Structural and functional characterization of the Sin Nombre virus L protein.

The Bunyavirales order is a large and diverse group of segmented negative-strand RNA viruses. Several virus families within this order contain important human pathogens, including Sin Nombre virus (SNV) of the Hantaviridae. Despite the high epidemic potential of bunyaviruses, specific medical countermeasures such as vaccines or antivirals are missing. The multifunctional ~250 kDa L protein of hantaviruses, amongst other functional domains, harbors the RNA-dependent RNA polymerase (RdRp) and an endonuclease and catalyzes transcription as well as replication of the viral RNA genome, making it a promising therapeutic target. The development of inhibitors targeting these key processes requires a profound understanding of the catalytic mechanisms. Here, we established expression and purification protocols of the full-length SNV L protein bearing the endonuclease mutation K124A. We applied different biochemical in vitro assays to provide an extensive characterization of the different enzymatic functions as well as the capacity of the hantavirus L protein to interact with the viral RNA. By using single-particle cryo-EM, we obtained a 3D model including the L protein core region containing the RdRp, in complex with the 5' promoter RNA. This first high-resolution model of a New World hantavirus L protein shows striking similarity to related bunyavirus L proteins. The interaction of the L protein with the 5' RNA observed in the structural model confirms our hypothesis of protein-RNA binding based on our biochemical data. Taken together, this study provides an excellent basis for future structural and functional studies on the hantavirus L protein and for the development of antiviral compounds.

Development of a High Min-Entropy Quantum Random Number Generator Based on Amplified Spontaneous Emission.

We present the theory, architecture, and performance characteristics of a quantum random number generator (QRNG) which operates in a PCI express form factor-compatible plug-and-play design. The QRNG relies on a thermal light source (in this case, amplified spontaneous emission), which exhibits photon bunching according to the Bose-Einstein (BE) statistics. We demonstrate that 98.7% of the unprocessed random bit stream min-entropy is traceable to the BE (quantum) signal. The classical component is then removed using a non-reuse shift-XOR protocol, and the final random numbers are generated at a 200 Mbps rate and shown to pass the statistical randomness test suites FIPS 140-2, Alphabit, SmallCrush, DIEHARD, and Rabbit of the TestU01 library.

Structural and functional characterization of the severe fever with thrombocytopenia syndrome virus L protein.

The Bunyavirales order contains several emerging viruses with high epidemic potential, including Severe fever with thrombocytopenia syndrome virus (SFTSV). The lack of medical countermeasures, such as vaccines and antivirals, is a limiting factor for the containment of any virus outbreak. To develop such antivirals a profound understanding of the viral replication process is essential. The L protein of bunyaviruses is a multi-functional and multi-domain protein performing both virus transcription and genome replication and, therefore, is an ideal drug target. We established expression and purification procedures for the full-length L protein of SFTSV. By combining single-particle electron cryo-microscopy and X-ray crystallography, we obtained 3D models covering ∼70% of the SFTSV L protein in the apo-conformation including the polymerase core region, the endonuclease and the cap-binding domain. We compared this first L structure of the Phenuiviridae family to the structures of La Crosse peribunyavirus L protein and influenza orthomyxovirus polymerase. Together with a comprehensive biochemical characterization of the distinct functions of SFTSV L protein, this work provides a solid framework for future structural and functional studies of L protein-RNA interactions and the development of antiviral strategies against this group of emerging human pathogens.

Structure and function of the Toscana virus cap-snatching endonuclease.

Toscana virus (TOSV) is an arthropod-borne human pathogen responsible for seasonal outbreaks of fever and meningoencephalitis in the Mediterranean basin. TOSV is a segmented negative-strand RNA virus (sNSV) that belongs to the genus phlebovirus (family Phenuiviridae, order Bunyavirales), encompassing other important human pathogens such as Rift Valley fever virus (RVFV). Here, we carried out a structural and functional characterization of the TOSV cap-snatching endonuclease, an N terminal domain of the viral polymerase (L protein) that provides capped 3'OH primers for transcription. We report TOSV endonuclease crystal structures in the apo form, in complex with a di-ketoacid inhibitor (DPBA) and in an intermediate state of inhibitor release, showing details on substrate binding and active site dynamics. The structure reveals substantial folding rearrangements absent in previously reported cap-snatching endonucleases. These include the relocation of the N terminus and the appearance of new structural motifs important for transcription and replication. The enzyme shows high activity rates comparable to other His+ cap-snatching endonucleases. Moreover, the activity is dependent on conserved residues involved in metal ion and substrate binding. Altogether, these results bring new light on the structure and function of cap-snatching endonucleases and pave the way for the development of specific and broad-spectrum antivirals.

