Systematic analysis of Kaposi's sarcoma (KS)-associated herpesvirus genomes from a KS case-control study in Cameroon: Evidence of dual infections but no association between viral sequence variation and KS risk.

In sub-Saharan Africa, Kaposi's sarcoma-associated herpesvirus (KSHV) is endemic, and Kaposi's sarcoma (KS) is a significant public health problem. Until recently, KSHV genotype analysis was performed using variable gene regions, representing a small fraction of the genome, and thus the contribution of sequence variation to viral transmission or pathogenesis are understudied. We performed near full-length KSHV genome sequence analysis on samples from 43 individuals selected from a large Cameroonian KS case-control study. KSHV genomes were obtained from 21 KS patients and 22 control participants. Phylogenetic analysis of the K1 region indicated the majority of sequences were A5 or B1 subtypes and all three K15 alleles were represented. Unique polymorphisms in the KSHV genome were observed including large gene deletions. We found evidence of multiple distinct KSHV genotypes in three individuals. Additionally, our analyses indicate that recombination is prevalent suggesting that multiple KSHV infections may not be uncommon overall. Most importantly, a detailed analysis of KSHV genomes from KS patients and control participants did not find a correlation between viral sequence variations and disease. Our study is the first to systematically compare near full-length KSHV genome sequences between KS cases and controls in the same endemic region to identify possible sequence variations associated with disease risk.

Gammaherpesvirus infection and malignant disease in rhesus macaques experimentally infected with SIV or SHIV.

Human gammaherpesviruses are associated with malignancies in HIV infected individuals; in macaques used in non-human primate models of HIV infection, gammaherpesvirus infections also occur. Limited data on prevalence and tumorigenicity of macaque gammaherpesviruses, mostly cross-sectional analyses of small series, are available. We comprehensively examine all three-rhesus macaque gammaherpesviruses -Rhesus rhadinovirus (RRV), Rhesus Lymphocryptovirus (RLCV) and Retroperitoneal Fibromatosis Herpesvirus (RFHV) in macaques experimentally infected with Simian Immunodeficiency Virus or Simian Human Immunodeficiency Virus (SIV/SHIV) in studies spanning 15 years at the AIDS and Cancer Virus Program of the Frederick National Laboratory for Cancer Research. We evaluated 18 animals with malignancies (16 lymphomas, one fibrosarcoma and one carcinoma) and 32 controls. We developed real time quantitative PCR assays for each gammaherpesvirus DNA viral load (VL) in malignant and non-tumor tissues; we also characterized the tumors using immunohistochemistry and in situ hybridization. Furthermore, we retrospectively quantified gammaherpesvirus DNA VL and SIV/SHIV RNA VL in longitudinally-collected PBMCs and plasma, respectively. One or more gammaherpesviruses were detected in 17 tumors; generally, one was predominant, and the relevant DNA VL in the tumor was very high compared to surrounding tissues. RLCV was predominant in tumors resembling diffuse large B cell lymphomas; in a Burkitt-like lymphoma, RRV was predominant; and in the fibrosarcoma, RFHV was predominant. Median RRV and RLCV PBMC DNA VL were significantly higher in cases than controls; SIV/SHIV VL and RLCV VL were independently associated with cancer. Local regressions showed that longitudinal VL patterns in cases and controls, from SIV infection to necropsy, differed for each gammaherpesvirus: while RFHV VL increased only slightly in all animals, RLCV and RRV VL increased significantly and continued to increase steeply in cases; in controls, VL flattened. In conclusion, the data suggest that gammaherpesviruses may play a significant role in tumorogenesis in macaques infected with immunodeficiency viruses.

Corrigendum to "YaliBricks, a versatile genetic toolkit for streamlined and rapid pathway engineering in Yarrowia lipolytica".

[This corrects the article DOI: 10.1016/j.meteno.2017.09.001.].

Genetic Tools for Streamlined and Accelerated Pathway Engineering in Yarrowia lipolytica.

Yarrowia lipolytica is an industrial oleaginous yeast that has many attractive physiological and metabolic characteristics for various biotechnological applications. Although it has a long history of industrial applications, the number of genetic tools available to effectively and efficiently engineer Y. lipolytica still falls behind the vast number of tools available for common organisms such as Escherichia coli and Saccharomyces cerevisiae. In this protocol, we have developed a complete and versatile genetic toolkit tailored for facile genetic manipulation in Y. lipolytica. We created a versatile DNA assembly platform YaliBrick, which can streamline the cloning of large multigene pathways with reused genetic parts. We established a sensitive luciferase reporter assay to characterize a set of 12 native promoters. In addition, we used YaliBrick to generate different gene configurations in multigene constructs. The five-gene biosynthetic pathway of the anticancer, antimicrobial pigment violacein was rapidly assembled in 1 week to demonstrate the simplicity and effectiveness of integrating pathway-balancing strategies with our YaliBrick vectors. In the end, we incorporated CRISPR-Cas9 into our YaliBrick vectors and achieved indel mutation and frameshift gene deletion at the CAN1 (arginine permease) genomic loci of Yarrowia lipolytica. The reported protocol provides a standard procedure to streamline and accelerate metabolic pathway engineering in Yarrowia lipolytica.

