Description: Targeting of transmembrane proteins to the endoplasmic reticulum (ER) proceeds via either the signal recognition particle (SRP) or the guided entry of tail-anchored proteins (GET) pathway, consisting of Get1 to -5 and Sgt2. While SRP cotranslationally targets membrane proteins containing one or multiple transmembrane domains, the GET pathway posttranslationally targets proteins containing a single C-terminal transmembrane domain termed the tail anchor. Here, we dissect the roles of the SRP and GET pathways in the sorting of homologous, two-membrane-spanning K + channel proteins termed Kcv, Kesv, and Kesv-VV. We show that Kcv is targeted to the ER cotranslationally via its N-terminal transmembrane domain, while Kesv-VV is targeted posttranslationally via its C-terminal transmembrane domain, which recruits Get4-5/Sgt2 and Get3. Unexpectedly, nascent Kcv recruited not only SRP but also the Get4-5 module of the GET pathway to ribosomes. Ribosome binding of Get4-5 was independent of Sgt2 and was strongly outcompeted by SRP. The combined data indicate a previously unrecognized cotranslational interplay between the SRP and GET pathways.

Description: Scrub typhus is a potentially lethal infection caused by the obligate intracellular bacterium Orientia tsutsugamushi . Reports on the emergence of doxycycline-resistant strains highlight the urgent need to develop novel antiinfectives against scrub typhus. Corallopyronin A (CorA) is a novel α-pyrone compound synthesized by the myxobacterium Corallococcus coralloides that was characterized as a noncompetitive inhibitor of the switch region of the bacterial RNA polymerase (RNAP). We investigated the antimicrobial action of CorA against the human-pathogenic Karp strain of O. tsutsugamushi in vitro and in vivo . The MIC of CorA against O. tsutsugamushi was remarkably low (0.0078 μg/ml), 16-fold lower than that against Rickettsia typhi . In the lethal intraperitoneal O. tsutsugamushi mouse infection model, a minimum daily dose of 100 μg CorA protected 100% of infected mice. Two days of treatment were sufficient to confer protection. In contrast to BALB/c mice, SCID mice succumbed to the infection despite treatment with CorA or tetracycline, suggesting that antimicrobial treatment required synergistic action of the adaptive immune response. Similar to tetracycline, CorA did not prevent latent infection of O. tsutsugamushi in vivo . However, latency was not caused by acquisition of antimicrobial resistance, since O. tsutsugamushi reisolated from latently infected BALB/c mice remained fully susceptible to CorA. No mutations were found in the CorA-binding regions of the β and β' RNAP subunit genes rpoB and rpoC . Inhibition of the RNAP switch region of O. tsutsugamushi by CorA is therefore a novel and highly potent target for antimicrobial therapy for scrub typhus.

Description: Zoonotic viruses circulate as swarms in animal reservoirs and can emerge into human populations, causing epidemics that adversely affect public health. Portable, safe, and effective vaccine platforms are needed in the context of these outbreak and emergence situations. In this work, we report the generation and characterization of an alphavirus replicon vaccine platform based on a non-select agent, attenuated Venezuelan equine encephalitis (VEE) virus vaccine, strain 3526 (VRP 3526). Using both noroviruses and coronaviruses as model systems, we demonstrate the utility of the VRP 3526 platform in the generation of recombinant proteins, production of virus-like particles, and in vivo efficacy as a vaccine against emergent viruses. Importantly, packaging under biosafety level 2 (BSL2) conditions distinguishes VRP 3526 from previously reported alphavirus platforms and makes this approach accessible to the majority of laboratories around the world. In addition, improved outcomes in the vulnerable aged models as well as against heterologous challenge suggest improved efficacy compared to that of previously attenuated VRP approaches. Taking these results together, the VRP 3526 platform represents a safe and highly portable system that can be rapidly deployed under BSL2 conditions for generation of candidate vaccines against emerging microbial pathogens. IMPORTANCE While VEE virus replicon particles provide a robust, established platform for antigen expression and vaccination, its utility has been limited by the requirement for high-containment-level facilities for production and packaging. In this work, we utilize an attenuated vaccine strain capable of use at lower biocontainment level but retaining the capacity of the wild-type replicon particle. Importantly, the new replicon platform provides equal protection for aged mice and following heterologous challenge, which distinguishes it from other attenuated replicon platforms. Together, the new system represents a highly portable, safe system for use in the context of disease emergence.

Description: Human papillomavirus (HPV) infection is the most common viral infection of the reproductive tract, with virtually all cases of cervical cancer being attributable to infection by oncogenic HPVs. However, the exact mechanism and receptors used by HPV to infect epithelial cells are controversial. The current entry model suggests that HPV initially attaches to heparan sulfate proteoglycans (HSPGs) at the cell surface, followed by conformational changes, cleavage by furin convertase, and subsequent transfer of the virus to an as-yet-unidentified high-affinity receptor. In line with this model, we established an in vitro infection system using the HSPG-deficient cell line pgsD677 together with HPV16 pseudovirions (HPV16-PsVs). While pgsD677 cells were nonpermissive for untreated HPV16-PsVs, furin cleavage of the particles led to a substantial increase in infection. Biochemical pulldown assays followed by mass spectrometry analysis showed that furin-precleaved HPV16-PsVs specifically interacted with surface-expressed vimentin on pgsD677 cells. We further demonstrated that both furin-precleaved and uncleaved HPV16-PsVs colocalized with surface-expressed vimentin on pgsD677, HeLa, HaCaT, and NIKS cells, while binding of incoming viral particles to soluble vimentin protein before infection led to a substantial decrease in viral uptake. Interestingly, decreasing cell surface vimentin by small interfering RNA (siRNA) knockdown in HeLa and NIKS cells significantly increased HPV16-PsV infectious internalization, while overexpression of vimentin had the opposite effect. The identification of vimentin as an HPV restriction factor enhances our understanding of the initial steps of HPV-host interaction and may lay the basis for the design of novel antiviral drugs preventing HPV internalization into epithelial cells. IMPORTANCE Despite HPV being a highly prevalent sexually transmitted virus causing significant disease burden worldwide, particularly cancer of the cervix, cell surface events preceding oncogenic HPV internalization are poorly understood. We herein describe the identification of surface-expressed vimentin as a novel molecule not previously implicated in the infectious internalization of HPV16. Contrary to our expectations, vimentin was found to act not as a receptor but rather as a restriction factor dampening the initial steps of HPV16 infection. These results importantly contribute to our current understanding of the molecular events during the infectious internalization of HPV16 and open a new direction in the development of alternative drugs to prevent HPV infection.