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The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) outbreak took the world by storm due to its rapid global spread and unpredictable disease outcomes. The extraordinary ascension of SARS-CoV-2 to pandemic status motivated a world-wide effort to rapidly develop vaccines that could effectively suppress virus spread and mitigate severe disease. These efforts culminated in the development and deployment of several highly effective vaccines that were heralded as the beginning-of-the-end of the pandemic. However, these successes were short lived due to the unexpected and continuous emergence of more transmissible and immune-evasive SARS-CoV-2 variants. Thus, attention has shifted toward developing novel vaccine platforms that elicit more robust and sustained neutralizing antibody responses. Recent findings by Muñoz-Alía and colleagues address this by combining a live recombinant measles vaccine platform with novel biochemical approaches to generate vaccine candidates that bolster the potency of neutralizing antibody responses against diverse SARS-CoV-2 spike proteins (M. Á. Muñoz-Alía, R. A. Nace, B. Balakrishnan, L. Zhang, et al., mBio 9:e02928-23, 2024, https://doi.org/10.1128/mbio.02928-23).

The single-stranded DNA cytosine-to-uracil deaminase APOBEC3B is an antiviral protein implicated in cancer. However, its substrates in cells are not fully delineated. Here APOBEC3B proteomics reveal interactions with a surprising number of R-loop factors. Biochemical experiments show APOBEC3B binding to R-loops in cells and in vitro. Genetic experiments demonstrate R-loop increases in cells lacking APOBEC3B and decreases in cells overexpressing APOBEC3B. Genome-wide analyses show major changes in the overall landscape of physiological and stimulus-induced R-loops with thousands of differentially altered regions, as well as binding of APOBEC3B to many of these sites. APOBEC3 mutagenesis impacts genes overexpressed in tumors and splice factor mutant tumors preferentially, and APOBEC3-attributed kataegis are enriched in RTCW motifs consistent with APOBEC3B deamination. Taken together with the fact that APOBEC3B binds single-stranded DNA and RNA and preferentially deaminates DNA, these results support a mechanism in which APOBEC3B regulates R-loops and contributes to R-loop mutagenesis in cancer. APOBEC3B interacts with R-loops and helps mediate their resolution in a deamination-dependent way. This association also renders R-loops susceptible to enhanced APOBEC3B-dependent mutagenesis.

Crystal structures of human APOBEC3A and a chimera of APOBEC3B and APOBEC3A bound to ssDNA reveal an unanticipated ‘U-shaped’ binding mode and provide insight into target selectivity. APOBEC-catalyzed cytosine-to-uracil deamination of single-stranded DNA (ssDNA) has beneficial functions in immunity and detrimental effects in cancer. APOBEC enzymes have intrinsic dinucleotide specificities that impart hallmark mutation signatures. Although numerous structures have been solved, mechanisms for global ssDNA recognition and local target-sequence selection remain unclear. Here we report crystal structures of human APOBEC3A and a chimera of human APOBEC3B and APOBEC3A bound to ssDNA at 3.1-Å and 1.7-Å resolution, respectively. These structures reveal a U-shaped DNA conformation, with the specificity-conferring −1 thymine flipped out and the target cytosine inserted deep into the zinc-coordinating active site pocket. The −1 thymine base fits into a groove between flexible loops and makes direct hydrogen bonds with the protein, accounting for the strong 5′-TC preference. These findings explain both conserved and unique properties among APOBEC family members, and they provide a basis for the rational design of inhibitors to impede the evolvability of viruses and tumors.

Emerging evidence indicates that HIV-1 hijacks host DNA damage repair (DDR) pathways to facilitate multiple facets of virus replication. Canonically, HIV-1 engages proviral DDR responses through the accessory protein Vpr, which induces constitutive activation of DDR kinases ATM and ATR. However, in response to prolonged DDR signaling, ATM directly induces pro-inflammatory NF-κB signaling and activates multiple members of the TRIM family of antiviral restriction factors, several of which have been previously implicated in antagonizing retroviral and lentiviral replication. Here, we demonstrate that the HIV-1 accessory protein Vif blocks ATM-directed DNA repair processes, activation of NF-κB signaling responses, and TRIM protein phosphorylation. Vif function in ATM antagonism occurs in clinical isolates and in common HIV-1 Group M subtypes/clades circulating globally. Pharmacologic and functional studies combine to suggest that Vif blocks Vpr-directed activation of ATM but not ATR, signifying that HIV-1 utilizes discrete strategies to fine-tune DDR responses that promote virus replication while simultaneously inhibiting immune activation.

