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Eastern equine encephalitis virus (EEEV) is the most virulent alphavirus that infects humans, and many survivors develop neurological sequelae, including paralysis and intellectual disability. Alphavirus spike proteins comprise trimers of heterodimers of glycoproteins E2 and E1 that mediate binding to cellular receptors and fusion of virus and host cell membranes during entry. We recently identified very-low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2) as cellular receptors for EEEV and a distantly related alphavirus, Semliki Forest virus (SFV). Here, we use single-particle cryo-electron microscopy (cryo-EM) to determine structures of the EEEV and SFV spike glycoproteins bound to the VLDLR ligand-binding domain and found that EEEV and SFV interact with the same cellular receptor through divergent binding modes. Our studies suggest that the ability of LDLR-related proteins to interact with viral spike proteins through very small footprints with flexible binding modes results in a low evolutionary barrier to the acquisition of LDLR-related proteins as cellular receptors for diverse sets of viruses.
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Microscopía por Crioelectrón , Virus de la Encefalitis Equina del Este , Receptores de LDL , Receptores de LDL/metabolismo , Receptores de LDL/química , Virus de la Encefalitis Equina del Este/metabolismo , Virus de la Encefalitis Equina del Este/ultraestructura , Humanos , Animales , Virus de los Bosques Semliki/metabolismo , Unión Proteica , Receptores Virales/metabolismo , Receptores Virales/química , Proteínas del Envoltorio Viral/metabolismo , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/ultraestructura , Modelos MolecularesRESUMEN
Western equine encephalitis virus (WEEV) is an arthropod-borne virus (arbovirus) that frequently caused major outbreaks of encephalitis in humans and horses in the early twentieth century, but the frequency of outbreaks has since decreased markedly, and strains of this alphavirus isolated in the past two decades are less virulent in mammals than strains isolated in the 1930s and 1940s1-3. The basis for this phenotypic change in WEEV strains and coincident decrease in epizootic activity (known as viral submergence3) is unclear, as is the possibility of re-emergence of highly virulent strains. Here we identify protocadherin 10 (PCDH10) as a cellular receptor for WEEV. We show that multiple highly virulent ancestral WEEV strains isolated in the 1930s and 1940s, in addition to binding human PCDH10, could also bind very low-density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2), which are recognized by another encephalitic alphavirus as receptors4. However, whereas most of the WEEV strains that we examined bind to PCDH10, a contemporary strain has lost the ability to recognize mammalian PCDH10 while retaining the ability to bind avian receptors, suggesting WEEV adaptation to a main reservoir host during enzootic circulation. PCDH10 supports WEEV E2-E1 glycoprotein-mediated infection of primary mouse cortical neurons, and administration of a soluble form of PCDH10 protects mice from lethal WEEV challenge. Our results have implications for the development of medical countermeasures and for risk assessment for re-emerging WEEV strains.
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Cadherinas , Virus de la Encefalitis Equina del Oeste , Receptores Virales , Animales , Ratones , Humanos , Receptores Virales/metabolismo , Cadherinas/metabolismo , Femenino , Receptores de LDL/metabolismo , Receptores de LDL/genética , Masculino , Encefalomielitis Equina/virología , Encefalomielitis Equina/transmisión , Encefalomielitis Equina/veterinariaRESUMEN
BACKGROUND: We investigated how a personalized care-planning software and linked mobile-app may aid people to self-manage their type 2 diabetes (T2D) more effectively. RESEARCH DESIGN AND METHODS: People with T2D and glycated hemoglobin (HbA1c) greater than 58 mmol/mol (7.5%) were randomized to either an intervention group receiving a personalized care plan, or the control group receiving usual care. Quality of life (QoL) was measured for both groups using validated questionnaires and one-on-one interviews with a subset of 12 participants from each group. RESULTS: QoL for the active treatment group increased, by their EQ -5D-5 L score increasing on average by 0.046, whereas it decreased for the control group on average by 0.009. The EQ Visual Analogue Score (VAS) of the intervention group also increased by 8.2%, whereas the control group had a reduction in EQ VAS score of 2.8% (p = 0.008 for difference). CONCLUSION: In this prospective RCT, the findings point to how the provision of personalized care plans can result in an improvement in individuals' self-rated QoL. This may lead to longer term health benefits.
