Biology subjects

Lennemann, N. J.

Publications and source records attributed to Lennemann, N. J..

6 recordsLinked to original sources

bioRxiv2025

Endoplasmic reticulum-anchored nonstructural proteins drive human astrovirus replication organelle formation

Human astroviruses (HAstV) are major cause of acute, non-bacterial gastroenteritis and have been implicated in severe infections of the nervous system. Despite global prevalence, there are no established treatments for HAstVs due to a lack of understanding of the fundamental biology of infection, including mechanisms of viral replication. Like all positive-stranded RNA (+ssRNA) viruses, infection induces remodeling of host membranes into replication organelles (ROs). However, the intracellular membrane source and viral proteins involved in the coordination of HAstV ROs remain poorly defined. Using immunofluorescence microscopy, we determined that HAstV1 infection drives extensive restructuring of the endoplasmic reticulum (ER) to concentrate RNA replication and virus packaging. Long-term, time-lapse imaging of the ER and time point transmission electron microscopy revealed that temporal manipulation of ER membrane corresponds with the emergence of ER-contiguous double membrane vesicles (DMV). The co-expression of transmembrane nonstructural proteins nsp1a/1 and nsp1a/2 established similar DMV networks in the absence of an active infection. Further, super resolution microscopy revealed the organization of these two viral proteins in RO-like arrangements within the perinuclear region of infected cells. Together, these findings enhance our understanding of HAstV1-induced RO biogenesis, highlighting nsp1a/1 and nsp1a/2 as exploitable targets for the design of antivirals restricting astrovirus replication. SignificanceHuman astroviruses (HAstV) are understudied, globally prevalent pathogens capable of causing potentially fatal infections of the central nervous system in children, the elderly, and immunocompromised individuals. Despite the capacity to cause devastating disease, there are no established vaccines, antivirals, or therapeutics available combat HAstV infections, in part due to a lack of knowledge for the functions of several viral proteins. This study furthers our understanding of HAstV manipulation of the host cell by identification of the viral proteins responsible for the biogenesis of virus-induced double membrane vesicles, which are a hallmark of replication.

microbiology↗

bioRxiv2025

Subcellular determinants of orthoflavivirus protease activity

Orthoflaviviruses are small, enveloped, positive-sense RNA viruses that cause over 500 million infections globally each year for which there are no antiviral treatments. The viral protease is an attractive target for therapeutics due to its critical functions throughout infection. Many studies have reported on the structure, function, and importance of orthoflavivirus proteases; However, the molecular determinants for cleavage of intracellular substrates by orthoflavivirus proteases and how these factors affect viral fitness are unknown. In this study, we used our fluorescent, protease-activity reporter system to investigate the subcellular determinants involved in orthoflavivirus protease cleavage. By modifying our reporter platform, we identified endoplasmic reticulum (ER) subdomain localization and membrane proximity of the substrate cut site as two previously uncharacterized molecular determinants for cleavage. We also altered the amino acid composition of the reporter cut site to introduce sequences present at the cytoplasmic junctions within orthoflavivirus polyproteins and found that each protease processed the sequence located at the junction between NS4A and the 2K peptide least efficiently. Live-cell imaging revealed that cleavage of the NS4A|2K sequence is significantly delayed compared to the capsid cleavage sequence. We further determined that introducing a more efficient cleavage sequence into the NS4A|2K junctions of orthoflavivirus infectious clones abolished virus recovery. Overall, this study identifies ER subdomain localization and membrane proximity of the cut site as molecular determinants for cleavage by orthoflavivirus proteases and provides insight into the role that sequence specificity plays in the coordinated processing of the viral polyprotein and establishing productive infections. ImportanceOrthoflaviviruses are the most prevalent and dangerous arthropod-borne viruses (arboviruses) leading to over 500 million global infections annually. Orthoflavivirus infection can cause severe pathologies, including hemorrhagic conditions and neurological disease, that lead to hundreds of thousands of deaths each year. The viral protease complex, responsible for processing the viral polyprotein into its functional subunits, is an attractive target for antiviral therapeutic development. Despite extensive research efforts on these viral protein complexes, all protease inhibitor candidates have fallen short of clinical efficacy, highlighting a considerable gap in knowledge of the viral proteases complex intracellular activity. The significance of our research is in characterizing the subcellular determinants associated with orthoflavivirus protease cleavage efficiency and how these factors can influence viral fitness. These findings contribute to closing this gap in knowledge of the mechanisms of orthoflavivirus proteases which can ultimately lead to the successful development of targeted antivirals.

