Yong Xiong, PhD

We strive to understand how host intrinsic antiviral proteins (restriction factors) suppress infections by viruses such as HIV and SARS-CoV-2, and how virus-encoded proteins subvert the host defense. To accomplish these goals, weapply structural biology and biochemical methods to interrogate diverse host-virus interactions, with key findings from our studies being validated in cell-based studies. We have made significant contributions to the field by furnishing structural and mechanistic results for a range of host-viral interaction systems. The results from our studies place us in an ideal position to continue elucidating the structural and biochemical principles of host restriction and viral immune evasion. We anticipate that insights stemming from our work will provide the intellectual basis for the development of new antiviral compounds and strategies. Moreover, the experimental and structural biology methods devised in our research will provide valuable new tools for the elucidation of host-viral interactions in general and of other challenging biological problems as well.

Our laboratory studies innate immune responses to viral infections. We use a variety of techniques, including X-ray crystallography, cryo-electron microscopy (cryo-EM) biochemistry, molecular biology, and computational biology. We also develop new X-ray crystallographic methods to facilitate the structural work.

Host interactions with SARS-CoV-2

Blocking virus entry

The SARS-CoV-2 virus gains cell entry through interaction between its spike glycoprotein and the human Angiotensin-converting enzyme 2 (ACE2). We aim to uncover the detailed mechanisms of ACE2-mediated viral entry, understand the processing of the viral spike glycoprotein by the cellular serine protease TMPRSS2, and identify antibodies and small molecules that disrupt the interaction between the spike protein and ACE2/TMPRSS2. We will use biochemical and structural techniques to uncover mechanistic insights of the spike protein interactions with these antibodies and small molecules, which will facilitate the design of vaccines and therapeutics to block the cell entry of coronavirus.

Nsp1-mediated reprogramming of cellular translation toward viral RNA

Nonstructural protein 1 (Nsp1) of SARS-CoV-2 plays a key role in suppressing host gene expression. We demonstrated that Nsp1 causes the most severe cytopathic effect and significantly alters multiple gene expression programs in human lung cells, physically blocks the mRNA entry channel and locks the 40S ribosomal subunit in a conformation incompatible with mRNA loading. Our goal is to establish mechanistic structural bases for how SARS-CoV-2 ensures translation of its own mRNA while global protein synthesis is shut down by Nsp1, and fully understand the cellular consequences of Nsp1 expression.

Inhibiting viral programmed ribosomal frameshifting

Programmed ribosomal frameshifting (PRF) is an indispensable feature of the SARS-CoV-2 life cycle wherein two separate sets of viral proteins are produced from the same viral mRNA. PRF is also a critical step in the life cycles of many other viruses, including HIV-1. A cellular innate immune factor called ‘Shiftless’ was recently identified to be a broad-spectrum inhibitor of programmed ribosomal frameshifting, but the structural and mechanistic details of its function are unknown. To unravel these mechanistic details, we are currently undertaking biochemical and structural studies of Shiftless with SARS-CoV-2/HIV-1 PRF signal RNAs and human 40S/60S/80S ribosomes.

Viral hijacking of host protein degradation pathways

Cellular protein degradation pathways are common targets that viruses hijack to escape host immune restrictions. Recent proteomic analyses have suggested that SARS-CoV-2 Orf10 specifically interacts with the human CRL2^ZYG11B complex, a member of the Cullin-RING E3 ubiquitin ligase family that plays a central role in proteasome-mediated degradation of cellular proteins. This interaction likely targets yet unidentified host restriction factors for degradation. We aim to identify the role of SARS-CoV-2 Orf10 during viral infection as well as molecular mechanisms by which it interacts with the human CRL2^ZYG11B complex. This information will be harnessed to screen small molecule inhibitors to disrupt the Orf10-CRL2^ZYG11B interaction, facilitating the development of novel drugs for COVID-19 therapy.

