Assessing nanobody interaction with SARS-CoV-2 Nsp9.

The interaction between SARS-CoV-2 non-structural protein Nsp9 and the nanobody 2NSP90 was investigated by NMR spectroscopy using the paramagnetic perturbation methodology PENELOP (Paramagnetic Equilibrium vs Nonequilibrium magnetization Enhancement or LOss Perturbation). The Nsp9 monomer is an essential component of the replication and transcription complex (RTC) that reproduces the viral gRNA for subsequent propagation. Therefore preventing Nsp9 recruitment in RTC would represent an efficient antiviral strategy that could be applied to different coronaviruses, given the Nsp9 relative invariance. The NMR results were consistent with a previous characterization suggesting a 4:4 Nsp9-to-nanobody stoichiometry with the occurrence of two epitope pairs on each of the Nsp9 units that establish the inter-dimer contacts of Nsp9 tetramer. The oligomerization state of Nsp9 was also analyzed by molecular dynamics simulations and both dimers and tetramers resulted plausible. A different distribution of the mapped epitopes on the tetramer surface with respect to the former 4:4 complex could also be possible, as well as different stoichiometries of the Nsp9-nanobody assemblies such as the 2:2 stoichiometry suggested by the recent crystal structure of the Nsp9 complex with 2NSP23 (PDB ID: 8dqu), a nanobody exhibiting essentially the same affinity as 2NSP90. The experimental NMR evidence, however, ruled out the occurrence in liquid state of the relevant Nsp9 conformational change observed in the same crystal structure.

Exposure of volunteers to microgravity by dry immersion bed over 21 days results in gene expression changes and adaptation of T cells.

The next steps of deep space exploration are manned missions to Moon and Mars. For safe space missions for crew members, it is important to understand the impact of space flight on the immune system. We studied the effects of 21 days dry immersion (DI) exposure on the transcriptomes of T cells isolated from blood samples of eight healthy volunteers. Samples were collected 7 days before DI, at day 7, 14, and 21 during DI, and 7 days after DI. RNA sequencing of CD3+ T cells revealed transcriptional alterations across all time points, with most changes occurring 14 days after DI exposure. At day 21, T cells showed evidence of adaptation with a transcriptional profile resembling that of 7 days before DI. At 7 days after DI, T cells again changed their transcriptional profile. These data suggest that T cells adapt by rewiring their transcriptomes in response to simulated weightlessness and that remodeling cues persist when reexposed to normal gravity.

Unconventional stabilization of the human T-cell leukemia virus type 1 immature Gag lattice.

Human T-cell leukemia virus type 1 (HTLV-1) has an atypical immature particle morphology compared to other retroviruses. This indicates that these particles are formed in a way that is unique. Here we report the results of cryo-electron tomography (cryo-ET) studies of HTLV-1 virus-like particles (VLPs) assembled in vitro, as well as derived from cells. This work shows that HTLV-1 employs an unconventional mechanism of Gag-Gag interactions to form the immature viral lattice. Analysis of high-resolution structural information from immature CA tubular arrays reveals that the primary stabilizing component in HTLV-1 is CA-NTD. Mutagenesis and biophysical analysis support this observation. This distinguishes HTLV-1 from other retroviruses, in which the stabilization is provided primarily by the CA-CTD. These results are the first to provide structural details of the quaternary arrangement of Gag for an immature deltaretrovirus, and this helps explain why HTLV-1 particles are morphologically distinct.

Approaching Protein Aggregation and Structural Dynamics by Equilibrium and Nonequilibrium Paramagnetic Perturbation.

PENELOP (Paramagnetic Equilibrium vs Nonequilibrium magnetization Enhancement or LOss Perturbation) is the presented nuclear magnetic resonance (NMR) approach to identify at once the location of proteins' exposed surface, hindered accessibility, and exchange processes occurring on a µs-ms time scale. In addition to mapping the protein surface accessibility, the application of this method under specific conditions makes it possible to distinguish conformational mobility and chemical exchange processes, thereby providing an alternative to characterization by more demanding techniques (transverse relaxation dispersion, saturation transfer, and high-pressure NMR). Moreover, its high sensitivity enables studying samples at low, physiologically more relevant concentrations. Association, dynamics, and oligomerization are addressed by PENELOP for a component of SARS-CoV-2 replication transcription complex and an amyloidogenic protein.

NMR-Based Analysis of Nanobodies to SARS-CoV-2 Nsp9 Reveals a Possible Antiviral Strategy Against COVID-19.

Following the entry into the host cell, SARS-CoV-2 replication is mediated by the replication transcription complex (RTC) assembled through a number of nonstructural proteins (Nsps). A monomeric form of Nsp9 is particularly important for RTC assembly and function. In the present study, 136 unique nanobodies targeting Nsp9 are generated. Several nanobodies belonging to different B-cell lineages are expressed, purified, and characterized. Results from immunoassays applied to purified Nsp9 and neat saliva from coronavirus disease (COVID-19) patients show that these nanobodies effectively and specifically recognize both recombinant and endogenous Nsp9. Nuclear magnetic resonance analyses supported by molecular dynamics reveal a composite Nsp9 oligomerization pattern and demonstrate that both nanobodies stabilize the tetrameric form of wild-type Nsp9 also identifying the epitopes on the tetrameric assembly. These results can have important implications in the potential use of these nanobodies to combat viral replication.

Structure of Nanobody Nb23.

BACKGROUND: Nanobodies, or VHHs, are derived from heavy chain-only antibodies (hcAbs) found in camelids. They overcome some of the inherent limitations of monoclonal antibodies (mAbs) and derivatives thereof, due to their smaller molecular size and higher stability, and thus present an alternative to mAbs for therapeutic use. Two nanobodies, Nb23 and Nb24, have been shown to similarly inhibit the self-aggregation of very amyloidogenic variants of ß2-microglobulin. Here, the structure of Nb23 was modeled with the Chemical-Shift (CS)-Rosetta server using chemical shift assignments from nuclear magnetic resonance (NMR) spectroscopy experiments, and used as prior knowledge in PONDEROSA restrained modeling based on experimentally assessed internuclear distances. Further validation was comparatively obtained with the results of molecular dynamics trajectories calculated from the resulting best energy-minimized Nb23 conformers. METHODS: 2D and 3D NMR spectroscopy experiments were carried out to determine the assignment of the backbone and side chain hydrogen, nitrogen and carbon resonances to extract chemical shifts and interproton separations for restrained modeling. RESULTS: The solution structure of isolated Nb23 nanobody was determined. CONCLUSIONS: The structural analysis indicated that isolated Nb23 has a dynamic CDR3 loop distributed over different orientations with respect to Nb24, which could determine differences in target antigen affinity or complex lability.