microbiology↗

bioRxiv2022

Dual-Fluorescent Reporter for Live-Cell Imaging of the ER During DENV Infection

Infection by flaviviruses leads to dramatic remodeling of the endoplasmic reticulum (ER). Viral replication occurs within virus-induced vesicular invaginations in the ER membrane. A hallmark of flavivirus infection is expansion of the ER membrane which can be observed at specific time points post infection. However, this process has not been effectively visualized in living cells throughout the course of infection at the single cell resolution. In this study, we developed a plasmid-based reporter system to monitor flavivirus infection and simultaneous virus-induced manipulation of single cells throughout the course of infection in real-time. This system requires viral protease cleavage to release an ER-anchored fluorescent protein infection reporter that is fused to a nuclear localization signal (NLS). This proteolytic cleavage allows for the translocation of the infection reporter signal to the nucleus while an ER-specific fluorescent marker remains localized in the lumen. Thus, the construct allows for the visualization of virus-dependent changes to the ER throughout the course of infection. In this study, we show that our reporter was efficiently cleaved upon the expression of multiple flavivirus proteases, including dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus (YFV). We also found that the DENV protease-dependent cleavage of our ER-anchored reporter exhibited more stringent cleavage sequence specificity than what has previously been shown with biochemical assays. Using this system for long term time-lapse imaging of living cells infected with DENV, we observed nuclear translocation of the reporter signal beginning approximately 8 hours post-infection, which continued to increase throughout the time course. Interestingly, we found that increased reporter signal translocation correlated with increased ER signal intensity, suggesting a positive association between DENV infection and ER expansion in a time-dependent manner. Overall, this report demonstrates that the FlavER platform provides a useful tool for monitoring flavivirus infection and simultaneously observing virus-dependent changes to the host cell ER, allowing for study of the temporal nature of virus-host interactions.

microbiology↗

bioRxiv2022

Construction and rescue of a DNA-launched DENV2 16681 infectious clone

Flaviviruses represent a large group of globally significant, insect-borne pathogens. For many of these viruses, there is a lack of antivirals and vaccines. Thus, there is a need to continue the development of tools to further advance our efforts to combat these pathogens, including reverse genetics techniques. Traditionally, reverse genetics methods for flaviviruses rely on producing infectious RNA from in vitro transcription reactions followed by electroporation or transfection into permissive cell lines. However, production of Zika virus has been successful from CMV-promoter driven expression plasmids, which provides cost and time advantages. In this report, we describe the design and construction of a DNA-launched infectious clone for dengue virus (DENV) serotype 2 strain 16681. An artificial intron was introduced in the nonstructural protein 1 segment of the viral genome to promote stability in bacteria. We found that rescued virus maintained similar replication kinetics in several cell lines commonly used in flavivirus research. Thus, we present a rapid and cost-effective method for producing DENV2 strain 16681 from plasmid DNA. This construct will be a useful platform for the continued development of anti-DENV therapeutics and vaccines.

microbiology↗

bioRxiv2020

BPIFB3 interacts with ARFGAP1 and TMED9 to regulate non-canonical autophagy and RNA virus infection

