Phenotypic lentivirus screens to identify functional single domain antibodies

Manipulation of proteins is key in assessing their in vivo function. Although genetic ablation is straightforward, reversible and specific perturbation of protein function remains a challenge. Single domain antibody fragments, such as camelid-derived VHHs, can serve as inhibitors or activators of intracellular protein function, but functional testing of identified VHHs is laborious. To address this challenge, we have developed a lentiviral screening approach to identify VHHs that elicit a phenotype when expressed intracellularly. We identified 19 antiviral VHHs that protect human A549 cells from lethal infection with influenza A virus (IAV) or vesicular stomatitis virus (VSV), respectively. Both negative-sense RNA viruses are vulnerable to VHHs uniquely specific for their respective nucleoproteins. Antiviral VHHs prevented nuclear import of viral ribonucleoproteins or mRNA transcription, respectively, and may provide clues for novel antiviral reagents. In principle, the screening approach described here should be applicable to identify inhibitors of any pathogen or biological pathway. To identify the proteins essential to a biological pathway, small-molecule inhibitors or activators may be used to manipulate protein function transiently. Alternatively, screens involving mutagenesis, a reduction in levels or complete elimination of gene products are common. As applied to mammalian cells, these methods usually seek to achieve the removal of a protein from its normal biological context. Many proteins are multifunctional, or are components of multi-subunit complexes. Depletion of any single component may cause unexpected phenotypes due to the collapse of entire protein complexes. Small-molecule inhibitors often lack specificity and at best can target a fraction of all the proteins of interest. The screening of chemically diverse libraries must be paired with sophisticated methods to identify the molecular targets of any hit identified. Antibodies have been used as intracellular perturbants of protein function after microinjection or cytosolic expression of single-chain variable antibody fragments, but technical challenges have limited their application to few selected cases. In addition to conventional antibodies, the immune system of camelids generates heavy-chain-only antibodies. Their antigen binding site only consists of the variable domain of the heavy chain. This domain can be expressed on its own and is referred to as a VHH or nanobody, an entity that can retain its function in the reducing environment of the cytosol, independent of glycosylation. Many VHHs bind to their targets with affinities comparable to conventional antibodies. VHHs expressed in the cytosol can therefore act as molecular perturbants by occluding the interfaces involved in protein–protein interactions, by binding in the active sites of enzymes, or through the recognition or stabilization of distinct conformations of their targets. Both phage and yeast display, as well as mass spectrometry in combination with high-throughput sequencing, allow the identification of VHHs based on their binding properties. However, the identification of inhibitory VHHs remains a time-consuming process. VHHs obtained through biochemical screening methods must be expressed individually in the relevant cell type to test for the functional consequences of VHH expression. To address this challenge, we developed a phenotypic VHH screening method in living cells.National Institutes of Health (U.S.) (Health Pioneer Award

Niemann-Pick C1 (NPC1)/NPC1-like1 Chimeras Define Sequences Critical for NPC1’s Function as a Filovirus Entry Receptor

We recently demonstrated that Niemann-Pick C1 (NPC1), a ubiquitous 13-pass cellular membrane protein involved in lysosomal cholesterol transport, is a critical entry receptor for filoviruses. Here we show that Niemann-Pick C1-like1 (NPC1L1), an NPC1 paralog and hepatitis C virus entry factor, lacks filovirus receptor activity. We exploited the structural similarity between NPC1 and NPC1L1 to construct and analyze a panel of chimeras in which NPC1L1 sequences were replaced with cognate sequences from NPC1. Only one chimera, NPC1L1 containing the second luminal domain (C) of NPC1 in place of its own, bound to the viral glycoprotein, GP. This engineered protein mediated authentic filovirus infection nearly as well as wild-type NPC1, and more efficiently than did a minimal NPC1 domain C-based receptor recently described by us. A reciprocal chimera, NPC1 containing NPC1L1’s domain C, was completely inactive. Remarkably, an intra-domain NPC1L1-NPC1 chimera bearing only a ~130-amino acid N–terminal region of NPC1 domain C could confer substantial viral receptor activity on NPC1L1. Taken together, these findings account for the failure of NPC1L1 to serve as a filovirus receptor, highlight the central role of the luminal domain C of NPC1 in filovirus entry, and reveal the direct involvement of N–terminal domain C sequences in NPC1’s function as a filovirus receptor

SARS-CoV-2 requires acidic pH to infect cells

Publisher Copyright: Copyright © 2022 the Author(s). Published by PNAS.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cell entry starts with membrane attachment and ends with spike (S) protein–catalyzed membrane fusion depending on two cleavage steps, namely, one usually by furin in producing cells and the second by TMPRSS2 on target cells. Endosomal cathepsins can carry out both. Using real-time three-dimensional single-virion tracking, we show that fusion and genome penetration require virion exposure to an acidic milieu of pH 6.2 to 6.8, even when furin and TMPRSS2 cleavages have occurred. We detect the sequential steps of S1-fragment dissociation, fusion, and content release from the cell surface in TMPRRS2-overexpressing cells only when exposed to acidic pH. We define a key role of an acidic environment for successful infection, found in endosomal compartments and at the surface of TMPRSS2-expressing cells in the acidic milieu of the nasal cavity.Peer reviewe

Bunyavirus requirement for endosomal K+ reveals new roles of cellular ion channels during infection

In order to multiply and cause disease a virus must transport its genome from outside the cell into the cytosol, most commonly achieved through the endocytic network. Endosomes transport virus particles to specific cellular destinations and viruses exploit the changing environment of maturing endocytic vesicles as triggers to mediate genome release. Previously we demonstrated that several bunyaviruses, which comprise the largest family of negative sense RNA viruses, require the activity of cellular potassium (K+) channels to cause productive infection. Specifically, we demonstrated a surprising role for K+ channels during virus endosomal trafficking. In this study, we have used the prototype bunyavirus, Bunyamwera virus (BUNV), as a tool to understand why K+ channels are required for progression of these viruses through the endocytic network. We report three major findings: First, the production of a dual fluorescently labelled bunyavirus to visualize virus trafficking in live cells. Second, we show that BUNV traffics through endosomes containing high [K+] and that these K+ ions influence the infectivity of virions. Third, we show that K+ channel inhibition can alter the distribution of K+ across the endosomal system and arrest virus trafficking in endosomes. These data suggest high endosomal [K+] is a critical cue that is required for virus infection, and is controlled by cellular K+ channels resident within the endosome network. This highlights cellular K+ channels as druggable targets to impede virus entry, infection and disease

Systematic analysis of SARS-CoV-2 infection of an ACE2-negative human airway cell

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) variants govern transmissibility, responsiveness to vaccination, and disease severity. In a screen for new models of SARS-CoV-2 infection, we identify human H522 lung adenocarcinoma cells as naturally permissive to SARS-CoV-2 infection despite complete absence of angiotensin-converting enzyme 2 (ACE2) expression. Remarkably, H522 infection requires the E484D S variant; viruses expressing wild-type S are not infectious. Anti-S monoclonal antibodies differentially neutralize SARS-CoV-2 E484D S in H522 cells as compared to ACE2-expressing cells. Sera from vaccinated individuals block this alternative entry mechanism, whereas convalescent sera are less effective. Although the H522 receptor remains unknown, depletion of surface heparan sulfates block H522 infection. Temporally resolved transcriptomic and proteomic profiling reveal alterations in cell cycle and the antiviral host cell response, including MDA5-dependent activation of type I interferon signaling. These findings establish an alternative SARS-CoV-2 host cell receptor for the E484D SARS-CoV-2 variant, which may impact tropism of SARS-CoV-2 and consequently human disease pathogenesis

Isolation of reconstructed functional ribonucleoprotein complexes of Machupo virus

Arenaviruses initiate infection by delivering a transcriptionally competent ribonucleoprotein (RNP) complex into the cytosol of host cells. The arenavirus RNP consists of the large (L) RNA-dependent RNA polymerase (RdRP) bound to a nucleoprotein (NP)-encapsidated genomic RNA (viral RNA [vRNA]) template. During transcription and replication, L must transiently displace RNA-bound NP to allow for template access into the RdRP active site. Concomitant with RNA replication, new subunits of NP must be added to the nascent complementary RNAs (cRNA) as they emerge from the product exit channel of L. Interactions between L and NP thus play a central role in arenavirus gene expression. We developed an approach to purify recombinant functional RNPs from mammalian cells in culture using a synthetic vRNA and affinity-tagged L and NP. Negative-stain electron microscopy of purified RNPs revealed they adopt diverse and flexible structures, like RNPs of othe

Structure of the Vesicular Stomatitis Virus L Protein in Complex with Its Phosphoprotein Cofactor

The large (L) proteins of non-segmented, negative-strand RNA viruses are multifunctional enzymes that produce capped, methylated, and polyadenylated mRNA and replicate the viral genome. A phosphoprotein (P), required for efficient RNA-dependent RNA polymerization from the viral ribonucleoprotein (RNP) template, regulates the function and conformation of the L protein. We report the structure of vesicular stomatitis virus L in complex with its P cofactor determined by electron cryomicroscopy at 3.0 A resolution, enabling us to visualize bound segments of P. The contacts of three P segments with multiple L domains show how P induces a closed, compact, initiation-competent conformation. Binding of P to L positions its N-terminal domain adjacent to a putative RNA exit channel for efficient encapsidation of newly synthesized genomes with the nucleoprotein and orients its C-terminal domain to interact with an RNP template. The model shows that a conserved tryptophan in the priming loop can support the initiating 5\u27 nucleotide

Repeatable population dynamics among vesicular stomatitis virus lineages evolved under high co-infection

Parasites and hosts can experience oscillatory cycles, where the densities of these interacting species dynamically fluctuate through time. Viruses with different replication strategies can also interact to produce cyclical dynamics. Frequent cellular co-infection can select for defective-interfering particles (DIPs): cheater viruses with shortened genomes that interfere with intracellular replication of full-length (ordinary) viruses. DIPs are positively selected when rare because they out-replicate ordinary viruses during co-infection, but DIPs are negatively selected when common because ordinary viruses become unavailable for intracellular exploitation via cheating. Here we tested whether oscillatory dynamics of ordinary viruses were similar across independently evolved populations of vesicular stomatitis virus (VSV). Results showed identical cyclical dynamics across populations in the first 10 experimental passages, which transitioned to repeatable dampened oscillations by passage 20. Genomic analyses revealed parallel molecular substitutions across populations, particularly novel mutations that became dominant by passage 10. Our study showed that oscillatory dynamics and molecular evolution of interacting viruses were highly repeatable in VSV populations passaged under frequent co-infection. Furthermore, our data suggested that frequent co-infection with DIPs caused lowered performance of full-length viruses, by reducing their population densities by orders of magnitude compared to reproduction of ordinary viruses during strictly clonal infections