Viral hijacking of host membrane trafficking pathways

SARS-CoV-2 and other viruses are tasked with subverting or hijacking cellular membrane trafficking pathways to remove membrane associated immune molecules from the site of action. One such host defense protein, BST2, serves to restrict viruses by acting as a molecular tether between the host cell membrane and budding viral membrane; thus, progeny virions are severely impaired in their ability to travel and infect new cells. We aim to understand how SARS-CoV-2 Orf7a antagonizes the effect of BST2 tethering during infection. In addition, we are investigating the roles of Nsp10 and the spike glycoprotein in modulating clathrin-mediated vesicular trafficking pathways to ensure efficient viral replication.

Innate defenses against HIV

HIV-1 capsid pattern sensing by cellular proteins

A major focus in the lab is investigating the interactions between the HIV and numerous cellular factors through the course of early HIV infection. The HIV capsid houses the viral genome and consists of over a thousand copies of capsid proteins (CA) arranged into hexamer and pentamer building blocks. These hexamers and pentamers further associate with each other to form a cone-shaped capsid structure. Cellular factors may interact with the capsid core to block infection (restriction factors), whereas others may be recruited by the virus to promote infection (cofactors).

A common theme of these interactions is the host pattern sensing of HIV capsid. Most capsid-binding proteins show relatively little affinity for the monomeric CA. Instead, they recognize the interfaces between multiple CA subunits, or more high-order interfaces between multiple hexamer/pentamer building blocks. However, this recognition mode has greatly limited high-resolution structural characterization of host-capsid interactions because it is difficult to reconstitute the pattern within the cone-shaped structure for uniform binding of host factors in vitro.

To overcome this major challenge, we have used a variety of protein engineering tools to build a library of assembled CA oligomers. These CA oligomers faithfully recreate the native high-order interfaces found between CA subunits and can be purified in large quality. These tools have enabled us to map how important viral restriction factors and co-factors bind the capsid. Among the host factors amenable to our engineered tools are TRIM5α, TRIMCyp, MxB, Fez1, SUN2, and numerous small-molecule compounds. Excitingly, we can use these tools to discover new host factors, and streamline their biochemical characterization. Furthermore, we are pursuing high-resolution structures of CA-host factor complexes using crystallography and cryo-EM.

A first line of defense: TRIM5 proteins

Retroviruses are often limited to a small number of host organisms due to species-specific cellular restriction factors. The tripartite motif proteins TRIM5α and TRIMCyp are important components of the cross-species barrier to HIV and many other retroviruses. TRIM5α inhibits retrovirus infection by directly recognizing retroviral capsid in a species-specific manner and eliciting premature disassembly of the capsid and activation of cellular innate immune signaling. Functioning as a viral capsid pattern sensor, TRIM5α binds only to the assembled capsid lattice. Our goal is to investigate TRIM5 protein-capsid interactions in vitro and establish the structural basis of these interactions.

Mutation of viral DNA by APOBEC3 proteins

APOBEC3G (A3G) is a host cytidine deaminase which extensively mutates viral DNA to restrict HIV infection. To elude this host defense, HIV virion infectivity factor (Vif) binds A3G and targets it for proteosomal degradation. Our goal is to first establish the mechanism by which A3G specifically recognizes viral DNA. At the same time, we’re also investigating the biochemical and structural principles underlying the interaction between Vif and A3G. Information gained from these studies will be used to direct structure-based design of anti-HIV drugs. Screening of Vif inhibitors is being carried out at the Yale Small Molecule Discovery Center.

Suppression of reverse transcription and regulation of cellular dNTPs by SAMHD1

SAMHD1, a deoxyribonucleoside triphosphate triphosphohydrolase (dNTPase), prevents the infection of non-dividing cells by retroviruses,including HIV, by depleting the cellular dNTP pool available for viral reverse transcription. SAMHD1 is a major regulator of cellular dNTP levels in mammalian cells. Mutations in SAMHD1 are associated with chronic lymphocytic leukemia (CLL) and the autoimmune condition Aicardi Goutières syndrome (AGS). We have obtained extensive biochemical and structural data that uncover an exquisite activation mechanism of SAMHD1 via combined action of both GTP and dNTPs at the eight allosteric and four catalytic sites of the SAMHD1 tetramer. These results establish a complete framework for our future studies of the important functions of SAMHD1 in the regulation of cellular dNTP levels, as well as in HIV restriction and the pathogenesis of CLL and AGS.