Autophagy is a degradative cellular pathway that targets cytoplasmic contents and organelles for turnover by the lysosome. Various autophagy pathways play key roles in the clearance of viral infections, and many families of viruses have developed unique methods for avoiding degradation. Some positive stranded RNA viruses, such as enteroviruses and flaviviruses, usurp the autophagic pathway to promote their own replication. We previously identified the endoplasmic reticulum-localized protein BPIFB3 as an important regulator of non-canonical autophagy that uniquely impacts the replication of enteroviruses and flaviviruses. Here, we find that many components of the canonical autophagy machinery are not required for BPIFB3-regulated autophagy and identify the host factors that facilitate its role in the replication of enteroviruses and flaviviruses. Using proximity-dependent biotinylation (BioID) followed by mass spectrometry, we identify ARFGAP1 and TMED9 as two cellular components that interact with BPIFB3 to regulate autophagy and viral replication. Importantly, our data demonstrate that non-canonical autophagy in mammalian cells can be controlled outside of the traditional pathway regulators and define the role of two proteins in BPIFB3-mediated non-canonical autophagy. Summary StatementBPIFB3 is a regulator of a non-canonical cellular autophagy pathway that impacts the replication of enteroviruses and flaviviruses. Here we define ARFGAP1 and TMED9 as essential components of this pathway.

cell biology↗

bioRxiv2018

BPIFB3 facilitates flavivirus infection by controlling RETREG1-dependent reticulophagy

The flavivirus genus, which includes dengue virus (DENV) and Zika virus (ZIKV), are significant human pathogens and the prevalence of infected vectors continues to geographically expand. Both DENV and ZIKV rely on expansion of the endoplasmic reticulum (ER) and the induction of autophagy to establish a productive viral infection. However, little is known regarding the interplay between the requirements for autophagy initiation during infection and the mechanisms used by these viruses to avoid clearance through the autophagic pathway. We recently showed that DENV and ZIKV inhibit reticulophagy (specific degradation of the ER through autophagy) by cleaving reticulophagy regulator 1 (RETREG1), an autophagy receptor responsible for targeted ER sheet degradation. These data suggest that DENV and ZIKV require specific autophagic pathways for their replication, while other autophagic pathways are antiviral. We previously identified BPI Fold Containing Family B Member 3 (BPIFB3) as a regulator of autophagy that negatively controls enterovirus replication. Here, we show that in contrast to enteroviruses, BPIFB3 functions as a positive regulator of DENV and ZIKV infection and that its RNAi-mediated silencing drastically inhibits the formation of viral replication organelles. We show that BPIFB3 depletion enhances ER fragmentation, while its overexpression protects against autophagy-induced ER degradation, demonstrating that BPIFB3 serves as a specific regulator of ER turnover. We further show that the antiviral effects of BPIFB3 depletion on flavivirus infection are reversed in RETREG1-depleted cells, and that BPIFB3 associates with RETREG1 within the ER, suggesting that BPIFB3 regulates a RETREG1-specific reticulophagy pathway. Collectively, these studies identify BPIFB3 as a regulator of the reticulophagy pathway and define the requirements for a novel host regulator of flavivirus infection.\n\nAuthor SummaryFlaviviruses and other arthropod transmitted viruses represent a widespread global health problem with limited treatment options currently available. Thus, greater knowledge of the host factors required for replication and transmission is needed to provide a better understanding of the cellular requirements for infection. Here, we show that the endoplasmic reticulum (ER) localized protein, BPIFB3 is required to facilitate flavivirus infection. Depletion of BPIFB3 in cells inhibits dengue virus and Zika virus infection prior to replication of the viral genome. Mechanistically, we show that BPIFB3 inhibits ER degradation in an autophagy-specific manner and that loss of BPIFB3 decreases the availability of ER membranes needed for flavivirus replication. We further show that BPIFB3 specifically regulates the RETREG1 pathway, but not other pathways of ER turnover. Together, our data define a previously uncharacterized method of regulating ER degradation and show that BPIFB3 is an essential host factor for a productive flavivirus infection.

microbiology↗