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Diabetes Mellitus Tipo 2 , Medición de Resultados Informados por el Paciente , Calidad de Vida , Humanos , Diabetes Mellitus Tipo 2/terapia , Masculino , Femenino , Persona de Mediana Edad , Anciano , Hemoglobina Glucada/análisis , Aplicaciones Móviles , Estudios Prospectivos , Adulto , Encuestas y Cuestionarios , Automanejo/métodosRESUMEN
Alphaviruses are arthropod-borne enveloped RNA viruses that include several important human pathogens with outbreak potential. Among them, eastern equine encephalitis virus (EEEV) is the most virulent, and many survivors develop neurological sequelae, including paralysis and intellectual disability. The spike proteins of alphaviruses comprise trimers of heterodimers of their envelope glycoproteins E2 and E1 that mediate binding to cellular receptors and fusion of virus and host cell membranes during entry. We recently identified very-low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2), two closely related proteins that are expressed in the brain, as cellular receptors for EEEV and a distantly related alphavirus, Semliki forest virus (SFV) 1 . The EEEV and SFV spike glycoproteins have low sequence homology, and how they have evolved to bind the same cellular receptors is unknown. Here, we used single-particle cryo-electron microscopy (cryo-EM) to determine structures of the EEEV and SFV spike glycoproteins bound to the VLDLR ligand-binding domain. The structures reveal that EEEV and SFV use distinct surfaces to bind VLDLR; EEEV uses a cluster of basic residues on the E2 subunit of its spike glycoprotein, while SFV uses two basic residues at a remote site on its E1 glycoprotein. Our studies reveal that different alphaviruses interact with the same cellular receptor through divergent binding modes. They further suggest that the ability of LDLR-related proteins to interact with viral spike proteins through very small footprints with flexible binding modes results in a low evolutionary barrier to the acquisition of LDLR-related proteins as cellular receptors for diverse sets of viruses.
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INTRODUCTION: Effective and scalable solutions to support management of Type 2 Diabetes (T2D) at a distance are a priority for health systems worldwide. The use of personalised care planning has been shown to be effective at improving the health outcomes and the experience of care amongst people with T2D and other long-term health conditions. Here we describe a specific example of such an intervention. METHODS: The sample comprised 197 participants with T2D randomised to either the active intervention group with digital health planning (App + usual care), with 115 participants, or the control group (usual care), with 82 participants. We analysed data in relation to changes in body mass index (BMI) and glycated haemoglobin (HbA1c) over a 6-month follow-up period. We also analysed responses to questionnaires sent out and held interviews with participants that were in the active treatment group and therefore had a care plan created and access to an app. RESULTS: The active treatment group had significant reductions in HbA1c (p < 0.01) and BMI (p < 0.037) vs the control group (no significant change). The average percentage change in HbA1c for the treatment group over 6 months was - 7.4% (± SE 1.4%), compared with 1.8% (± SE 2.1%) for the control group. The average percentage change in BMI for the treatment group was - 0.7% (± SE 0.4%) and it was - 0.2% (± SE 0.5%) for the control group. A higher percentage of the active treatment group reduced their HbA1c and BMI than the control group. For HbA1c, 72.4% of the active treatment group reduced their HbA1c, compared to 41.5% of the control group. For BMI, 52.7% of the active treatment group experienced a reduction, compared to 42.9% for the control group. Self-measured quality of life (QoL) improved for patients in the active treatment group, shown by an increase in their pre-trial to post-trial EQ-5D-5L rating by an average of 0.0464 (± SE 0.0625), compared to a decrease of 0.0086 (± SE 0.0530) for the control group. The average EQ VAS score also increased pre- to post-trial for the active treatment group, on average by 8.2%, whereas it decreased by an average of - 2.8% for the control group. CONCLUSION: These findings point to how the provision of personalised plans of care, support and education linked to a mobile app, can result in HbA1c and BMI reduction for many individuals with T2D. The use of a patient management app as well as a personalised care plan also led to an improvement in patient self-rated QoL and engagement.
