Alternative Open Reading Frame Research Articles

PB1-F2 Amino Acids Regulate Influenza A Viral Polymerase Activity

In 2001, the 11th influenza A viral protein PB1-F2 was detected and found to be encoded by an alternative open reading frame in the PB1 polymerase gene. PB1-F2 has several unique functions, including roles in promoting apoptosis, increasing inflammation, and regulating viral polymerase activity. This study focused on a single PB1-F2 function: regulation of polymerase activity. We constructed a minigenome system to determine the influence of PB1-F2 amino acid (aa) mutations on polymerase activity. We examined four types of aa mutations: three species-specific aa mutations and one mutation that alters pathogenicity in mice. We discovered that an arginine (R) residue at aa position 29 is highly conserved in avian-derived virus strains. Introducing this mutation into mammalian strain A/WSN/33 (H1N1) led to a marked increase in polymerase activity in mammalian cells.These findings suggest that as PB1-F2 in H5N1 viruses regulates viral polymerase activity, it could be targeted for control of avian influenza infection and drug discovery.

Molecular Profile of Human Serine Palmitoyltransferase-1 Proximate of Chromosome 9 Disease Susceptibility Gene Cluster in Inflammatory Cancer Cell Lines

Background: Over 1100 genes have been annotated for human chromosome 9, including disease genes implicated in inflammation, atherosclerosis, cancer and neurodegeneration. The serine palmitoyltransferase-1, SPTLC1, gene is at the 9q22.2 cytogenetic band, a high G+C content region with common genetic alterations sufficient to modify cellular behavior. The sequence is highly conserved among diverse species from bacteria to humans, including a recently discovered 126 nucleotide alternate open reading frame, AltORF. The protein encoded by the reading frames has domains of biological interest and considerable overlapping molecular functions associated with cellular behavior and cancer progression. Methods: Here we examined molecular features of SPTLC1 in a group of inflammation associated cancer cell lines SKN-SH, MDA-PCa, Glioma LN18, PC3 and 647V. Subcellular localization of SPTLC1 was assessed by immunofluorescence microscopy and recombinant green fluorescent protein expression. In addition, PCR, DNA sequencing and bioinformatics analysis were used for molecular profiling of the SPTLC1 genomic and reverse transcribed cDNA fragments. Results: SPTLC1 is detected in all cell lines examined, with intense peri-nuclear staining, consistent with localization in the cytoplasm. Genomic DNA sample, but not the cD NA of SKN cells could be amplified with an AltORF primer set. The PC3 and MDA-PCa cancer cell lines which are both of prostate origin, show differences in SPTLC1 PCR amplification. Similar levels of SPTLC1 AltORF transcripts were detected by quantitative RT-PCR in all cell lines, except the PC3 cell line with low transcript level whose cDNA did not generate nucleotide base sequence information. Conclusions: This is the first reported transcriptional expression of the SPTLC1 AltORF for the inflammation associated human cancer cell lines. Interestingly, it is proximate of oncogenic cancer susceptibility genes and distal of tumor suppressor genes, the high content of short nucleotide repeats in the SPTLC1 AltORF sequence suggesting the region may be genetically unstable. This nominal functional genomics report on the human SPTLC1 AltORF will contribute to compiling a more detailed SPTLC1 gene ontology and is expected to help shed more insight into unique molecular attributes of SPTLC1 in the context of cancer cell behavior, malignant progression and the design of treatment for inflammation associated cancers.

Purifying selection in porcine reproductive and respiratory syndrome virus ORF5a protein influences variation in envelope glycoprotein 5 glycosylation

Porcine reproductive and respiratory syndrome virus ORF5a protein is encoded in an alternate open reading frame upstream of the major envelope glycoprotein (GP5) in subgenomic mRNA5. Bioinformatic analysis of 3466 type 2 PRRSV sequences showed that the two proteins have co-evolved through a fine balance of purifying codon usage to maintain a conserved RQ-rich motif in ORF5a protein, while eliciting a variable N-linked glycosylation motif in the alternative GP5 reading frame. Conservation of the ORF5a protein RQ-motif also explains an anomalous uracil desert in GP5 hypervariable glycosylation region. The N-terminus of the mature GP5 protein was confirmed to start with amino acid 32, the hypervariable region of the ectodomain. Since GP5 glycosylation variability is assumed to result from immunological selection against neutralizing antibodies, these findings show that an alternative possibility unrelated to immunological selection not only exists, but provides a foundation for investigating previously unsuspected aspects of PRRSV biology. Understanding functional consequences of subtle nucleotide sequence modifications in the region responsible for critical function in ORF5a protein and GP5 glycosylation is essential for rational design of new vaccines against PRRS.

