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Paul Ahlquist

Publications and source records attributed to Paul Ahlquist.

At least 19 recordsLinked to original sources
PubMed2025

MPAC: a computational framework for inferring pathway activities from multi-omic data.

MOTIVATION: Fully capturing cellular state requires examining genomic, epigenomic, transcriptomic, proteomic, and other assays for a biological sample and comprehensive computational modeling to reason with the complex and sometimes conflicting measurements. Modeling these so-called multi-omic data is especially beneficial in disease analysis, where observations across omic data types may reveal unexpected patient groupings and inform clinical outcomes and treatments. RESULTS: We present Multi-omic Pathway Analysis of Cells (MPAC), a computational framework that interprets multi-omic data through prior knowledge from biological pathways. MPAC leverages network relationships encoded in pathways through a factor graph to infer consensus activity levels for proteins and associated pathway entities from multi-omic data, runs permutation testing to eliminate spurious activity predictions, and groups biological samples by pathway activities to allow identifying and prioritizing proteins with potential clinical relevance, e.g. associated with patient prognosis. Using DNA copy number alteration and RNA-seq data from head and neck squamous cell carcinoma patients from The Cancer Genome Atlas as an example, we demonstrate that MPAC predicts a patient subgroup related to immune responses not identified by analysis with either input omic data type alone. Key proteins identified via this subgroup have pathway activities related to clinical outcome as well as immune cell composition. Our MPAC R package enables similar multi-omic analyses on new datasets. AVAILABILITY AND IMPLEMENTATION: The MPAC package is available at Bioconductor https://bioconductor.org/packages/MPAC.

Humans↗
PubMed2025

MPAC: a computational framework for inferring pathway activities from multi-omic data.

Fully capturing cellular state requires examining genomic, epigenomic, transcriptomic, proteomic, and other assays for a biological sample and comprehensive computational modeling to reason with the complex and sometimes conflicting measurements. Modeling these so-called multi-omic data is especially beneficial in disease analysis, where observations across omic data types may reveal unexpected patient groupings and inform clinical outcomes and treatments. We present Multi-omic Pathway Analysis of Cells (MPAC), a computational framework that interprets multi-omic data through prior knowledge from biological pathways. MPAC leverages network relationships encoded in pathways through a factor graph to infer consensus activity levels for proteins and associated pathway entities from multi-omic data, runs permutation testing to eliminate spurious activity predictions, and groups biological samples by pathway activities to allow identifying and prioritizing proteins with potential clinical relevance, e.g., associated with patient prognosis. Using DNA copy number alteration and RNA-seq data from head and neck squamous cell carcinoma patients from The Cancer Genome Atlas as an example, we demonstrate that MPAC predicts a patient subgroup related to immune responses not identified by analysis with either input omic data type alone. Key proteins identified via this subgroup have pathway activities related to clinical outcome as well as immune cell compositions. Our MPAC R package, available at https://bioconductor.org/packages/MPAC, enables similar multi-omic analyses on new datasets.

Journal Article↗
PubMed2006

Genome-wide expression profiling reveals EBV-associated inhibition of MHC class I expression in nasopharyngeal carcinoma.

To identify the molecular mechanisms by which EBV-associated epithelial cancers are maintained, we measured the expression of essentially all human genes and all latent EBV genes in a collection of 31 laser-captured, microdissected nasopharyngeal carcinoma (NPC) tissue samples and 10 normal nasopharyngeal tissues. Global gene expression profiles clearly distinguished tumors from normal healthy epithelium. Expression levels of six viral genes (EBNA1, EBNA2, EBNA3A, EBNA3B, LMP1, and LMP2A) were correlated among themselves and strongly inversely correlated with the expression of a large subset of host genes. Among the human genes whose inhibition was most strongly correlated with increased EBV gene expression were multiple MHC class I HLA genes involved in regulating immune response via antigen presentation. The association between EBV gene expression and inhibition of MHC class I HLA expression implies that antigen display is either directly inhibited by EBV, facilitating immune evasion by tumor cells, and/or that tumor cells with inhibited presentation are selected for their ability to sustain higher levels of EBV to take maximum advantage of EBV oncogene-mediated tumor-promoting actions. Our data clearly reflect such tumor promotion, showing that deregulation of key proteins involved in apoptosis (BCL2-related protein A1 and Fas apoptotic inhibitory molecule), cell cycle checkpoints (AKIP, SCYL1, and NIN), and metastasis (matrix metalloproteinase 1) is closely correlated with the levels of EBV gene expression in NPC.