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As of June 2022, the GISAID database contains more than 11 million SARS-CoV-2 genomes, including several thousand nucleotide sequences for the most common variants such as delta or omicron. These SARS-CoV-2 strains have been collected from patients around the world since the beginning of the pandemic. We start by assessing the similarity of all pairs of nucleotide sequences using the Jaccard index and principal component analysis. As shown previously in the literature, an unsupervised cluster analysis applied to the SARS-CoV-2 genomes results in clusters of sequences according to certain characteristics such as their strain or their clade. Importantly, we observe that nucleotide sequences of common variants are often outliers in clusters of sequences stemming from variants identified earlier on during the pandemic. Motivated by this finding, we are interested in applying outlier detection to nucleotide sequences. We demonstrate that nucleotide sequences of common variants (such as alpha, delta, or omicron) can be identified solely based on a statistical outlier criterion. We argue that outlier detection might be a useful surveillance tool to identify emerging variants in real time as the pandemic progresses.
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COVID-19 , Humanos , Secuencia de Bases , SARS-CoV-2 , Análisis por Conglomerados , Bases de Datos FactualesRESUMEN
Introduction: The use of personalised care planning has been effective at improving health outcomes for people with long-term health conditions. Methods: We analysed data in relation to changes in BMI/HbA1c. The sample was made up of (n = 36) participants randomised to either the active intervention group (App+usual care) or the control group (usual care). Results: The average HbA1c percentage change for the treatment group was 9.5%, but just -2% for the control (usual care) group (P = 0.015 for the difference). The average percentage change in BMI for the treatment group was -0.4%, but 0.1% for the control group (P = 0.03 for the difference). Conclusion: These preliminary findings point to how the provision of personalised plans of care, support and education linked to a mobile app, can result in HbA1c and BMI reduction over a 6-month period. While the results are preliminary, they portend the potential for digital plans of care to enhance T2DM management.
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Humoral immunity to SARS-CoV-2 can be supplemented with polyclonal sera from convalescent donors or an engineered monoclonal antibody (mAb) product. While pentameric IgM antibodies are responsible for much of convalescent sera's neutralizing capacity, all available mAbs are based on the monomeric IgG antibody subtype. We now show that IgM mAbs derived from immune memory B cell receptors are potent neutralizers of SARS-CoV-2. IgM mAbs outperformed clonally identical IgG antibodies across a range of affinities and SARS-CoV-2 receptor-binding domain epitopes. Strikingly, efficacy against SARS-CoV-2 viral variants was retained for IgM but not for clonally identical IgG. To investigate the biological role for IgM memory in SARS-CoV-2, we also generated IgM mAbs from antigen-experienced IgM+ memory B cells in convalescent donors, identifying a potent neutralizing antibody. Our results highlight the therapeutic potential of IgM mAbs and inform our understanding of the role for IgM memory against a rapidly mutating pathogen.