Direct Detection of Alternative Open Reading Frames Translation Products in Human Significantly Expands the Proteome

A fully mature mRNA is usually associated to a reference open reading frame encoding a single protein. Yet, mature mRNAs contain unconventional alternative open reading frames (AltORFs) located in untranslated regions (UTRs) or overlapping the reference ORFs (RefORFs) in non-canonical +2 and +3 reading frames. Although recent ribosome profiling and footprinting approaches have suggested the significant use of unconventional translation initiation sites in mammals, direct evidence of large-scale alternative protein expression at the proteome level is still lacking. To determine the contribution of alternative proteins to the human proteome, we generated a database of predicted human AltORFs revealing a new proteome mainly composed of small proteins with a median length of 57 amino acids, compared to 344 amino acids for the reference proteome. We experimentally detected a total of 1,259 alternative proteins by mass spectrometry analyses of human cell lines, tissues and fluids. In plasma and serum, alternative proteins represent up to 55% of the proteome and may be a potential unsuspected new source for biomarkers. We observed constitutive co-expression of RefORFs and AltORFs from endogenous genes and from transfected cDNAs, including tumor suppressor p53, and provide evidence that out-of-frame clones representing AltORFs are mistakenly rejected as false positive in cDNAs screening assays. Functional importance of alternative proteins is strongly supported by significant evolutionary conservation in vertebrates, invertebrates, and yeast. Our results imply that coding of multiple proteins in a single gene by the use of AltORFs may be a common feature in eukaryotes, and confirm that translation of unconventional ORFs generates an as yet unexplored proteome.

Evasion of Antiviral Innate Immunity by Theiler's Virus L* Protein through Direct Inhibition of RNase L

Theiler's virus is a neurotropic picornavirus responsible for chronic infections of the central nervous system. The establishment of a persistent infection and the subsequent demyelinating disease triggered by the virus depend on the expression of L*, a viral accessory protein encoded by an alternative open reading frame of the virus. We discovered that L* potently inhibits the interferon-inducible OAS/RNase L pathway. The antagonism of RNase L by L* was particularly prominent in macrophages where baseline oligoadenylate synthetase (OAS) and RNase L expression levels are elevated, but was detectable in fibroblasts after IFN pretreatment. L* mutations significantly affected Theiler's virus replication in primary macrophages derived from wild-type but not from RNase L-deficient mice. L* counteracted the OAS/RNase L pathway through direct interaction with the ankyrin domain of RNase L, resulting in the inhibition of this enzyme. Interestingly, RNase L inhibition was species-specific as Theiler's virus L* protein blocked murine RNase L but not human RNase L or RNase L of other mammals or birds. Direct RNase L inhibition by L* and species specificity were confirmed in an in vitro assay performed with purified proteins. These results demonstrate a novel viral mechanism to elude the antiviral OAS/RNase L pathway. By targeting the effector enzyme of this antiviral pathway, L* potently inhibits RNase L, underscoring the importance of this enzyme in innate immunity against Theiler's virus.

