Discovery of frameshifting in Alphavirus 6K resolves a 20-year enigma.

BACKGROUND: The genus Alphavirus includes several potentially lethal human viruses. Additionally, species such as Sindbis virus and Semliki Forest virus are important vectors for gene therapy, vaccination and cancer research, and important models for virion assembly and structural analyses. The genome encodes nine known proteins, including the small '6K' protein. 6K appears to be involved in envelope protein processing, membrane permeabilization, virion assembly and virus budding. In protein gels, 6K migrates as a doublet--a result that, to date, has been attributed to differing degrees of acylation. Nonetheless, despite many years of research, its role is still relatively poorly understood. RESULTS: We report that ribosomal -1 frameshifting, with an estimated efficiency of approximately 10-18%, occurs at a conserved UUUUUUA motif within the sequence encoding 6K, resulting in the synthesis of an additional protein, termed TF (TransFrame protein; approximately 8 kDa), in which the C-terminal amino acids are encoded by the -1 frame. The presence of TF in the Semliki Forest virion was confirmed by mass spectrometry. The expression patterns of TF and 6K were studied by pulse-chase labelling, immunoprecipitation and immunofluorescence, using both wild-type virus and a TF knockout mutant. We show that it is predominantly TF that is incorporated into the virion, not 6K as previously believed. Investigation of the 3' stimulatory signals responsible for efficient frameshifting at the UUUUUUA motif revealed a remarkable diversity of signals between different alphavirus species. CONCLUSION: Our results provide a surprising new explanation for the 6K doublet, demand a fundamental reinterpretation of existing data on the alphavirus 6K protein, and open the way for future progress in the further characterization of the 6K and TF proteins. The results have implications for alphavirus biology, virion structure, viroporins, ribosomal frameshifting, and bioinformatic identification of novel frameshift-expressed genes, both in viruses and in cellular organisms

Genetic vaccination against acute viral disease

This thesis describes the development of recombinant vaccines based on the Semliki Forest virus (SFV) expression system. Immunisation of mice with recombinant virus particles, a layered DNA/RNA plasmid vector, and recombinant self-replicating RNA were carried out and the protective effect of these recombinant vaccines against viral challenge were examined. The construction of a full-length infectious clone formed the basis for the SFV expression system which has previously been described. In the expression system, genes coding for the SFV structural proteins are replaced with a multiple cloning site where the gene coding for a foreign antigen can be inserted. Transfection of cells with RNA transcribed in vitro from such vectors results in transient high level production of the antigen followed by death of the cells as a result of the induction of apoptosis. Recombinant virus particles can be made by co-transfecting cells with expression vector RNA and a defective helper RNA coding for the SFV structural proteins. Recombinant SFV (rSFV) particles lack the genes for the structural proteins and thus undergo only a single round of non-productive infection after entry into a cell. Mice immunised with rSFV encoding influenza A virus (FLU) nucleoprotein (NP) or E.coli LacZ protein were shown to develop antigen-specific IgG and CTL responses that persisted for over one year. Humoral and cellular immune responses could be induced in mice following vaccination by peripheral and mucosal routes. Examination of IgG1/IgG2a antibody isotypes indicated that predominantly a T-helper type 1 response was induced. Importantly, this study showed that development of immune responses against the vector itself did not significantly inhibit responses following booster immunisations with rSFV. This is important for recombinant vaccine design as the use of many recombinant virus vaccines is hampered due to pre-existing immunity to the vectors. Expression of envelope proteins, prME, and a nonstructural protein, NS1, of the tick-borne flavivirus, louping ill virus (LIV). from rSFV in cell culture was characterised. both proteins were shown to be correctly processed and secreted from transfected cells. Humoral and T-cell proliferative responses were induced in mice immunised with rSFV particles encoding LIV antigens. Mice vaccinated with rSFV` particles coding for FLU or LIV antigens were protected from challenge with FLU or from challenge with two strains of LIV. the prototype strain LI/31, and LI/l, a naturally occurring antibody escape variant. A layered DNA/RNA vector based on the SFV replicon (pBK-SFV) was developed. A cytomegalovirus (CMV) promoter drives production, not of the antigen-encoding gene, but of the recombinant SFV RNA. In comparison to a conventional DNA vector coding for the same antigen, vaccination of mice with pBK-SFV DNA was shown to elicit stronger humoral and cellular immune responses. Mice vaccinated with pBK-SFV` encoding FLU antigens were shown to be protected against influenza A virus challenge. In order to compare the protective efficacy of rSFV vaccines, a triple challenge model based on three strains of LIV with graded virulence was established. Results from challenge experiments showed that rSFV particles induced better protective immunity than plasmid vaccines (conventional or SFV-based) or the commercially available LIV inactivated vaccine. Antigen-specific humoral and cellular immune responses were also elicited in mice following immunisation with naked rSFV RNA encoding FLU, LIV, or respiratory syncytial virus (RSV) antigens. Mice vaccinated with rSFV RNA were protected from challenge with FLU. LIV or RSV. Studies showed that bone marrow derived dendritic cells (BMDDC) from mice are unable to be productively infected by rSFV particles encoding GFP (green fluorescent protein) as de novo synthesis of GFP could not be detected in these cells. However, RT-PCR analysis of rSFV` infected BMDDC showed that negative strand RNA intermediates of rSFV infection could be detected. indicating that rSFV RNA replication had been initiated in these cells. rnRNA coding for interferon-[alpha]-induced antiviral proteins and the cytokines TNF-[alpha] and IL-12 rnRNA were also upregulated in these cells. This suggests that cytokine signals required for DC maturation and activation were turned on as a result of the initiation of viral RNA replication. Uptake of antigen (GFP) by BMDDC from rSFV infected syngeneic B 16 cells was shown by confocal microscopy. Thus, induction of apoptosis in rSFV transfected cells could facilitate antigen transfer to professional antigen presenting cells for subsequent cross-priming, and might thereby explain the efficient immune responses induced following immunisation with rSFV vaccines