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COVID-19 , SARS-CoV-2 , Anticuerpos Monoclonales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , COVID-19/terapia , Humanos , Inmunización Pasiva , Inmunoglobulina G , Inmunoglobulina M , Células B de Memoria , Glicoproteína de la Espiga del Coronavirus , Sueroterapia para COVID-19Asunto(s)
Anticuerpos Monoclonales , Antineoplásicos Inmunológicos , Evolución Biológica , COVID-19 , Virus , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Antivirales/genética , Antineoplásicos Inmunológicos/uso terapéutico , COVID-19/genética , COVID-19/terapia , Humanos , SARS-CoV-2/genética , Fenómenos Fisiológicos de los Virus/genética , Virus/genética , Virus/inmunologíaAsunto(s)
Anticuerpos Monoclonales , Antineoplásicos Inmunológicos , COVID-19 , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Neutralizantes , Antineoplásicos Inmunológicos/farmacología , Antineoplásicos Inmunológicos/uso terapéutico , COVID-19/prevención & control , Semivida , Humanos , Inmunoglobulina GRESUMEN
SARS-CoV-2 can cause acute respiratory distress and death in some patients1. Although severe COVID-19 is linked to substantial inflammation, how SARS-CoV-2 triggers inflammation is not clear2. Monocytes and macrophages are sentinel cells that sense invasive infection to form inflammasomes that activate caspase-1 and gasdermin D, leading to inflammatory death (pyroptosis) and the release of potent inflammatory mediators3. Here we show that about 6% of blood monocytes of patients with COVID-19 are infected with SARS-CoV-2. Monocyte infection depends on the uptake of antibody-opsonized virus by Fcγ receptors. The plasma of vaccine recipients does not promote antibody-dependent monocyte infection. SARS-CoV-2 begins to replicate in monocytes, but infection is aborted, and infectious virus is not detected in the supernatants of cultures of infected monocytes. Instead, infected cells undergo pyroptosis mediated by activation of NLRP3 and AIM2 inflammasomes, caspase-1 and gasdermin D. Moreover, tissue-resident macrophages, but not infected epithelial and endothelial cells, from lung autopsies from patients with COVID-19 have activated inflammasomes. Taken together, these findings suggest that antibody-mediated SARS-CoV-2 uptake by monocytes and macrophages triggers inflammatory cell death that aborts the production of infectious virus but causes systemic inflammation that contributes to COVID-19 pathogenesis.
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COVID-19 , Inflamación , Monocitos , Receptores de IgG , SARS-CoV-2 , COVID-19/virología , Caspasa 1/metabolismo , Proteínas de Unión al ADN , Humanos , Inflamasomas/metabolismo , Inflamación/metabolismo , Inflamación/virología , Monocitos/metabolismo , Monocitos/virología , Proteína con Dominio Pirina 3 de la Familia NLR , Proteínas de Unión a Fosfato , Proteínas Citotóxicas Formadoras de Poros , Receptores de IgG/metabolismoRESUMEN
The use of anti-spike (S) serologic assays as surrogate measurements of SARS-CoV-2 vaccine induced immunity will be an important clinical and epidemiological tool. The characteristics of a commercially available anti-S antibody assay (Roche Elecsys anti-SARS-CoV-2 S) were evaluated in a cohort of vaccine recipients. Levels were correlated with pseudotype neutralizing antibodies (NAb) across SARS-CoV-2 variants. We recruited adults receiving a two-dose series of mRNA-1273 or BNT162b2 and collected serum at scheduled intervals up to 8 months post-first vaccination. Anti-S and NAb levels were measured, and correlation was evaluated by (i) vaccine type and (ii) SARS-CoV-2 variant (wild-type, Alpha, Beta, Gamma, and three constructs Day 146*, Day 152*, and RBM-2). Forty-six mRNA vaccine recipients were enrolled. mRNA-1273 vaccine recipients had higher peak anti-S and NAb levels compared with BNT162b2 (P < 0.001 for anti-S levels; P < 0.05 for NAb levels). When anti-S and NAb levels were compared, there was good correlation (all r values ≥ 0.85) in both BNT162b2 and mRNA-1273 vaccine recipients across all evaluated variants; however, these correlations were nonlinear in nature. Lower correlation was identified between anti-S and NAb for the Beta variant (r = 0.88) compared with the wild-type (WT) strain (r = 0.94). Finally, the degree of neutralizing activity at any given anti-S level was lower for each variant compared with that of the WT strain, (P < 0.001). Although the Roche anti-S assay correlates well with NAb levels, this association is affected by vaccine type and SARS-CoV-2 variant. These variables must be considered when interpreting anti-S levels. IMPORTANCE We evaluated anti-spike antibody concentrations in healthy mRNA vaccinated individuals and compared these concentrations to values obtained from pseudotype neutralization assays targeting SARS-CoV-2 variants of concern to determine how well anti-spike antibodies correlate with neutralizing titers, which have been used as a marker of immunity from COVID-19 infection. We found high peak anti-spike concentrations in these individuals, with significantly higher levels seen in mRNA-1273 vaccine recipients. When we compared anti-spike and pseudotype neuralization titers, we identified good correlation; however, this correlation was affected by both vaccine type and variant, illustrating the difficulty of applying a "one size fits all" approach to anti-spike result interpretation. Our results support CDC recommendations to discourage anti-spike antibody testing to assess for immunity after vaccination and cautions providers in their interpretations of these results as a surrogate of protection in COVID-vaccinated individuals.