Biopsy↗
PubMed2006

Parallels among positive-strand RNA viruses, reverse-transcribing viruses and double-stranded RNA viruses.

Viruses are divided into seven classes on the basis of differing strategies for storing and replicating their genomes through RNA and/or DNA intermediates. Despite major differences among these classes, recent results reveal that the non-virion, intracellular RNA-replication complexes of some positive-strand RNA viruses share parallels with the structure, assembly and function of the replicative cores of extracellular virions of reverse-transcribing viruses and double-stranded RNA viruses. Therefore, at least four of seven principal virus classes share several underlying features in genome replication and might have emerged from common ancestors. This has implications for virus function, evolution and control.

Biological Evolution↗
PubMed2006

Inducible yeast system for Viral RNA recombination reveals requirement for an RNA replication signal on both parental RNAs.

To facilitate RNA recombination studies, we tested whether Saccharomyces cerevisiae, which supports brome mosaic virus (BMV) replication, also supports BMV RNA recombination. Yeast strains expressing BMV RNA replication proteins 1a and 2a(pol) were engineered to transiently coexpress two independently inducible, overlapping, nonreplicating derivatives of BMV genomic RNA3. B3Delta3' lacked the coat protein gene and negative-strand RNA promoter. B3Delta5' lacked the positive-strand RNA promoter and had the coat gene replaced by the selectable URA3 gene. After 12 to 72 h of induction, B3Delta3' and B3Delta5' transcription was repressed and Ura(+) yeast cells were selected. All Ura(+) cells contained recombinant RNA3 replicons expressing URA3. Most replicons arose by intermolecular homologous recombination between B3Delta3' and B3Delta5'. Such recombinants were isolated only when 1a and 2a(pol) were expressed and after transient transcription of both B3Delta3' and B3Delta5', showing that recombination occurred at the RNA, not DNA, level. A minority of URA3-expressing replicons were derived from B3Delta5', independently of B3Delta3', by 5' truncation and modification, generating novel positive-strand promoters and demonstrating that BMV can give rise to subgenomic RNA replicons. Intermolecular B3Delta3'-B3Delta5' recombination occurred only when both parental RNAs bore a functional, cis-acting template recognition and recruitment element targeting viral RNAs to replication complexes. The results imply that recombination occurred in RNA replication complexes to which parental RNAs were independently recruited. Moreover, the ability to obtain intermolecular recombinants at precisely measurable, reproducible frequencies, to control genetic background and induction conditions, and other features of this system will facilitate further studies of virus and host functions in RNA recombination.

Base Sequence↗
PubMed2006

Host deadenylation-dependent mRNA decapping factors are required for a key step in brome mosaic virus RNA replication.