Translation regulation gets its ‘omics’ moment

The fate of cellular RNA is largely determined by complex networks of protein-RNA interactions through ribonucleoprotein (RNP) complexes. Despite their relatively short half-life, transcripts associate with many different proteins that process, modify, translate, and degrade the RNA. Following biogenesis some mRNPs are immediately directed to translation and produce proteins, but many are diverted and regulated by processes including miRNA-mediated mechanisms, transport and localization, as well as turnover. Because of this complex interplay estimates of steady-state expression by methods such as RNAseq alone cannot capture critical aspects of cellular fate, environmental response, tumorigenesis, or gene expression regulation. More selective and integrative tools are needed to measure protein-RNA complexes and the regulatory processes involved. One focus area is measurements of the transcriptome associated with ribosomes and translation. These so-called polysome or ribosome profiling techniques can evaluate translation efficiency as well as the interplay between translation initiation, elongation, and termination-subject areas not well understood at a systems biology level. Ribosome profiling is a highly promising technique that provides mRNA positional information of ribosome occupancy, potentially bridging the gap between gene expression (i.e., RNAseq and microarray analysis) and protein quantification (i.e., mass spectrometry). In combination with methods such as RNA immunoprecipitation, miRNA profiling, or proteomics, we obtain a fresh view of global post-transcriptional and translational gene regulation. In addition, these techniques also provide new insight into new regulatory elements, such as alternative open reading frames, and translation regulation under different conditions.

Both core and F proteins of hepatitis C virus could enhance cell proliferation in transgenic mice

The role of the protein encoded by the alternative open reading frame (ARF/F/core+1) of the Hepatitis C virus (HCV) genome in viral pathogenesis remains unknown. The different forms of ARF/F/core+1 protein were labile in cultured cells, a myc-tag fused at the N-terminus of the F protein made it more stable. To determine the role of core and F proteins in HCV pathogenesis, transgenic mice with either protein expression under the control of Albumin promoter were generated. Expression of core protein and F protein with myc tag (myc-F) could be detected by Western blotting analysis in the livers of these mice. The ratio of liver to body weight is increased for both core and myc-F transgenic mice compared to that of wild type mice. Indeed, the proliferating cell nuclear antigen protein, a proliferation marker, was up-regulated in the transgenic mice with core or myc-F protein. Further analyses by microarray and Western blotting suggested that β-catenin signaling pathway was activated by either core or myc-F protein in the transgenic mice.These transgenic mice were further treated with either Diethynitrosamine (a tumor initiator) or Phenobarbital (a tumor promoter). Phenobarbital but not Diethynitrosamine treatment could increase the liver/body weight ratio of these mice. However, no tumor formation was observed in these mice. In conclusion, HCV core and myc-F proteins could induce hepatocyte proliferation in the transgenic mice possibly through β-catenin signaling pathway.

West Nile alternative open reading frame (N-NS4B/WARF4) is produced in infected West Nile Virus (WNV) cells and induces humoral response in WNV infected individuals

BackgroundWest Nile Virus (WNV) is a flavivirus that requires an efficient humoral and cellular host response for the control of neuroinvasive infection. We previously reported the existence of six alternative open reading frame proteins in WNV genome, one of which entitled WARF4 is exclusively restricted to the lineage I of the virus. WARF4 is able to elicit antibodies in WNV infected horses; however, there was no direct experimental proof of the existence of this novel protein. The purpose of this study was to demonstrate the in vitro production of WARF4 protein following WNV infection of cultured VERO cells and its immunity in WNV infected individuals.ResultsWe produced a monoclonal antibody against WARF4 protein (MAb 3A12) which detected the novel protein in WNV lineage I-infected, cultured VERO cells while it did not react with WNV lineage II infected cells. MAb 3A12 specificity to WARF4 protein was confirmed by its reactivity to only one peptide among four analyzed that cover the full WARF4 amino acids sequence. In addition, WARF4 protein was expressed in the late phase of WNV lineage I infection. Western blotting and bioinformatics analyses strongly suggest that the protein could be translated by programmed −1 ribosomal frameshifting process. Since WARF4 is embedded in the NS4B gene, we rename this novel protein N-NS4B/WARF4. Furthermore, serological analysis shows that N-NS4B/WARF4 is able to elicit antibodies in WNV infected individuals.ConclusionsN-NS4B/WARF4 is the second Alternative Reading Frame (ARF) protein that has been demonstrated to be produced following WNV infection and might represent a novel tool for a better characterization of immune response in WNV infected individuals. Further serological as well as functional studies are required to characterize the function of the N-NS4B/WARF4 protein. Since the virus might actually make an extensive use of ARFs, it appears important to investigate the novel six ARF putative proteins of WNV.

