Biology subjects

Guereca, M.

Publications and source records attributed to Guereca, M..

4 recordsLinked to original sources

bioRxiv2025

Engineering gene expression dynamics via self-amplifying RNA with drug-responsive non-structural proteins

The design of gene therapies with drug-regulatable expression of therapeutic payloads is of interest for diverse applications. We hypothesized that a regulated expression system based on alphavirus-derived self-amplifying RNAs (saRNAs), which encode 4 non-structural proteins (nsPs) that copy the RNA backbone to enable sustained expression, would have advantages in safety and simplicity of delivery. Here we designed saRNAs where payload expression is regulated by the FDA-approved drug trimethoprim (TMP), by fusing TMP-responsive degradation domains (DDs) to nsPs to regulate RNA self-amplification. Screening a library of nsP-DD fusions, we identified an optimal design with DDs fused to nsP2, nsP3, and the payload, achieving a high fold-change in expression level in response to TMP and low expression in the off state. In mice, this saRNA circuit enabled diverse dynamic expression patterns in response to oral TMP. Implementing this circuit for controlled expression of an HIV antigen, an escalating TMP regimen significantly enhanced germinal center responses critical for B cell affinity maturation. This drug- regulated RNA technology holds potential for vaccines, immunotherapies, and gene therapies.

bioengineering↗

bioRxiv2024

Efficient genome replication in influenza A virus requires NS2 and sequence beyond the canonical promoter

Influenza A virus encodes promoters in both the sense and antisense orientations. These support the generation of new genomes, antigenomes, and mRNA transcripts. Using minimal replication assays--transfections with viral polymerase, nucleoprotein, and a genomic template--the influenza promoter sequences were identified as 13nt at the 5 end of the viral genomic RNA (U13) and 12nt at the 3 end (U12). Other than the fourth 3 nucleotide, the U12 and U13 sequences are identical between all eight RNA molecules that comprise the segmented influenza genome. Despite possessing identical promoters, individual segments can exhibit different transcriptional dynamics during infection. However flu promoter sequences were defined in experiments without influenza NS2, a protein which modulates transcription and replication differentially between genomic segments. This suggests that the identity of the "complete" promoter may depend on NS2. Here we assess how internal sequences of two genomic segments, HA and PB1, may contribute to NS2-dependent replication as well as map such interactions down to individual nucleotides in PB1. We find that the expression of NS2 significantly alters sequence requirements for efficient replication beyond the identical U12 and U13 sequence, providing a mechanism for the divergent replication and transcription dynamics across the influenza A virus genome.

microbiology↗

bioRxiv2023

Cryptic proteins translated from deletion-containing viral genomes dramatically expand the influenza virus proteome

Productive infections by RNA viruses require faithful replication of the entire genome. Yet many RNA viruses also produce deletion-containing viral genomes (DelVGs), aberrant replication products with large internal deletions. DelVGs interfere with the replication of wild-type virus and their presence in patients is associated with better clinical outcomes as they. The DelVG RNA itself is hypothesized to confer this interfering activity. DelVGs antagonize replication by out-competing the full-length genome and triggering innate immune responses. Here, we identify an additionally inhibitory mechanism mediated by a new class of viral proteins encoded by DelVGs. We identified hundreds of cryptic viral proteins translated from DelVGs. These DelVG-encoded proteins (DPRs) include canonical viral proteins with large internal deletions, as well as proteins with novel C-termini translated from alternative reading frames. Many DPRs retain functional domains shared with their full-length counterparts, suggesting they may have activity during infection. Mechanistic studies of DPRs derived from the influenza virus protein PB2 showed that they poison replication of wild-type virus by acting as dominant-negative inhibitors of the viral polymerase. These findings reveal that DelVGs have a dual inhibitory mechanism, acting at both the RNA and protein level. They further show that DPRs have the potential to dramatically expand the functional proteomes of diverse RNA viruses.

microbiology↗

bioRxiv2023

Identifying the core genome of the nucleus-forming bacteriophage family and characterization of Erwinia phage RAY

We recently discovered that some bacteriophages establish a nucleus-like replication compartment (phage nucleus), but the core genes that define nucleus-based phage replication and their phylogenetic distribution were unknown. By studying phages that encode the major phage nucleus protein chimallin, including previously sequenced yet uncharacterized phages, we discovered that chimallin-encoding phages share a set of 72 highly conserved genes encoded within seven distinct gene blocks. Of these, 21 core genes are unique to this group, and all but one of these unique genes encode proteins of unknown function. We propose that phages with this core genome comprise a novel viral family we term Chimalliviridae. Fluorescence microscopy and cryo-electron tomography studies of Erwinia phage vB_EamM_RAY confirm that many of the key steps of nucleus-based replication encoded in the core genome are conserved among diverse chimalliviruses, and reveal that non-core components can confer intriguing variations on this replication mechanism. For instance, unlike previously studied nucleus-forming phages, RAY doesnt degrade the host genome, and its PhuZ homolog appears to form a five-stranded filament with a lumen. This work expands our understanding of phage nucleus and PhuZ spindle diversity and function, providing a roadmap for identifying key mechanisms underlying nucleus-based phage replication.

microbiology↗