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COVID-19 , SARS-CoV-2 , Vacuna nCoV-2019 mRNA-1273 , Adulto , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Vacuna BNT162 , COVID-19/diagnóstico , COVID-19/prevención & control , Vacunas contra la COVID-19 , Humanos , SARS-CoV-2/genética , Vacunas Sintéticas , Vacunas de ARNmRESUMEN
Enterococci are a part of human microbiota and a leading cause of multidrug resistant infections. Here, we identify a family of Enterococcus pore-forming toxins (Epxs) in E. faecalis, E. faecium, and E. hirae strains isolated across the globe. Structural studies reveal that Epxs form a branch of ß-barrel pore-forming toxins with a ß-barrel protrusion (designated the top domain) sitting atop the cap domain. Through a genome-wide CRISPR-Cas9 screen, we identify human leukocyte antigen class I (HLA-I) complex as a receptor for two members (Epx2 and Epx3), which preferentially recognize human HLA-I and homologous MHC-I of equine, bovine, and porcine, but not murine, origin. Interferon exposure, which stimulates MHC-I expression, sensitizes human cells and intestinal organoids to Epx2 and Epx3 toxicity. Co-culture with Epx2-harboring E. faecium damages human peripheral blood mononuclear cells and intestinal organoids, and this toxicity is neutralized by an Epx2 antibody, demonstrating the toxin-mediated virulence of Epx-carrying Enterococcus.
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Toxinas Bacterianas/metabolismo , Enterococcus , Leucocitos Mononucleares , Factores de Virulencia/metabolismo , Animales , Bovinos , Enterococcus/metabolismo , Enterococcus/patogenicidad , Caballos , Ratones , Pruebas de Sensibilidad Microbiana , PorcinosRESUMEN
Five New World mammarenaviruses (NWMs) cause life-threatening hemorrhagic fever (HF). Cellular entry by these viruses is mediated by human transferrin receptor 1 (hTfR1). Here, we demonstrate that an antibody (ch128.1/IgG1) which binds the apical domain of hTfR1, potently inhibits infection of attenuated and pathogenic NWMs in vitro. Computational docking of the antibody Fab crystal structure onto the known structure of hTfR1 shows an overlapping receptor-binding region shared by the Fab and the viral envelope glycoprotein GP1 subunit that binds hTfR1, and we demonstrate competitive inhibition of NWM GP1 binding by ch128.1/IgG1 as the principal mechanism of action. Importantly, ch128.1/IgG1 protects hTfR1-expressing transgenic mice against lethal NWM challenge. Additionally, the antibody is well-tolerated and only partially reduces ferritin uptake. Our findings provide the basis for the development of a novel, host receptor-targeted antibody therapeutic broadly applicable to the treatment of HF of NWM etiology.
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Antígenos CD/metabolismo , Arenaviridae/metabolismo , Fiebre Hemorrágica Americana/metabolismo , Receptores de Transferrina/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Células A549 , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/farmacología , Antígenos CD/inmunología , Arenaviridae/efectos de los fármacos , Arenaviridae/fisiología , Chlorocebus aethiops , Fiebre Hemorrágica Americana/prevención & control , Fiebre Hemorrágica Americana/virología , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Virus Junin/efectos de los fármacos , Virus Junin/fisiología , Ratones Endogámicos C57BL , Ratones Transgénicos , Simulación del Acoplamiento Molecular , Unión Proteica/efectos de los fármacos , Receptores de Transferrina/antagonistas & inhibidores , Receptores de Transferrina/inmunología , Células VeroRESUMEN
Alphaviruses, like many other arthropod-borne viruses, infect vertebrate species and insect vectors separated by hundreds of millions of years of evolutionary history. Entry into evolutionarily divergent host cells can be accomplished by recognition of different cellular receptors in different species, or by binding to receptors that are highly conserved across species. Although multiple alphavirus receptors have been described1-3, most are not shared among vertebrate and invertebrate hosts. Here we identify the very low-density lipoprotein receptor (VLDLR) as a receptor for the prototypic alphavirus Semliki forest virus. We show that the E2 and E1 glycoproteins (E2-E1) of Semliki forest virus, eastern equine encephalitis virus and Sindbis virus interact with the ligand-binding domains (LBDs) of VLDLR and apolipoprotein E receptor 2 (ApoER2), two closely related receptors. Ectopic expression of either protein facilitates cellular attachment, and internalization of virus-like particles, a VLDLR LBD-Fc fusion protein or a ligand-binding antagonist block Semliki forest virus E2-E1-mediated infection of human and mouse neurons in culture. The administration of a VLDLR LBD-Fc fusion protein has protective activity against rapidly fatal Semliki forest virus infection in mouse neonates. We further show that invertebrate receptor orthologues from mosquitoes and worms can serve as functional alphavirus receptors. We propose that the ability of some alphaviruses to infect a wide range of hosts is a result of their engagement of evolutionarily conserved lipoprotein receptors and contributes to their pathogenesis.