Properties of the Adeno-Associated Virus Assembly-Activating Protein

Adeno-associated virus (AAV) capsid assembly requires expression of the assembly-activating protein (AAP) together with capsid proteins VP1, VP2, and VP3. AAP is encoded by an alternative open reading frame of the cap gene. Sequence analysis and site-directed mutagenesis revealed that AAP contains two hydrophobic domains in the N-terminal part of the molecule that are essential for its assembly-promoting activity. Mutation of these sequences reduced the interaction of AAP with the capsid proteins. Deletions and a point mutation in the capsid protein C terminus also abolished capsid assembly and strongly reduced the interaction with AAP. Interpretation of these observations on a structural basis suggests an interaction of AAP with the VP C terminus, which forms the capsid protein interface at the 2-fold symmetry axis. This interpretation is supported by a decrease in the interaction of monoclonal antibody B1 with VP3 under nondenaturing conditions in the presence of AAP, indicative of steric hindrance of B1 binding to its C-terminal epitope by AAP. In addition, AAP forms high-molecular-weight oligomers and changes the conformation of nonassembled VP molecules as detected by conformation-sensitive monoclonal antibodies A20 and C37. Combined, these observations suggest a possible scaffolding activity of AAP in the AAV capsid assembly reaction.

Predicting Statistical Properties of Open Reading Frames in Bacterial Genomes

An analytical model based on the statistical properties of Open Reading Frames (ORFs) of eubacterial genomes such as codon composition and sequence length of all reading frames was developed. This new model predicts the average length, maximum length as well as the length distribution of the ORFs of 70 species with GC contents varying between 21% and 74%. Furthermore, the number of annotated genes is predicted with high accordance. However, the ORF length distribution in the five alternative reading frames shows interesting deviations from the predicted distribution. In particular, long ORFs appear more often than expected statistically. The unexpected depletion of stop codons in these alternative open reading frames cannot completely be explained by a biased codon usage in the +1 frame. While it is unknown if the stop codon depletion has a biological function, it could be due to a protein coding capacity of alternative ORFs exerting a selection pressure which prevents the fixation of stop codon mutations. The comparison of the analytical model with bacterial genomes, therefore, leads to a hypothesis suggesting novel gene candidates which can now be investigated in subsequent wet lab experiments.

Influenza Virus Protein PB1-F2 Inhibits the Induction of Type I Interferon by Binding to MAVS and Decreasing Mitochondrial Membrane Potential

PB1-F2 is a small, 87- to 90-amino-acid-long protein encoded by the +1 alternate open reading frame of the PB1 gene of most influenza A virus strains. It has been shown to contribute to viral pathogenicity in a host- and strain-dependent manner, and we have previously discovered that a serine at position 66 (66S) in the PB1-F2 protein increases virulence of the 1918 and H5N1 pandemic viruses. Recently, we have shown that PB1-F2 inhibits the induction of type I interferon (IFN) at the level of the MAVS adaptor protein. However, the molecular mechanism for the IFN antagonist function of PB1-F2 has remained unclear. In the present study, we demonstrated that the C-terminal portion of the PB1-F2 protein binds to MAVS in a region that contains the transmembrane domain. Strikingly, PB1-F2 66S was observed to bind to MAVS more efficiently than PB1-F2 66N. We also tested the effect of PB1-F2 on the IFN antagonist functions of the polymerase proteins PB1, PB2, and PA and observed enhanced IFN inhibition by the PB1 and PB2 proteins in combination with PB1-F2 but not by the PA protein. Using a flow cytometry-based assay, we demonstrate that the PB1-F2 protein inhibits MAVS-mediated IFN synthesis by decreasing the mitochondrial membrane potential (MMP). Interestingly, PB1-F2 66S affected the MMP more efficiently than wild-type PB1-F2. In summary, the results of our study identify the molecular mechanism by which the influenza virus PB1-F2 N66S protein increases virulence.