Corrigendum to "Rapid evolution of regulatory element libraries for tunable transcriptional and translational control of gene expression" [Synth Syst Biotechnol 2 (4) (2017) 295-301].

[This corrects the article DOI: 10.1016/j.synbio.2017.10.003.].

Rapid evolution of regulatory element libraries for tunable transcriptional and translational control of gene expression.

Engineering cell factories for producing biofuels and pharmaceuticals has spurred great interests to develop rapid and efficient synthetic biology tools customized for modular pathway engineering. Along the way, combinatorial gene expression control through modification of regulatory element offered tremendous opportunity for fine-tuning gene expression and generating digital-like genetic circuits. In this report, we present an efficient evolutionary approach to build a range of regulatory control elements. The reported method allows for rapid construction of promoter, 5'UTR, terminator and trans-activating RNA libraries. Synthetic overlapping oligos with high portion of degenerate nucleotides flanking the regulatory element could be efficiently assembled to a vector expressing fluorescence reporter. This approach combines high mutation rate of the synthetic DNA with the high assembly efficiency of Gibson Mix. Our constructed library demonstrates broad range of transcriptional or translational gene expression dynamics. Specifically, both the promoter library and 5'UTR library exhibits gene expression dynamics spanning across three order of magnitude. The terminator library and trans-activating RNA library displays relatively narrowed gene expression pattern. The reported study provides a versatile toolbox for rapidly constructing a large family of prokaryotic regulatory elements. These libraries also facilitate the implementation of combinatorial pathway engineering principles and the engineering of more efficient microbial cell factory for various biomanufacturing applications.

YaliBricks, a versatile genetic toolkit for streamlined and rapid pathway engineering in Yarrowia lipolytica.

Effective metabolic engineering of microorganisms relies on balanced expression of both heterologous and endogenous genes to channel metabolic flux towards products of interest while achieving reasonable biomass buildup. To facilitate combinatorial pathway engineering and facile genetic operation, we engineered a set of modular cloning vectors compatible with BioBrick standards, called YaliBricks, to allow for rapid assembly of multigene pathways with customized genetic control elements (promoters, intronic sequences and terminators) in the oleaginous yeast Yarrowia lipolytica. We established a sensitive luciferase reporter and characterized a set of 12 native promoters to expand the oleaginous yeast genetic toolbox for transcriptional fine-tuning. We harnessed the intron alternative splicing mechanism and explored three unique gene configurations that allow us to encode genetic structural variations into metabolic function. We elucidated the role of how these genetic structural variations affect gene expression. To demonstrate the simplicity and effectiveness of streamlined genetic operations, we assembled the 12 kb five-gene violacein biosynthetic pathway in one week. We also expanded this set of vectors to accommodate self-cleavage ribozymes and efficiently deliver guide RNA (gRNA) for targeted genome-editing with a codon-optimized CRISPR-Cas9 nuclease. Taken together, the tools built in this study provide a standard procedure to streamline and accelerate metabolic pathway engineering and genetic circuits construction in Yarrowia lipolytica.

T-cell responses to KSHV infection: a systematic approach.

Prior studies of T-cell responses to KSHV have included relatively few participants and focused on relatively few KSHV antigens. To provide a more comprehensive analysis, we investigated T-cell responses to the whole KSHV proteome using IFN-γ ELISpot. Using ∼7,500 overlapping 15mer peptides we generated one to three peptide pools for each of the 82 KSHV ORFs. IFN-γ ELISpot analysis of PBMCs from 19 patients with a history of KSHV-associated disease and 24 healthy donors (11 KSHV seropositive) detected widely varied responses. Fifty six of the 82 ORFs were recognized by at least one individual but there was little overlap between participants. Responses to at least one ORF pool were observed in all 19 patients and in 7 seropositive donors. Four seropositive donors and 10 seronegative donors had no detectable responses while 3 seronegative donors had weak responses to one ORF. Patients recognised more ORFs than the donors (p=0.04) but the response intensity (spot forming units: SFU per million cells) was similar in the two groups. In four of the responding donors, individual peptides eliciting the predominant responses were identified: three donors responded to only one peptide per ORF, while one recognized five. Using intracellular cytokine staining in four participant samples, we detected peptide-induced IFN-γ, MIP1-ß, and TNF-α as well as CD107a degranulation, consistent with multifunctional effector responses in CD8+ and CD4+ T cells. Sequence analysis of TCRs present in peptide specific T-cell clones generated from two participants showed both mono- and multi-clonotypic responses. Finally, we molecularly cloned the KSHV specific TCRs and incorporated the sequences into retroviral vectors to transfer the specificities to fresh donor cells for additional studies. This study suggests that KSHV infected individuals respond to diverse KSHV antigens, consistent with a lack of shared immunodominance and establishes useful tools to facilitate KSHV immunology studies.