In situ product recovery of single-chain antibodies in a membrane bioreactor.

The demand for biopharmaceuticals, such as monoclonal antibodies, has risen significantly over the last years. To be competitive, continuous production processes that yield consistent product quality and an economic advantage are desirable. In this study, an in situ product recovery process is described, involving use of submerged membranes to recover single-chain antibodies from a continuous fermentation of Hansenula polymorpha yeast cells.Reverse-flow diafiltration (RFD) was applied to prevent cake layer formation. Optimal flux ranges for this process could be identified by a systematic flux step method. The RFD process was optimized, preventing mixing of permeate and unreacted substrate: the space-time yield of antibodies using RFD could be tripled. Increase of the fouling related transmembrane pressure was below 45 Pa min(-1) for all applied dilution rates, indicating that the filtration process was stable. The membrane as well as the feeding mode of RFD did not influence cell viability nor product concentration. A wide range of dilution rates was successfully tested, demonstrating that this process is suitable for industrial applications.

A Comparative Assessment of the Pathogenic Potential of Newly Discovered Henipaviruses.

Recent advances in high-throughput sequencing technologies have led to the discovery of a plethora of previously unknown viruses in animal samples. Some of these newly detected viruses are closely related to human pathogens. A prime example are the henipaviruses. Both Nipah (NiV) and Hendra virus (HeV) cause severe disease in humans. Henipaviruses are of zoonotic origin, and animal hosts, including intermediate hosts, play a critical role in viral transmission to humans. The natural reservoir hosts of NiV and HeV seem to be restricted to a few fruit bat species of the Pteropus genus in distinct geographic areas. However, the recent discovery of novel henipa- and henipa-like viruses suggests that these viruses are far more widespread than was originally thought. To date, these new viruses have been found in a wide range of animal hosts, including bats, shrews, and rodents in Asia, Africa, Europe, and South America. Since these viruses are closely related to human pathogens, it is important to learn whether they pose a threat to human health. In this article, we summarize what is known about the newly discovered henipaviruses, highlight differences to NiV and HeV, and discuss their pathogenic potential.

Correction: Meier et al. Hantavirus Replication Cycle-An Updated Structural Virology Perspective. Viruses 2021, 13, 1561.

There was an error in the original publication [...].

Hantavirus Replication Cycle-An Updated Structural Virology Perspective.

Hantaviruses infect a wide range of hosts including insectivores and rodents and can also cause zoonotic infections in humans, which can lead to severe disease with possible fatal outcomes. Hantavirus outbreaks are usually linked to the population dynamics of the host animals and their habitats being in close proximity to humans, which is becoming increasingly important in a globalized world. Currently there is neither an approved vaccine nor a specific and effective antiviral treatment available for use in humans. Hantaviruses belong to the order Bunyavirales with a tri-segmented negative-sense RNA genome. They encode only five viral proteins and replicate and transcribe their genome in the cytoplasm of infected cells. However, many details of the viral amplification cycle are still unknown. In recent years, structural biology methods such as cryo-electron tomography, cryo-electron microscopy, and crystallography have contributed essentially to our understanding of virus entry by membrane fusion as well as genome encapsidation by the nucleoprotein. In this review, we provide an update on the hantavirus replication cycle with a special focus on structural virology aspects.

Equine MX2 is a restriction factor of equine infectious anemia virus (EIAV).

Human myxovirus resistance protein B (hMXB) is a restriction factor of HIV-1 that also inhibits a variety of retroviruses. However, hMXB is not antiviral against equine infectious anemia virus (EIAV). We show here that equine MX2 (eMX2) potently restricts EIAV in vitro. Additionally, eMX2 inhibits HIV-1 and other lentiviruses, including murine leukemia virus. Previously, it was reported that hMXB repression is reduced in hMXB Δ1-25, but not in GTP-binding mutant K131A and GTP-hydrolysis mutant T151A. In contrast to this phenomenon, our study indicates that eMX2 restriction is not diminished in eMX2 Δ1-25, but is in eMX2 K127A and T147A, which correspond to hMXB K131A and T151A, respectively. Thus, eMX2 may inhibit retroviral replication by a novel mechanism that differs from that of hMXB.