HAltORF: a database of predicted out-of-frame alternative open reading frames in human

Human alternative open reading frames (HAltORF) is a publicly available and searchable online database referencing putative products of out-of-frame alternative translation initiation (ATI) in human mRNAs. Out-of-frame ATI is a process by which a single mRNA encodes independent proteins, when distinct initiation codons located in different reading frames are recognized by a ribosome to initiate translation. This mechanism is largely used in viruses to increase the coding potential of small viral genomes. There is increasing evidence that out-of-frame ATI is also used in eukaryotes, including human, and may contribute to the diversity of the human proteome. HAltORF is the first web-based searchable database that allows thorough investigation in the human transcriptome of out-of-frame alternative open reading frames with a start codon located in a strong Kozak context, and are thus the more likely to be expressed. It is also the first large scale study on the human transcriptome to successfully predict the expression of out-of-frame ATI protein products that were previously discovered experimentally. HAltORF will be a useful tool for the identification of human genes with multiple coding sequences, and will help to better define and understand the complexity of the human proteome.Database URL: http://haltorf.roucoulab.com/.

Generation of protective memory CD8 T cells specific for a retroviral cryptic epitope (105.25)

Abstract Our lab has shown that cytotoxic CD8 T cells specific for immunodominant cryptic retroviral epitopes are capable of protecting against retrovirus-induced disease. LP-BM5 murine retrovirus causes a profound and progressive immunodeficiency in strains of mice that are unable to produce a protective CD8-T cell response. In resistant BALB/c mice, our lab has identified a Kd-restricted LP-BM5 immunodominant epitope, SYNTGRFPPL, within a previously unrecognized alternative open reading frame of the retroviral gag gene. Upon adoptive transfer, CD8 T cells which recognize SYNTGRFPPL mediate an acute, effector-phase response to LP-BM5, but are inefficient at generating long-term immunological memory to LP-BM5-induced disease. Rapamycin, IL-15 and high-dose IL-2 have been shown to increase the generation and survival of memory CD8 T cells. Using the CD8 T-cell adoptive transfer system employed in our lab to demonstrate acute protection to LP-BM5-induced disease, the aim was to engineer a protective, SYNTGRFPPL-specific memory response by including rapamycin, IL-15, or high-dose IL-2 at the in vitro CD8 T-cell expansion phase of adoptive cellular immunotherapy. The optimization of both acute and long-term memory responses via adoptive transfer of protective CD8 T cells specific for cryptic retroviral epitopes has implications for immunotherapeutic treatment of retrovirus-induced disease.

Analysis of PSPHL as a Candidate Gene Influencing the Racial Disparity in Endometrial Cancer

Endometrial cancer is the most commonly diagnosed gynecologic malignancy in the United States. A well recognized disparity by race in both incidence and survival outcome exists for this cancer. Specifically Caucasians are about two times more likely to develop endometrial cancer than are African-Americans. However, African-American women are more likely to die from this disease than are Caucasians. The basis for this disparity remains unknown. Previous studies have identified differences in the types and frequencies of gene mutations among endometrial cancers from Caucasians and African-Americans suggesting that the tumors from these two groups might have differing underlying genetic defects. We performed a gene expression microarray study in an effort to identify differentially expressed transcripts between African-American and Caucasian women’s endometrial cancers. Our gene expression screen identified a list of potential biomarkers that are differentially expressed between these two groups of cancers. Of these we identified a poorly characterized transcript with a region of homology to phospho serine phosphatase (PSPH) and designated phospho serine phosphatase like (PSPHL) as the most differentially over-expressed gene in cancers from African-Americans. We further clarified the nature of expressed transcripts. Northern blot analysis confirmed the message was limited to a transcript of under 1 kB. Sequence analysis of transcripts confirmed two alternate open reading frame (ORF) isoforms due to alternative splicing events. Splice specific primer sets confirmed both isoforms were differentially expressed in tissues from Caucasians and African-Americans. We further examined the expression in other tissues from women to include normal endometrium, normal and malignant ovary. In all cases PSPHL expression was more often present in tissues from African-Americans than Caucasians. Our data confirm the African-American based expression of the PSPHL transcript in endometrial cancer and also identify its expression in other tissues from African-Americans including ovary and ovarian cancer. PSPHL represents a candidate gene that might influence the observed racial disparity in endometrial and other cancers.