The genomes of positive-strand RNA [+RNA] viruses perform two mutually exclusive functions: they act as mRNAs for the translation of viral proteins and as templates for viral replication. A universal key step in the replication of +RNA viruses is the coordinated transition of the RNA genome from the cellular translation machinery to the viral replication complex. While host factors are involved in this step, their nature is largely unknown. By using the ability of the higher eukaryotic +RNA virus brome mosaic virus (BMV) to replicate in yeast, we previously showed that the host Lsm1p protein is required for efficient recruitment of BMV RNA from translation to replication. Here we show that in addition to Lsm1p, all tested components of the Lsm1p-7p/Pat1p/Dhh1p decapping activator complex, which functions in deadenylation-dependent decapping of cellular mRNAs, are required for BMV RNA recruitment for RNA replication. In contrast, other proteins of the decapping machinery, such as Edc1p and Edc2p from the deadenylation-dependent decapping pathway and Upf1p, Upf2p, and Upf3p from the deadenylation-independent decapping pathway, had no significant effects. The dependence of BMV RNA recruitment on the Lsm1p-7p/Pat1p/Dhh1p complex was linked exclusively to the 3' noncoding region of the BMV RNA. Collectively, our results suggest that the Lsm1p-7p/Pat1p/Dhh1p complex that transfers cellular mRNAs from translation to degradation might act as a key regulator in the switch from BMV RNA translation to replication.

Adenosine Monophosphate↗
PubMed2006

Genes involved in DNA repair and nitrosamine metabolism and those located on chromosome 14q32 are dysregulated in nasopharyngeal carcinoma.

Polymorphisms in nitrosamine metabolism, DNA repair, and immune response genes have been associated with nasopharyngeal carcinoma (NPC). Studies have suggested chromosomal regions involved in NPC. To shed light on NPC etiology, we evaluated host gene expression patterns in 31 NPC and 10 normal nasopharyngeal tissue specimens using the Affymetrix Human Genome U133 Plus 2.0 Array. We focused on genes in five a priori biological pathways and chromosomal locations. Rates of differential expression within these prespecified lists and overall were tested using a bootstrap method. Differential expression was observed for 7.6% of probe sets overall. Elevations in rate of differential expression were observed within the DNA repair (13.7%; P = 0.01) and nitrosamine metabolism (17.5%; P = 0.04) pathways. Differentially expressed probe sets within the DNA repair pathway were consistently overexpressed (93%), with strong effects observed for PRKDC, PCNA, and CHEK1. Differentially expressed probe sets within the nitrosamine metabolism pathway were consistently underexpressed (100%), with strong effects observed for NQ01, CYP2B6, and CYP2E1. No significant evidence of increases in rate of differential expression was seen within the immune/inflammatory pathway. A significant elevation in rate of differential expression was noted for chromosome 4p15.1-4q12 (13.0%; P = 0.04); both overexpression and underexpression were evident (38% and 62%, respectively). An elevation in the rate of differential expression on chromosome 14q32 was observed (11.3%; P = 0.06) with a consistent pattern of gene underexpression (100%; P < 0.0001). These effects were similar when excluding late-stage tumors. Our results suggest that nitrosamine activation and DNA repair are important in NPC. The consistent down-regulation of expression on chromosome 14q32 suggests loss of heterozygosity in this region.

Biopsy↗
PubMed2005

Virus evolution: fitting lifestyles to a T.

The structure of a double-stranded RNA virus outer shell has revealed unexpected similarities with virions of positive-strand RNA viruses. These similarities intersect with emerging parallels in RNA replication to create intriguing evolutionary possibilities.

Biological Evolution↗
PubMed2005

Production of infectious human papillomavirus independently of viral replication and epithelial cell differentiation.

Papillomaviruses are small DNA viruses that are associated with benign and malignant epithelial lesions, including >95% of cervical cancers and approximately 20% of head and neck cancers. Because papillomavirus replication and virion production are tied to epithelial cell differentiation, infectious papillomavirus virion production has been limited to cumbersome organotypic cultures and mouse xenografts. Consequent difficulties in obtaining useful amounts of wild-type or mutant human papillomavirus (HPV) virions have greatly limited studies on many aspects of papillomavirus biology. To overcome these limitations, we developed a system to encapsidate the full-length papillomaviral genome into infectious virions, independently of viral DNA replication and epithelial differentiation. This transient-transfection-based system produces >1,000 times more infectious virus per cell culture dish than the much more labor-intensive organotypic culture. Furthermore, we show that this method allows the facile generation of infectious particles containing wild-type, mutant, or chimeric papillomaviral genomes, overcoming barriers to studying many facets of replication, host interactions, and vaccine and drug development, which has been limited by the insufficient availability of infectious virions.