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Mosquitos Vectores , Virus de los Bosques Semliki , Animales , Proteínas Relacionadas con Receptor de LDL , Ligandos , Ratones , Receptores de LDL , Virus de los Bosques Semliki/metabolismo , Virus Sindbis/fisiologíaRESUMEN
Many studies have examined the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants on neutralizing antibody activity after they have become dominant strains. Here, we evaluate the consequences of further viral evolution. We demonstrate mechanisms through which the SARS-CoV-2 receptor binding domain (RBD) can tolerate large numbers of simultaneous antibody escape mutations and show that pseudotypes containing up to seven mutations, as opposed to the one to three found in previously studied variants of concern, are more resistant to neutralization by therapeutic antibodies and serum from vaccine recipients. We identify an antibody that binds the RBD core to neutralize pseudotypes for all tested variants but show that the RBD can acquire an N-linked glycan to escape neutralization. Our findings portend continued emergence of escape variants as SARS-CoV-2 adapts to humans.
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Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Evasión Inmune , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/inmunología , Enzima Convertidora de Angiotensina 2/química , Enzima Convertidora de Angiotensina 2/metabolismo , Vacuna BNT162/inmunología , Betacoronavirus/inmunología , COVID-19/inmunología , COVID-19/virología , Reacciones Cruzadas , Microscopía por Crioelectrón , Cristalografía por Rayos X , Epítopos , Evolución Molecular , Humanos , Modelos Moleculares , Mutación , Polisacáridos/análisis , Unión Proteica , Dominios Proteicos , Receptores de Coronavirus/química , Receptores de Coronavirus/metabolismo , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genética , Pseudotipado ViralRESUMEN
Herpesviruses comprise a family of DNA viruses that cause a variety of human and veterinary diseases. During productive infection, mammalian, avian, and reptilian herpesviruses replicate their genomes using a set of conserved viral proteins that include a two subunit DNA polymerase. This enzyme is both a model system for family B DNA polymerases and a target for inhibition by antiviral drugs. This chapter reviews the structure, function, and mechanisms of the polymerase of herpes simplex viruses 1 and 2 (HSV), with only occasional mention of polymerases of other herpesviruses such as human cytomegalovirus (HCMV). Antiviral polymerase inhibitors have had the most success against HSV and HCMV. Detailed structural information regarding HSV DNA polymerase is available, as is much functional information regarding the activities of the catalytic subunit (Pol), which include a DNA polymerization activity that can utilize both DNA and RNA primers, a 3'-5' exonuclease activity, and other activities in DNA synthesis and repair and in pathogenesis, including some remaining to be biochemically defined. Similarly, much is known regarding the accessory subunit, which both resembles and differs from sliding clamp processivity factors such as PCNA, and the interactions of this subunit with Pol and DNA. Both subunits contribute to replication fidelity (or lack thereof). The availability of both pharmacologic and genetic tools not only enabled the initial identification of Pol and the pol gene, but has also helped dissect their functions. Nevertheless, important questions remain for this long-studied enzyme, which is still an attractive target for new drug discovery.