Norovirus Regulation of the Innate Immune Response and Apoptosis Occurs via the Product of the Alternative Open Reading Frame 4

Small RNA viruses have evolved many mechanisms to increase the capacity of their short genomes. Here we describe the identification and characterization of a novel open reading frame (ORF4) encoded by the murine norovirus (MNV) subgenomic RNA, in an alternative reading frame overlapping the VP1 coding region. ORF4 is translated during virus infection and the resultant protein localizes predominantly to the mitochondria. Using reverse genetics we demonstrated that expression of ORF4 is not required for virus replication in tissue culture but its loss results in a fitness cost since viruses lacking the ability to express ORF4 restore expression upon repeated passage in tissue culture. Functional analysis indicated that the protein produced from ORF4 antagonizes the innate immune response to infection by delaying the upregulation of a number of cellular genes activated by the innate pathway, including IFN-Beta. Apoptosis in the RAW264.7 macrophage cell line was also increased during virus infection in the absence of ORF4 expression. In vivo analysis of the WT and mutant virus lacking the ability to express ORF4 demonstrated an important role for ORF4 expression in infection and virulence. STAT1-/- mice infected with a virus lacking the ability to express ORF4 showed a delay in the onset of clinical signs when compared to mice infected with WT virus. Quantitative PCR and histopathological analysis of samples from these infected mice demonstrated that infection with a virus not expressing ORF4 results in a delayed infection in this system. In light of these findings we propose the name virulence factor 1, VF1 for this protein. The identification of VF1 represents the first characterization of an alternative open reading frame protein for the calicivirus family. The immune regulatory function of the MNV VF1 protein provide important perspectives for future research into norovirus biology and pathogenesis.

Immunotherapy of Murine Retrovirus-Induced Acquired Immunodeficiency by CD4 T Regulatory Cell Depletion and PD-1 Blockade

LP-BM5 retrovirus induces a complex disease featuring an acquired immunodeficiency syndrome termed murine AIDS (MAIDS) in susceptible strains of mice, such as C57BL/6 (B6). CD4 T helper effector cells are required for MAIDS induction and progression of viral pathogenesis. CD8 T cells are not needed for viral pathogenesis, but rather, are essential for protection from disease in resistant strains, such as BALB/c. We have discovered an immunodominant cytolytic T lymphocyte (CTL) epitope encoded in a previously unrecognized LP-BM5 retroviral alternative (+1 nucleotide [nt]) gag translational open reading frame. CTLs specific for this cryptic gag epitope are the basis of protection from LP-BM5-induced immunodeficiency in BALB/c mice, and the inability of B6 mice to mount an anti-gag CTL response appears critical to the initiation and progression of LP-BM5-induced MAIDS. However, uninfected B6 mice primed by LP-BM5-induced tumors can generate CTL responses to an LP-BM5 retrovirus infection-associated epitope(s) that is especially prevalent on such MAIDS tumor cells, indicating the potential to mount a protective CD8 T-cell response. Here, we utilized this LP-BM5 retrovirus-induced disease system to test whether modulation of normal immune down-regulatory mechanisms can alter retroviral pathogenesis. Thus, following in vivo depletion of CD4 T regulatory (Treg) cells and/or selective interruption of PD-1 negative signaling in the CD8 T-cell compartment, retroviral pathogenesis was significantly decreased, with the combined treatment of CD4 Treg cell depletion and PD-1 blockade working in a synergistic fashion to substantially reduce the induction of MAIDS.

Deep sequencing-based identification of small non-coding RNAs in Streptomyces coelicolor

Streptomyces coelicolor is considered the model organism among Gram positive, GC rich bacteria. Its genome has been sequenced but little is known about the occurrence and distribution of small non-coding RNAs in this biotechnologically relevant organism. Using deep sequencing we analyzed the transcriptome at the end of exponential growth, which corresponds to the onset of secondary metabolism. We mapped 193 transcriptional start sites of mRNA genes and identified putative new and alternative open reading frames. We identified 63 non-coding RNAs including 29 cis encoded antisense RNAs, and confirmed expression for 11, most of them being growth-phase dependent. A comparison between the sequencing results and bioinformatic sRNA predictions using Dynalign and RNAz revealed only a small overlap between the different approaches.