3T3 Cells↗
PubMed2005

Viral and host determinants of RNA virus vector replication and expression.

Positive-strand RNA viruses have proven to be valuable vectors for delivery and expression of antigens for direct vaccination of animals and vaccine production in plants. However, optimal use of these viruses as vectors for vaccine and other purposes is limited by incomplete understanding of their replication pathways and associated constraints on inserted foreign genes. Further insights into RNA virus vector design and optimization are emerging from recent advances on the function of viral RNA replication factors, the nature of the viral RNA replication complex as a membrane-bounded compartment sequestering replication components from competing processes and host defenses, and identification of surprisingly diverse host genes contributing to many virus replication steps.

Animals↗
PubMed2005

Detecting protein-protein interaction in live yeast by flow cytometry.

BACKGROUND: The yeast Saccharomyces cerevisiae is the most commonly used organism for studying protein- protein interactions. In this report we demonstrate the use of flow cytometry in observing fluorescence resonance energy transfer (FRET) between cyan and yellow fluorescent fusion proteins (CFP and YFP, respectively) as a marker for protein interaction in live yeast cells. Probability binning is also employed to provide a statistical confirmation of our observations. METHODS: We coexpressed CFP and YFP fusions containing the N-terminal transmembrane domain (NTM) of Tom70p in yeast and analyzed FRET in live cells with a multilaser flow cytometer. The Tom70p NTM was previously shown to be sufficient for mitochondrial localization and protein-protein interaction (Millar and Shore, 1994, J Biol Chem 269:12229-12232). RESULTS: FRET was observed only in cells that expressed CFP and YFP fusions that each contained the wild-type NTM. The introduction of mutations previously shown to disrupt NTM interaction eliminated FRET. Probability binning confirmed that differences between the FRET channels of experimental and control samples were statistically and physiologically significant. CONCLUSION: Flow cytometric analysis of FRET in yeast is a powerful technique for studying protein-protein interactions. The use of flow cytometry allows FRET data to be gathered from a large number of individual cells, thus providing important advantages unavailable to other techniques. Its application to yeast presents a new method to a popular system widely used in proteomic studies.

Bacterial Proteins↗
PubMed2005

In vivo self-interaction of nodavirus RNA replicase protein a revealed by fluorescence resonance energy transfer.

Flock house virus (FHV) is the best-characterized member of the Nodaviridae, a family of small, positive-strand RNA viruses. Unlike most RNA viruses, FHV encodes only a single polypeptide, protein A, that is required for RNA replication. Protein A contains a C-proximal RNA-dependent RNA polymerase domain and localizes via an N-terminal transmembrane domain to the outer mitochondrial membrane, where FHV RNA replication takes place in association with invaginations referred to as spherules. We demonstrate here that protein A self-interacts in vivo by using flow cytometric analysis of fluorescence resonance energy transfer (FRET), spectrofluorometric analysis of bioluminescence resonance energy transfer, and coimmunoprecipitation. Several nonoverlapping protein A sequences were able to independently direct protein-protein interaction, including an N-terminal region previously shown to be sufficient for localization to the outer mitochondrial membrane (D. J. Miller and P. Ahlquist, J. Virol. 76:9856-9867, 2000). Mutations in protein A that diminished FRET also diminished FHV RNA replication, a finding consistent with an important role for protein A self-interaction in FHV RNA synthesis. Thus, the results imply that FHV protein A functions as a multimer rather than as a monomer at one or more steps in RNA replication.

Animals↗
PubMed2005

Isolation of cell lines that show novel, murine leukemia virus-specific blocks to early steps of retroviral replication.

