NLPs were more resistant to digestion by trypsin than the CPDN5 but were more sensitive to denaturation by heat. We discuss the possible role of K97 and E128 in the assembly of PapMV.Abbreviations CP, coat protein; NLP, nucleocapsid like particles; nt, nucleotide; PapMV, Papaya mosaic potexvirus.
The development of versatile vaccine platforms is a priority that is recognized by health authorities worldwide; such platforms should induce both arms of the immune system, the humoral and cytotoxic-Tlymphocyte responses. In this study, we have established that a vaccine platform based on the coat protein of papaya mosaic virus (PapMV CP), previously shown to induce a humoral response, can induce major histocompatibility complex (MHC) class I cross-presentation of HLA-A*0201 epitopes from gp100, a melanoma antigen, and from influenza virus M1 matrix protein. PapMV proteins were able to assemble into stable virus-like particles (VLPs) in a crystalline and repetitive structure. When we pulsed HLA-A*0201؉ antigenpresenting cells (APCs) with the recombinant PapMV FLU or gp100, we noted that antigen-specific CD8 ؉ T cells were highly reactive to these APCs, demonstrating that the epitope from the VLPs were processed and loaded on the MHC class I complex. APCs were preincubated with two different proteasome inhibitors, which did not affect the efficiency of peptide presentation on MHC class I. Classical presentation from an endogenous antigen was abolished in the same conditions. Clearly, antigen presentation mediated by the PapMV system was proteasome independent. Finally, PapMV-pulsed APCs had the capacity to expand highly avid antigenspecific T cells against the influenza virus M1 HLA-A*0201 epitope when cocultured with autologous peripheral blood mononuclear cells. This study demonstrates the potential of PapMV for MHC class I crosspresentation and for the expansion of human antigen-specific T cells. It makes VLPs from PapMV CP a very attractive platform to trigger cellular responses for vaccine development against chronic infectious diseases and cancers.
Background The aim of this study was to establish the prevalence of lipodystrophy and its association to cumulative exposure to antiretroviral drugs. Method We conducted a cross sectional study in all HIV- infected patients attending the HIV clinic in the Centre hospitalier universitaire de Montréal (CHUM) with DEXA scan. Lipodystrophy was defined as a trunk/limb fat ratio ≥ 1.5. Association between cumulative exposure to antiretroviral (measured in years of use) with trunk/limb fat ratio (coded as a continuous variable) was assessed using univariate and multivariate linear regression for each antiretroviral drug with at least 40 exposed patients. Results One hundred sixty-six patients were included. Seventy-five percent were male, median age was 56 years, 67% were Caucasian. Overall, prevalence of lipodystrophy was 47%, with a mean trunk/limb fat ratio of 1.87, SD = 1.03, min = 0.6 and max = 5.87. Each 10-year increase in age and HIV infection duration was associated with an average increase of 0.24 and 0.34 for the trunk/limb fat ratio respectively. (p = 0.003, p = 0.002, respectively) Patients classified as lipodystrophic were more likely to be diabetic (50 vs. 28%, p = 0.07) and to have dyslipidemia (47 vs. 19%, p = 0.01). According to viral load at DEXA test, each one log increase was associated with less probability (0.7) of lipodystrophy. (p = 0.01) Among ARV drugs tested, there was an association between years of use of d4T, ritonavir and raltegravir and higher trunk/limb fat ratio (indicating more lipodystrophy) (p < 0.05). Conclusion Lipodystrophy is very common in HIV infected patients and is correlated with duration of some new antiretroviral drugs.
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