An overlapping reading frame in the PRNP gene encodes a novel polypeptide distinct from the prion protein

The prion protein gene PRNP directs the synthesis of one of the most intensively studied mammalian proteins, the prion protein (PrP). Yet the physiological function of PrP has remained elusive and has created controversies in the literature. We found a downstream alternative translation initiation AUG codon surrounded by an optimal Kozak sequence in the +3 reading frame of PRNP. The corresponding alternative open reading frame encodes a polypeptide termed alternative prion protein (AltPrP) with a completely different amino acid sequence from PrP. We introduced a hemagglutinin (HA) tag in frame with AltPrP in PrP cDNAs from different species to test the expression of this novel polypeptide using anti-HA antibodies. AltPrP is constitutively coexpressed with human, bovine, sheep, and deer PrP. AltPrP is localized at the mitochondria and is up-regulated by endoplasmic reticulum stress and proteasomal inhibition. Generation of anti-AltPrP antibodies allowed us to test for endogenous expression of AltPrP in wild-type human cells expressing PrP. By transfecting cells with siRNA against PrP mRNA, we repressed expression of both PrP and AltPrP, confirming endogenous expression of AltPrP from PRNP. AltPrP was also detected in human brain homogenate, primary neurons, and peripheral blood mononuclear cells. These results demonstrate an unexpected function for PRNP, which, in addition to plasma membrane-anchored PrP, also encodes a second polypeptide termed AltPrP.

Immunotherapy of murine retrovirus-induced acquired immunodeficiency by CD4 T regulatory cell depletion and PD-1 blockade (154.37)

Abstract LP-BM5 retrovirus induces a disease featuring an acquired immunodeficiency syndrome termed murine AIDS (MAIDS) in susceptible strains of mice, such as C57BL/6 (B6). CD4 T helper effector cells are critical to MAIDS induction and progression of viral pathogenesis. CD8 T cells are not needed for viral pathogenesis, but rather are essential for protection from disease in resistant strains, such as BALB/c. We have discovered an immunodominant cytolytic T lymphocyte (CTL) epitope encoded in a previously un-recognized LP-BM5 retroviral alternative (+1 NT) gag translational open reading frame. CTLs specific for this cryptic gag epitope are the basis of protection from LP-BM5-induced immunodeficiency in BALB/c mice, and the inability of B6 mice to mount an anti-gag CTL response is critical to MAIDS pathogenesis. However, uninfected B6 mice can generate CTL responses to LP-BM5 retrovirus-associated epitope(s) especially prevalent on LP-BM5-induced tumors. Here, we utilized this LP-BM5 retrovirus-induced disease model to test whether modulation of normal immune down-regulatory mechanisms can modulate retroviral pathogenesis. Following in vivo depletion of CD4 T regulatory (Treg) cells, and/or selective interruption of PD-1 negative signaling in the CD8 T-cell compartment, retroviral pathogenesis, but not viral load, was substantially decreased, with the combined treatment of CD4 Treg depletion and PD-1 blockade working in a synergistic fashion to eliminate induction of MAIDS.

Theiler's Virus L* Protein Is Targeted to the Mitochondrial Outer Membrane

The L* protein encoded by Theiler's murine encephalomyelitis virus (TMEV) is a unique example of a picornaviral protein encoded by an alternative open reading frame. This protein is an important determinant of TMEV persistence in the mouse central nervous system. We showed that in infected cells, L* is partitioned between the cytosol and the mitochondria. In mitochondria, L* is anchored in the outer membrane and exposed to the cytosol. The targeting of L* to mitochondria is independent of other viral components and likely depends on a conformational signal. L* targeting to mitochondria might involve chaperones of the Hsp70 family, as these proteins are shown to interact.