MyD88 Expression Is Required for Efficient Cross-Presentation of Viral Antigens from Infected Cells

While virus-infected dendritic cells (DCs) in certain instances have the capacity to activate naïve T cells by direct priming, cross-priming by DCs via the uptake of antigens from infected cells has lately been recognized as another important pathway for the induction of antiviral immunity. During cross-priming, danger and stranger signals play important roles in modulating immune responses. Analogous to what has been shown for other microbial infections, virally infected cells may contain several pathogen-associated molecular patterns that are recognized by Toll-like receptors (TLRs). We analyzed whether the efficient presentation of antigens derived from infected cells requires the usage of MyD88, which is a common adaptor molecule used by all TLRs. For this study, we used murine DCs that were wild type or deficient in MyD88 expression and fibroblasts that were infected with an alphavirus replicon to answer this question. Our results show that when DCs are directly infected, they are able to activate antigen-specific CD8(+) T cells in a MyD88-independent manner. In contrast, a strict requirement of MyD88 for cross-priming was observed when virally infected cells were used as a source of antigen in vitro and in vivo. This indicates that the effects of innate immunity stimulation via the MyD88 pathway control the efficiency of cross-presentation, but not direct presentation or DC maturation, and have important implications in the development of cytotoxic T lymphocyte responses against alphaviral replicon infections

The pyrrolo-1,5-benzoxazepine,PBOX-6, inhibits the growth of breast cancer cells in vitro independent of estrogen receptor status and inhibits breast tumor growth in vivo

Members of a novel series of pyrrolo-1,5-benzoxazepine (PBOX) compounds have been shown to induce apoptosis in a number of human leukemia cell lines of different haematological lineage, suggesting their potential as anti-cancer agents. In this study, we sought to determine if PBOX-6, a well characterised member of the PBOX series of compounds, is also an effective inhibitor of breast cancer growth. Two estrogen receptor (ER)-positive (MCF-7 and T-47-D) and two ER-negative (MDA-MB-231 and SK-BR-3) cell lines were examined. The 3,4,5-dimethylthiazol-2-yl-2,5-diphenyl-tetrazolium bromide (MTT) assay was used to determine reduction in cell viability. PBOX-6 reduced the cell viability of all four cell lines tested, regardless of ER status, with IC(50) values ranging from 1.0 to 2.3 microM. PBOX-6 was most effective in the SK-BR-3 cells, which express high endogenous levels of the HER-2 oncogene. Overexpression of the HER-2 oncogene has been associated with aggressive disease and resistance to chemotherapy. The mechanism of PBOX-6-induced cell death was due to apoptosis, as indicated by the increased proportion of cells in the pre-G1 peak and poly(ADP-ribose) polymerase (PARP) cleavage. Moreover, intratumoural administration of PBOX-6 (7.5 mg/kg) significantly inhibited tumour growth in vivo in a mouse mammary carcinoma model (p=0.04, n=5, Student's t-test). Thus, PBOX-6 could be a promising anti-cancer agent for both hormone-dependent and -independent breast cancers.</p

Monocyte-derived dendritic cells early exposed to Mycobacterium tuberculosis induce an enhanced T helper 17 response and transfer mycobacterial antigens.

Tuberculosis (TB) is a complex disease, and the success of the bacterium depends on its ability to evade the immune response. Previously, we determined that Mycobacterium tuberculosis (Mtb) impairs the function of dendritic cells (DC), promoting the generation of cells that are poor stimulators of mycobacterial antigen-specific CD4T cells, which are required to control this persistent infection. In this study, we aimed to determine the mechanisms by which monocyte-derived DCs differentiated in the presence of Mtb (MtbDC) may impact on the proliferation of specific anti-mycobacterial T cells. We found that the presence of Mtb during monocyte-derived DC differentiation favours T helper (Th) 2 and Th17 polarization, in detriment of a Th1 response, compared to DC mature with Mtb. The bias on T cell polarization was associated to the profile of C-type lectin receptors expression found in MtbDC (DC-SIGNlow/MRlow/Dectin-1high). Alternatively, MtbDC release Mtb antigens (Ag) that can be taken up and presented by bystander DC, promoting the proliferation of CD4T cells, but to a lesser extent than direct presentation by Mtb-matured DC. In summary, we have further characterized the generation of MtbDC as an effective evasion strategy driven by the pathogen, leading to the inhibition of Ag-presentation and bias of T cell polarization towards Th2 and Th17 profiles, features which partially explain the persistence of Mtb in the host.Fil: Balboa, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Kviatcovsky, Denise. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Schierloh, Luis Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: García, Marina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: de la Barrera, Silvia Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Sasiain, María del Carmen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentin