De novo-designed transmembrane proteins bind and regulate a cytokine receptor.

Transmembrane (TM) domains as simple as a single span can perform complex biological functions using entirely lipid-embedded chemical features. Computational design has the potential to generate custom tool molecules directly targeting membrane proteins at their functional TM regions. Thus far, designed TM domain-targeting agents have been limited to mimicking the binding modes and motifs of natural TM interaction partners. Here, we demonstrate the design of de novo TM proteins targeting the erythropoietin receptor (EpoR) TM domain in a custom binding topology competitive with receptor homodimerization. The TM proteins expressed in mammalian cells complex with EpoR and inhibit erythropoietin-induced cell proliferation. In vitro, the synthetic TM domain complex outcompetes EpoR homodimerization. Structural characterization reveals that the complex involves the intended amino acids and agrees with our designed molecular model of antiparallel TM helices at 1:1 stoichiometry. Thus, membrane protein TM regions can now be targeted in custom-designed topologies.

Virology-the path forward.

In the United States (US), biosafety and biosecurity oversight of research on viruses is being reappraised. Safety in virology research is paramount and oversight frameworks should be reviewed periodically. Changes should be made with care, however, to avoid impeding science that is essential for rapidly reducing and responding to pandemic threats as well as addressing more common challenges caused by infectious diseases. Decades of research uniquely positioned the US to be able to respond to the COVID-19 crisis with astounding speed, delivering life-saving vaccines within a year of identifying the virus. We should embolden and empower this strength, which is a vital part of protecting the health, economy, and security of US citizens. Herein, we offer our perspectives on priorities for revised rules governing virology research in the US.

Allosteric inhibition of the T cell receptor by a designed membrane ligand.

The T cell receptor (TCR) is a complex molecular machine that directs the activation of T cells, allowing the immune system to fight pathogens and cancer cells. Despite decades of investigation, the molecular mechanism of TCR activation is still controversial. One of the leading activation hypotheses is the allosteric model. This model posits that binding of pMHC at the extracellular domain triggers a dynamic change in the transmembrane (TM) domain of the TCR subunits, which leads to signaling at the cytoplasmic side. We sought to test this hypothesis by creating a TM ligand for TCR. Previously we described a method to create a soluble peptide capable of inserting into membranes and binding to the TM domain of the receptor tyrosine kinase EphA2 (Alves et al., eLife, 2018). Here, we show that the approach is generalizable to complex membrane receptors, by designing a TM ligand for TCR. We observed that the designed peptide caused a reduction of Lck phosphorylation of TCR at the CD3ζ subunit in T cells. As a result, in the presence of this peptide inhibitor of TCR (PITCR), the proximal signaling cascade downstream of TCR activation was significantly dampened. Co-localization and co-immunoprecipitation in diisobutylene maleic acid (DIBMA) native nanodiscs confirmed that PITCR was able to bind to the TCR. AlphaFold-Multimer predicted that PITCR binds to the TM region of TCR, where it interacts with the two CD3ζ subunits. Our results additionally indicate that PITCR disrupts the allosteric changes in the compactness of the TM bundle that occur upon TCR activation, lending support to the allosteric TCR activation model. The TCR inhibition achieved by PITCR might be useful to treat inflammatory and autoimmune diseases and to prevent organ transplant rejection, as in these conditions aberrant activation of TCR contributes to disease.

Sequence-independent activity of a predicted long disordered segment of the human papillomavirus type 16 L2 capsid protein during virus entry.

The activity of proteins is thought to be invariably determined by their amino acid sequence or composition, but we show that a long segment of a viral protein can support infection independent of its sequence or composition. During virus entry, the papillomavirus L2 capsid protein protrudes through the endosome membrane into the cytoplasm to bind cellular factors such as retromer required for intracellular virus trafficking. Here, we show that an ~110 amino acid segment of L2 is predicted to be disordered and that large deletions in this segment abolish infectivity of HPV16 pseudoviruses by inhibiting cytoplasmic protrusion of L2, association with retromer, and proper virus trafficking. The activity of these mutants can be restored by insertion of protein segments with diverse sequences, compositions, and chemical properties, including scrambled amino acid sequences, a tandem array of a short sequence, and the intrinsically disordered region of an unrelated cellular protein. The infectivity of mutants with small in-frame deletions in this segment directly correlates with the size of the segment. These results indicate that the length of the disordered segment, not its sequence or composition, determines its activity during HPV16 pseudovirus infection. We propose that a minimal length of L2 is required for it to protrude far enough into the cytoplasm to bind cytoplasmic trafficking factors, but the sequence of this segment is largely irrelevant. Thus, protein segments can carry out complex biological functions such as Human papillomavirus pseudovirus infection in a sequence-independent manner. This finding has important implications for protein function and evolution.

Noncanonical Rab9a action supports retromer-mediated endosomal exit of human papillomavirus during virus entry.

Rab GTPases play key roles in controlling intracellular vesicular transport. GTP-bound Rab proteins support vesicle trafficking. Here, we report that, unlike cellular protein cargos, retromer-mediated delivery of human papillomaviruses (HPV) into the retrograde transport pathway during virus entry is inhibited by Rab9a in its GTP-bound form. Knockdown of Rab9a inhibits HPV entry by modulating the HPV-retromer interaction and impairing retromer-mediated endosome-to-Golgi transport of the incoming virus, resulting in the accumulation of HPV in the endosome. Rab9a is in proximity to HPV as early as 3.5 h post-infection, prior to the Rab7-HPV interaction, and HPV displays increased association with retromer in Rab9a knockdown cells, even in the presence of dominant negative Rab7. Thus, Rab9a can regulate HPV-retromer association independently of Rab7. Surprisingly, excess GTP-Rab9a impairs HPV entry, whereas excess GDP-Rab9a reduces association between L2 and Rab9a and stimulates entry. These findings reveal that HPV and cellular proteins utilize the Rab9a host trafficking machinery in distinct ways during intracellular trafficking.

Noncanonical Rab9a action supports endosomal exit of human papillomavirus during virus entry.

Rab GTPases play key roles in controlling intracellular vesicular transport. GTP-bound Rab proteins support vesicle trafficking. Here, we report that, unlike cellular protein cargos, the delivery of human papillomaviruses (HPV) into the retrograde transport pathway during virus entry is inhibited by Rab9a in its GTP-bound form. Knockdown of Rab9a hampers HPV entry by regulating the HPV-retromer interaction and impairing retromer-mediated endosome-to-Golgi transport of the incoming virus, resulting in the accumulation of HPV in the endosome. Rab9a is in proximity to HPV as early as 3.5 h post-infection, prior to the Rab7-HPV interaction. HPV displays increased association with retromer in Rab9a knockdown cells, even in the presence of dominant negative Rab7. Thus, Rab9a can regulate HPV-retromer association independently of Rab7. Surprisingly, excess GTP-Rab9a impairs HPV entry, whereas excess GDP-Rab9a stimulates entry. These findings reveal that HPV employs a trafficking mechanism distinct from that used by cellular proteins.

Sequence independent activity of a predicted long disordered segment of the human papillomavirus L2 capsid protein during virus entry.

The papillomavirus L2 capsid protein protrudes through the endosome membrane into the cytoplasm during virus entry to bind cellular factors required for intracellular virus trafficking. Cytoplasmic protrusion of HPV16 L2, virus trafficking, and infectivity are inhibited by large deletions in an ∻110 amino acid segment of L2 that is predicted to be disordered. The activity of these mutants can be restored by inserting protein segments with diverse compositions and chemical properties into this region, including scrambled sequences, a tandem array of a short sequence, and the intrinsically disordered region of a cellular protein. The infectivity of mutants with small in-frame insertions and deletions in this segment directly correlates with the size of the segment. These results indicate that the length of the disordered segment, not its sequence or its composition, determines its activity during virus entry. Sequence independent but length dependent activity has important implications for protein function and evolution.

Designed Transmembrane Proteins Inhibit the Erythropoietin Receptor in a Custom Binding Topology.

Transmembrane (TM) domains as simple as a single span can perform complex biological functions using entirely lipid-embedded chemical features. Computational design has potential to generate custom tool molecules directly targeting membrane proteins at their functional TM regions. Thus far, designed TM domain-targeting agents have been limited to mimicking binding modes and motifs of natural TM interaction partners. Here, we demonstrate the design of de novo TM proteins targeting the erythropoietin receptor (EpoR) TM domain in a custom binding topology competitive with receptor homodimerization. The TM proteins expressed in mammalian cells complex with EpoR and inhibit erythropoietin-induced cell proliferation. In vitro, the synthetic TM domain complex outcompetes EpoR homodimerization. Structural characterization reveals that the complex involves the intended amino acids and agrees with our designed molecular model of antiparallel TM helices at 1:1 stoichiometry. Thus, membrane protein TM regions can now be targeted in custom designed topologies.

HPV is a cargo for the COPI sorting complex during virus entry.

During entry, human papillomavirus (HPV) traffics from the cell surface to the endosome and then to the trans-Golgi network (TGN) and Golgi apparatus. HPV must transit across the TGN/Golgi and exit these compartments to reach the nucleus to cause infection, although how these steps are accomplished is unclear. Combining cellular fractionation, unbiased proteomics, and gene knockdown strategies, we identified the coat protein complex I (COPI), a highly conserved protein complex that facilitates retrograde trafficking of cellular cargos, as a host factor required for HPV infection. Upon TGN/Golgi arrival, the cytoplasmic segment of HPV L2 binds directly to COPI. COPI depletion causes the accumulation of HPV in the TGN/Golgi, resembling the fate of a COPI binding-defective L2 mutant. We propose that the L2-COPI interaction drives HPV trafficking through the TGN and Golgi stacks during virus entry. This shows that an incoming virus is a cargo of the COPI complex.

Virology under the Microscope-a Call for Rational Discourse.

Viruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals. Despite this long history, the COVID-19 pandemic has brought unprecedented attention to the field of virology. Some of this attention is focused on concern about the safe conduct of research with human pathogens. A small but vocal group of individuals has seized upon these concerns - conflating legitimate questions about safely conducting virus-related research with uncertainties over the origins of SARS-CoV-2. The result has fueled public confusion and, in many instances, ill-informed condemnation of virology. With this article, we seek to promote a return to rational discourse. We explain the use of gain-of-function approaches in science, discuss the possible origins of SARS-CoV-2 and outline current regulatory structures that provide oversight for virological research in the United States. By offering our expertise, we - a broad group of working virologists - seek to aid policy makers in navigating these controversial issues. Balanced, evidence-based discourse is essential to addressing public concern while maintaining and expanding much-needed research in virology.

Virology under the Microscope-a Call for Rational Discourse.

Viruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals. Despite this long history, the COVID-19 pandemic has brought unprecedented attention to the field of virology. Some of this attention is focused on concern about the safe conduct of research with human pathogens. A small but vocal group of individuals has seized upon these concerns - conflating legitimate questions about safely conducting virus-related research with uncertainties over the origins of SARS-CoV-2. The result has fueled public confusion and, in many instances, ill-informed condemnation of virology. With this article, we seek to promote a return to rational discourse. We explain the use of gain-of-function approaches in science, discuss the possible origins of SARS-CoV-2 and outline current regulatory structures that provide oversight for virological research in the United States. By offering our expertise, we - a broad group of working virologists - seek to aid policy makers in navigating these controversial issues. Balanced, evidence-based discourse is essential to addressing public concern while maintaining and expanding much-needed research in virology.

Virology under the Microscope-a Call for Rational Discourse.

Viruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals. Despite this long history, the COVID-19 pandemic has brought unprecedented attention to the field of virology. Some of this attention is focused on concern about the safe conduct of research with human pathogens. A small but vocal group of individuals has seized upon these concerns - conflating legitimate questions about safely conducting virus-related research with uncertainties over the origins of SARS-CoV-2. The result has fueled public confusion and, in many instances, ill-informed condemnation of virology. With this article, we seek to promote a return to rational discourse. We explain the use of gain-of-function approaches in science, discuss the possible origins of SARS-CoV-2 and outline current regulatory structures that provide oversight for virological research in the United States. By offering our expertise, we - a broad group of working virologists - seek to aid policy makers in navigating these controversial issues. Balanced, evidence-based discourse is essential to addressing public concern while maintaining and expanding much-needed research in virology.

Human Papillomavirus L2 Capsid Protein Stabilizes γ-Secretase during Viral Infection.

Intracellular trafficking of human papillomavirus (HPV) during virus entry requires γ-secretase, a cellular protease consisting of a complex of four cellular transmembrane (TM) proteins. γ-secretase typically cleaves substrate proteins but it plays a non-canonical role during HPV entry. γ-secretase binds to the HPV minor capsid protein L2 and facilitates its insertion into the endosomal membrane. After insertion, L2 protrudes into the cytoplasm, which allows HPV to bind other cellular factors required for proper virus trafficking into the retrograde transport pathway. Here, we further characterize the interaction between γ-secretase and HPV L2. We show that γ-secretase is required for cytoplasmic protrusion of L2 and that L2 associates strongly with the PS1 catalytic subunit of γ-secretase and stabilizes the γ-secretase complex. Mutational studies revealed that a putative TM domain in HPV16 L2 cannot be replaced by a foreign TM domain, that infectivity of HPV TM mutants is tightly correlated with γ-secretase binding and stabilization, and that the L2 TM domain is required for protrusion of the L2 protein into the cytoplasm. These results provide new insight into the interaction between γ-secretase and L2 and highlight the importance of the native HPV L2 TM domain for proper virus trafficking during entry.

Traptamer screening: a new functional genomics approach to study virus entry and other cellular processes.

Historically, the genetic analysis of mammalian cells entailed the isolation of randomly arising mutant cell lines with altered properties, followed by laborious genetic mapping experiments to identify the mutant gene responsible for the phenotype. In recent years, somatic cell genetics has been revolutionized by functional genomics screens, in which expression of every protein-coding gene is systematically perturbed, and the phenotype of the perturbed cells is determined. We outline here a novel functional genomics screening strategy that differs fundamentally from commonly used approaches. In this strategy, we express libraries of artificial transmembrane proteins named traptamers and select rare cells with the desired phenotype because, by chance, a traptamer specifically perturbs the expression or activity of a target protein. Active traptamers are then recovered from the selected cells and can be used as tools to dissect the biological process under study. We also briefly describe how we have used this new strategy to provide insights into the complex process by which human papillomaviruses enter cells.

Cancer Microbiology.

Microbes play important roles in cancer from direct carcinogenic effects to their use in treatment. Cancers caused by microorganisms account for approximately 15% of cancers, primarily in low- and middle-income countries. Unique features of infectious carcinogens include their transmissibility, mutability, and specific immune interactions, which provide challenges and opportunities for cancer prevention and treatment. For these agents, infection control through exposure reduction, antivirals, antibiotics, and vaccines is cancer control. In addition, developing evidence suggests that microorganisms including the human microbiome can indirectly modulate cancer formation and influence the effectiveness and toxicity of cancer treatments. Finally, microorganisms themselves can be used to prevent or treat cancer. The convergence of these factors signals the emergence of a new field, cancer microbiology. Recognition of cancer microbiology will spur research, stimulate cross-disciplinary training, inform drug development, and improve public health.