Hijacking of host DNA damage repair (DDR) pathways to facilitate virus replication is broadly conserved amongst diverse viral families. It has been well established that the HIV-1 accessory protein Vpr induces constitutive DDR signaling and G2/M cell cycle arrest, but the virologic function of this activity remains unclear. Here, we use a combination of functional, pharmacologic, biochemical, and genetic approaches to establish that virion-associated and de novo Vpr proteins induce DDR responses that trigger global epigenetic remodeling and activation of transcription programs to enhance HIV-1 promoter activity during acute infection and reactivation from latency. Functional, genetic, and bimolecular fluorescence complementation experiments reveal that Vpr segregates into two functionally discrete pools—a multimeric pool in the nucleus associated with chromatin and a monomeric pool in the cytoplasm associated with a host E3-ubiquitin ligase. Vpr-induced DDR and epigenetic remodeling activities are present in common HIV-1 subtypes circulating globally and in patient-derived isolates.

FLAGELLIN-SENSING2 (FLS2) is the plant cell surface receptor that perceives bacterial flagellin or flg22 peptide, initiates flg22-signaling responses, and contributes to bacterial growth restriction. Flg22 elicitation also leads to ligand-induced endocytosis and degradation of FLS2 within 1 h. Why plant cells remove this receptor precisely at the time during which its function is required remains mainly unknown. Here, we assessed in planta flg22-signaling competency in the context of ligand-induced degradation of endogenous FLS2 and chemical interference known to impede flg22-dependent internalization of FLS2 into endocytic vesicles. Within 1 h after an initial flg22 treatment, Arabidopsis (Arabidopsis thaliana) leaf tissue was unable to reelicit flg22 signaling in ligand-, time-, and dose-dependent manner. These results indicate that flg22-induced degradation of endogenous FLS2 may serve to desensitize cells to the same stimulus (homologous desensitization), likely to prevent continuous signal output upon repetitive flg22 stimulation. In addition to impeding ligand-induced FLS2 degradation, pretreatment with the vesicular trafficking inhibitors Wortmannin or Tyrphostin A23 impaired flg22-elicited reactive oxygen species production that was partially independent of BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1. Interestingly, these inhibitors did not affect flg22-induced mitogenactivated protein kinase phosphorylation, indicating the ability to utilize vesicular trafficking inhibitors to target different flg22-signaling responses. For Tyrphostin A23, reduced flg22-induced reactive oxygen species could be separated from the defect in FLS2 degradation. At later times (> 2 h) after the initial flg22 elicitation, recovery of FLS2 protein levels positively correlated with resensitization to flg22, indicating that flg22-induced new synthesis of FLS2 may prepare cells for a new round of monitoring the environment for flg22.

The family of forkhead box O (FoxO) transcription factors regulate cellular processes involved in glucose metabolism, stress resistance, DNA damage repair, and tumor suppression. FoxO transactivation activity is tightly regulated by a complex network of signaling pathways and post-translational modifications. While it has been well established that phosphorylation promotes FoxO cytoplasmic retention and inactivation, the mechanism underlying dephosphorylation and nuclear translocation is less clear. Here, we investigate the role of protein phosphatase 2A (PP2A) in regulating this process. We demonstrate that PP2A and AMP-activated protein kinase (AMPK) combine to regulate nuclear translocation of multiple FoxO family members following inhibition of metabolic signaling or induction of oxidative stress. Moreover, chemical inhibitor studies indicate that nuclear accumulation of FoxO proteins occurs through inhibition of nuclear export as opposed to promoting nuclear import as previously speculated. Functional, genetic, and biochemical studies combine to identify the PP2A complexes that regulate FoxO nuclear translocation, and the binding motif required. Mutating the FoxO-PP2A interface to enhance or diminish PP2A binding alters nuclear translocation kinetics accordingly. Together, these studies shed light on the molecular mechanisms regulating FoxO nuclear translocation and provide insights into how FoxO regulation is integrated with metabolic and stress-related stimuli.