In order to identify cellular proteins required for early stages of retroviral replication, a high volume screening with mammalian somatic cells was performed. Ten pools of chemically mutagenized Chinese hamster ovary (CHO-K1) cells were challenged with a murine leukemia virus (MLV) vector pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G), and cells that failed to be transduced were enriched by cell sorting. Each pool yielded a clonally derived cell line with a 5-fold or greater resistance to virus infection, and five cell lines exhibited a >50-fold resistance. These five cell lines were efficiently infected by a human immunodeficiency virus vector pseudotyped with VSV-G. When engineered to express the TVA receptor for subgroup A avian sarcoma and leukosis virus (ASLV-A), the five cell lines were resistant to infection with a MLV vector pseudotyped with the ASLV-A envelope protein but were fully susceptible to infection with an ASLV-A vector. Thus, the defect in these cells resides after virus-cell membrane fusion and, unlike those in other mutant cell lines that have been described, is specific for the MLV core. To identify the specific stages of MLV infection that are impaired in the resistant cell lines, real-time quantitative PCR analyses were employed and two phenotypic groups were identified. Viral infection of three cell lines was restricted before reverse transcription; in the other two cell lines, it was blocked after reverse transcription, nuclear localization, and two-long terminal repeat circle formation but before integration. These data provide genetic evidence that at least two distinct intracellular gene products are required specifically for MLV infection. These cell lines are important tools for the biochemical and genetic analysis of early stages in retrovirus infection.

Animals↗
PubMed2005

Brome mosaic virus 1a nucleoside triphosphatase/helicase domain plays crucial roles in recruiting RNA replication templates.

Positive-strand RNA virus RNA replication is invariably membrane associated and frequently involves viral proteins with nucleoside triphosphatase (NTPase)/helicase motifs or activities. Brome mosaic virus (BMV) encodes two RNA replication factors: 1a has a C-terminal NTPase/helicase-like domain, and 2a(pol) has a central polymerase domain. 1a accumulates on endoplasmic reticulum membranes, recruits 2a(pol), and induces 50- to 70-nm membrane invaginations (spherules) serving as RNA replication compartments. 1a also recruits BMV replication templates such as genomic RNA3. In the absence of 2a(pol), 1a dramatically stabilizes RNA3 by transferring RNA3 to a membrane-associated, nuclease-resistant state that appears to correspond to the interior of the 1a-induced spherules. Prior results show that the 1a NTPase/helicase-like domain contributes to RNA recruitment. Here, we tested mutations in the conserved helicase motifs of 1a to further define the roles of this domain in RNA template recruitment. All 1a helicase mutations tested showed normal 1a accumulation, localization to perinuclear endoplasmic reticulum membranes, and recruitment of 2a(pol). Most 1a helicase mutants also supported normal spherule formation. Nevertheless, these mutations severely inhibited RNA replication and 1a-induced stabilization of RNA3 in vivo. For such 1a mutants, the membrane-associated RNA3 pool was both reduced and highly susceptible to added nuclease. Thus, 1a recruitment of viral RNA templates to a membrane-associated, nuclease-resistant state requires additional functions beyond forming spherules and recruiting RNA to membranes, and these functions depend on the 1a helicase motifs. The possibility that, similar to some double-stranded RNA viruses, the 1a NTPase/helicase-like domain may be involved in importing viral RNAs into a preformed replication compartment is discussed.

Bromovirus↗
PubMed2005

Mutual interference between genomic RNA replication and subgenomic mRNA transcription in brome mosaic virus.