Newly designed and validated impedance spectroscopy setup in microtiter plates successfully monitors viable biomass online.

In microtiter plates, conventional online monitoring of biomass concentration based on optical measurements is limited to transparent media: It also cannot differentiate between dead or viable biomass or suspended particles. To address this limitation, this study introduces and validates a new online monitoring setup based on impedance spectroscopy for detecting only viable biomass in 48- and 96-well microtiter plates. The setup was first validated electronically and characterized by determining the cell constants of the measuring geometry. Defined cell suspensions of Ustilago maydis, Hansenula polymorpha, Escherichia coli and Bacillus licheniformis were characterized to find, among other parameters, the most suitable frequency range and the characteristic frequency of ß-dispersion for each organism. Finally, the setup was exemplarily applied to monitor the growth of Hansenula polymorpha online. As reference, three different parallel cultures were performed in established cultivation systems. This new online monitoring setup based on impedance spectroscopy is robust and enables precise measurements of microbial biomass concentration. It is promising for future high-throughput applications.

Improvement and scale-down of a Trichoderma reesei shake flask protocol to microtiter plates enables high-throughput screening.

Nowadays, high-throughput screening is essential for determining the best microbial strains and fermentation conditions. Although microtiter plates allow higher throughput in screening than shake flasks, they do not guarantee sufficient oxygen supply if operated at unsuitable conditions. This is especially the case in viscous fermentations, potentially leading to poor liquid movement and surface growth. Therefore, in this study, two aims were pursued. First, an industrial Trichoderma reesei shake flask protocol is improved with respect to oxygen supply and production. Second, this improved shake flask protocol is scaled down into microtiter plate under consideration of similar oxygen supply. For this purpose, the respiration activity monitoring system (RAMOS) was applied. An approach based on a sulfite system was introduced to ensure equal maximum oxygen transfer capacities (OTRmax) in microtiter plates and shake flasks. OTRmax-values of 250 mL shake flasks and 24-well microtiter plates were determined in a wide range of operating conditions. These sulfite datasets were used to identify operating conditions leading to the same oxygen supply for T. reesei in shake flasks and 24-well microtiter plates. For 24-well microtiter plates, the shake flask OTRmax of 20 mmol/L/h of an industrial protocol was obtained under the following optimal operating conditions: 1 mL filling volume per well, 200 rpm shaking frequency and 50 mm shaking diameter. With these conditions almost identical oxygen transfer rates and product concentrations were measured in both scales. The proposed approach is a fast and accurate means to scale-down established screening procedures into microtiter plates to achieve high-throughput.

In situ cell retention of a CHO culture by a reverse-flow diafiltration membrane bioreactor.

Heterogeneities occur in various bioreactor designs including cell retention devices. Whereas in external devices changing environmental conditions cannot be prevented, cells are retained in their optimal environment in internal devices. Conventional reverse-flow diafiltration utilizes an internal membrane device, but pulsed feeding causes temporal heterogeneities. In this study, the influence of conventional reverse-flow diafiltration on the yeast Hansenula polymorpha is investigated. Alternating 180 s of feeding with 360 s of non-feeding at a dilution rate of 0.2 h(-1) results in an oscillating DOT signal with an amplitude of 60%. Thereby, induced short-term oxygen limitations result in the formation of ethanol and a reduced product concentration of 25%. This effect is enforced at increased dilution rate. To overcome this cyclic problem, sequential operation of three membranes is introduced. Thus, quasi-continuous feeding is achieved reducing the oscillation of the DOT signal to an amplitude of 20% and 40% for a dilution rate of 0.2 h(-1) and 0.5 h(-1) , respectively. Fermentation conditions characterized by complete absence of oxygen limitation and without formation of overflow metabolites could be obtained for dilution rates from 0.1 h(-1) - 0.5 h(-1) . Thus, sequential operation of three membranes minimizes oscillations in the DOT signal providing a nearly homogenous culture over time.