Integration of the reverse-transcribed viral DNA into host chromosomes is a critical step in the life-cycle of retroviruses, including an oncogenic delta(δ)-retrovirus human T-cell leukemia virus type-1 (HTLV-1). Retroviral integrase forms a higher order nucleoprotein assembly (intasome) to catalyze the integration reaction, in which the roles of host factors remain poorly understood. Here, we use cryo-electron microscopy to visualize the HTLV-1 intasome at 3.7-Å resolution. The structure together with functional analyses reveal that the B56γ (B’γ) subunit of an essential host enzyme, protein phosphatase 2 A (PP2A), is repurposed as an integral component of the intasome to mediate HTLV-1 integration. Our studies reveal a key host-virus interaction underlying the replication of an important human pathogen and highlight divergent integration strategies of retroviruses. Integration of the reverse-transcribed viral DNA into host chromosomes is a critical step in the life-cycle of retroviruses such as the human T-cell leukemia virus type-1 (HTLV-1). Here the authors describe the structural role of the host co-factor protein phosphatase 2A B56γ subunit in supporting integration as part of the HTLV-1 intasome.

Powassan virus (POWV) is an emerging tick-borne Flavivirus that causes lethal encephalitis and long-term neurologic damage. Currently, there are no POWV therapeutics, licensed vaccines, or reverse genetics systems for producing infectious POWVs from recombinant DNA. Using a circular polymerase extension reaction (CPER), we generated recombinant LI9 (recLI9) POWVs with attenuating NS1 protein mutations and a recLI9-split-eGFP reporter virus. NS1 proteins are highly conserved glycoproteins that regulate replication, spread, and neurovirulence. POWV NS1 contains three putative N-linked glycosylation sites that we modified individually in infectious recLI9 mutants (N85Q, N208Q, and N224Q). NS1 glycosylation site mutations reduced replication kinetics and were attenuated, with 1–2 log decreases in titer. Severely attenuated recLI9-N224Q exhibited a 2- to 3-day delay in focal cell-to-cell spread and reduced NS1 secretion but was lethal when intracranially inoculated into suckling mice. However, footpad inoculation of recLI9-N224Q resulted in the survival of 80% of mice and demonstrated that NS1-N224Q mutations reduce POWV neuroinvasion in vivo . To monitor NS1 trafficking, we CPER fused a split GFP11-tag to the NS1 C-terminus and generated an infectious reporter virus, recLI9-NS1-GFP11. Cells infected with recLI9-NS1-GFP11 revealed NS1 trafficking in live cells and the novel formation of large NS1-lined intracellular vesicles. An infectious recLI9-NS1-GFP11 reporter virus permits real-time analysis of NS1 functions in POWV replication, assembly, and secretion and provides a platform for evaluating antiviral compounds. Collectively, our robust POWV reverse genetics system permits analysis of viral spread and neurovirulence determinants in vitro and in vivo and enables the rational genetic design of live attenuated POWV vaccines. Our findings newly establish a mechanism for genetically modifying Powassan viruses (POWVs), systematically defining pathogenic determinants and rationally designing live attenuated POWV vaccines. This initial study demonstrates that mutating POWV NS1 glycosylation sites attenuates POWV spread and neurovirulence in vitro and in vivo . Our findings validate a robust circular polymerase extension reaction approach as a mechanism for developing, and evaluating, attenuated genetically modified POWVs. We further designed an infectious GFP-tagged reporter POWV that permits us to monitor secretory trafficking of POWV in live cells, which can be applied to screen potential POWV replication inhibitors. This robust system for modifying POWVs provides the ability to define attenuating POWV mutations and create genetically attenuated recPOWV vaccines.

Human APOBEC3H (A3H) is a single-stranded DNA cytosine deaminase that inhibits HIV-1. Seven haplotypes (I–VII) and four splice variants (SV154/182/183/200) with differing antiviral activities and geographic distributions have been described, but the genetic and mechanistic basis for variant expression and function remains unclear. Using a combined bioinformatic/experimental analysis, we find that SV200 expression is specific to haplotype II, which is primarily found in sub-Saharan Africa. The underlying genetic mechanism for differential mRNA splicing is an ancient intronic deletion [del(ctc)] within A3H haplotype II sequence. We show that SV200 is at least fourfold more HIV-1 restrictive than other A3H splice variants. To counteract this elevated antiviral activity, HIV-1 protease cleaves SV200 into a shorter, less restrictive isoform. Our analyses indicate that, in addition to Vif-mediated degradation, HIV-1 may use protease as a  counter-defense mechanism against A3H in >80% of sub-Saharan African populations. Human APOBEC3H has several haplotypes and splice variants with distinct anti-HIV-1 activities, but the genetics underlying the expression of these variants are unclear. Here, the authors identify an intronic deletion in A3H haplotype II resulting in production of the most active splice variant, which is counteracted by HIV-1 protease.