Replication by many positive-strand RNA viruses includes genomic RNA amplification and subgenomic mRNA (sgRNA) transcription. For brome mosaic virus (BMV), both processes occur in virus-induced, membrane-associated compartments, require BMV replication factors 1a and 2a, and use negative-strand RNA3 as a template for genomic RNA3 and sgRNA syntheses. To begin elucidating their relations, we examined the interaction of RNA3 replication and sgRNA transcription in Saccharomyces cerevisiae expressing 1a and 2a, which support the full RNA3 replication cycle. Blocking sgRNA transcription stimulated RNA3 replication by up to 350%, implying that sgRNA transcription inhibits RNA3 replication. Such inhibition was independent of the sgRNA-encoded coat protein and operated in cis. We further found that sgRNA transcription inhibited RNA3 replication at a step or steps after negative-strand RNA3 synthesis, implying competition with positive-strand RNA3 synthesis for negative-strand RNA3 templates, viral replication factors, or common host components. Consistent with this, sgRNA transcription was stimulated by up to 400% when mutations inhibiting positive-strand RNA3 synthesis were introduced into the RNA3 5'-untranslated region. Thus, BMV subgenomic and genomic RNA syntheses mutually interfered with each other, apparently by competition for one or more common factors. In plant protoplasts replicating all three BMV genomic RNAs, mutations blocking sgRNA transcription often had lesser effects on RNA3 accumulation, possibly because RNA3 also competed with RNA1 and RNA2 replication templates and because any increase in RNA3 replication at the expense of RNA1 and RNA2 would be self-limited by decreased 1a and 2a expression from RNA1 and RNA2.

Bromovirus↗
PubMed2005

Lack of association between EBV and breast carcinoma.

Multiple conflicting findings have been presented which indicate that EBV may be found in anywhere from 0% to 51% of breast carcinomas. When EBV has been found causally associated with other human cancers, its DNA and one or more of its viral products have been detected in most tumor cells of a given biopsy. To test whether EBV has such an association with breast cancer, we measured the number of viral DNA molecules per cell in matched normal and tumor biopsies from 45 patients using real-time quantitative PCR. In no case could EBV DNA consistently be detected, with either of two different probes, at levels above 0.1 molecules per cell in two sections of the tumor samples. These levels of detection match those detected in EBV-negative cell lines and therefore likely represent noise in the assays. Equally importantly, the distribution of these low signals was the same between tumors and their matched normal controls. We conclude that EBV does not contribute to the development of breast cancers as it does to epithelial cancers such as nasopharyngeal and gastric carcinomas or to Burkitt's and Hodgkin's lymphomas.

Actins↗
PubMed2004

Alternate, virus-induced membrane rearrangements support positive-strand RNA virus genome replication.

All positive-strand RNA [(+)RNA] viruses replicate their RNA on intracellular membranes, often in association with spherular invaginations of the target membrane. For brome mosaic virus, we previously showed that such spherules serve as compartments or mini-organelles for RNA replication and that their assembly, structure, and function have similarities to the replicative cores of retrovirus and double-stranded RNA virus virions. Some other (+)RNA viruses conduct RNA replication in association with individual or clustered double-membrane vesicles, appressed double membranes, or other structures whose possible relationships to the spherular invaginations are unclear. Here we show that modulating the relative levels and interactions of brome mosaic virus replication factors 1a and 2a polymerase (2apol) shifted the membrane rearrangements associated with RNA replication from small invaginated spherules to large, karmellae-like, multilayer stacks of appressed double membranes that supported RNA replication as efficiently as spherules. Spherules were induced by expressing 1a, which has functional similarities to retrovirus virion protein Gag, or 1a plus low levels of 2apol. Double-membrane layers were induced by 1a plus higher levels of 2apol and were suppressed by deleting the major 1a-interacting domain from 2apol. The stacked, double-membrane layers alternated with spaces that, like spherule interiors, were 50-60 nm wide, connected to the cytoplasm, and contained 1a and 2apol. These and other results suggest that seemingly diverse membrane rearrangements associated with RNA replication by varied (+)RNA viruses may represent topologically and functionally related structures formed by similar protein-protein and protein-membrane interactions and interconverted by altering the balances among those interactions.

Bromovirus↗