Receptor tyrosine kinase inhibitors block multiple steps of influenza a virus replication.

Host signaling pathways play important roles in the replication of influenza virus, but their functional effects remain to be characterized at the molecular level. Here we identify two receptor tyrosine kinase inhibitors (RTKIs) of the tyrphostin class that exhibit robust antiviral activity against influenza A virus replication in cultured cells. One of these (AG879) is a selective inhibitor of the nerve growth factor receptor and human epidermal growth factor receptor 2 (TrkA/HER2) signaling; the other, tyrphostin A9 (A9), inhibits the platelet-derived growth factor receptor (PDGFR) pathway. We find that each inhibits at least three postentry steps of the influenza virus life cycle: AG879 and A9 both strongly inhibit the synthesis of all three influenza virus RNA species, block Crm1-dependent nuclear export, and also prevent the release of viral particles through a pathway that is modulated by the lipid biosynthesis enzyme farnesyl diphosphate synthase (FPPS). Tests of short hairpin RNA (shRNA) knockdown and additional small-molecule inhibitors confirmed that interventions targeting TrkA can suppress influenza virus replication. Our study suggests that host cell receptor tyrosine kinase signaling is required for maximal influenza virus RNA synthesis, viral ribonucleoprotein (vRNP) nuclear export, and virus release and that specific RTKIs hold promise as novel anti-influenza virus therapeutics.

Receptor tyrosine kinase inhibitors that block replication of influenza a and other viruses.

We have previously reported that two receptor tyrosine kinase inhibitors (RTKIs), called AG879 and tyrphostin A9 (A9), can each block the replication of influenza A virus in cultured cells. In this study, we further characterized the in vitro antiviral efficacies and specificities of these agents. The 50% effective concentration (EC(50)) of each against influenza A was found to be in the high nanomolar range, and the selectivity index (SI = 50% cytotoxic concentration [CC(50)]/EC(50)) was determined to be >324 for AG879 and 50 for A9, indicating that therapeutically useful concentrations of each drug produce only low levels of cytotoxicity. Each compound showed efficacy against representative laboratory strains of both human influenza A (H1N1 or H3N2) and influenza B viruses. Importantly, no drug-resistant influenza virus strains emerged even after 25 viral passages in the presence of AG879, whereas viruses resistant to amantadine appeared after only 3 passages. AG879 and A9 each also exhibited potent inhibitory activity against a variety of other RNA and DNA viruses, including Sendai virus (Paramyxoviridae), herpes simplex virus (Herpesviridae), mouse hepatitis virus (Coronaviridae), and rhesus rotavirus (Reoviridae), but not against Pichinde virus (Arenaviridae). These results together suggest that RTKIs may be useful as therapeutics against viral pathogens, including but not limited to influenza, due to their high selectivity indices, low frequency of drug resistance, and broad-spectrum antiviral activities.

Mutational analyses of packaging signals in influenza virus PA, PB1, and PB2 genomic RNA segments.

The influenza A virus genome consists of eight negative-sense RNA segments that must each be packaged to produce an infectious virion. We have previously mapped the minimal cis-acting regions necessary for efficient packaging of the PA, PB1, and PB2 segments, which encode the three protein subunits of the viral RNA polymerase. The packaging signals in each of these RNAs lie within two separate regions at the 3' and 5' termini, each encompassing the untranslated region and extending up to 80 bases into the adjacent coding sequence. In this study, we introduced scanning mutations across the coding regions in each of these RNA segments in order to finely define the packaging signals. We found that mutations producing the most severe defects were confined to a few discrete 5' sites in the PA or PB1 coding regions but extended across the entire (80-base) 5' coding region of PB2. In sequence comparisons among more than 580 influenza A strains from diverse hosts, these highly deleterious mutations were each found to affect one or more conserved bases, though they did not all lie within the most broadly conserved portions of the regions that we interrogated. We have introduced silent and conserved mutations to the critical packaging sites, which did not affect protein function but impaired viral replication at levels roughly similar to those of their defects in RNA packaging. Interestingly, certain mutations showed strong tendencies to revert to wild-type sequences, which implies that these putative packaging signals are critical for the influenza life cycle.

Nucleocapsid protein-mediated maturation of dimer initiation complex of full-length SL1 stemloop of HIV-1: sequence effects and mechanism of RNA refolding.

Specific binding of HIV-1 viral protein NCp7 to a unique 35-base RNA stem-loop SL1 is critical for formation and packaging of the genomic RNA dimer found within HIV-1 virions. NCp7 binding stimulates refolding of SL1 from a metastable kissing dimer (KD) into thermodynamically stable linear dimer (LD). Using UV melting, gel electrophoresis and heteronuclear NMR, we investigated effects of various site-specific mutations within the full-length SL1 on temperature- or NCp7-induced refolding in vitro. Refolding involved intramolecular melting of SL1 stems but not dissociation of the intermolecular KD interface. Refolding required only two NCp7 molecules per KD but was limited by the amount of NCp7 present, implying that the protein does not catalytically promote refolding. Efficient refolding depended strictly on the presence and, to a lesser degree, on sequence of a highly conserved G-rich internal loop that normally limits thermal stability of the SL1 stem. Adding two base pairs to the lower stem created a hyperstable SL1 mutant that failed to refold, even when bound by NCp7 at high stoichiometries. NMR analysis of these kinetically trapped mutant RNA-protein complexes indicated that NCp7 initiates refolding by dissociating base pairs in the upper stem of SL1. This study illuminates structural transitions critical for HIV-1 assembly and replication.

Outsourcing of Academic Clinical Laboratories: Experiences and Lessons From the Association of Pathology Chairs Laboratory Outsourcing Survey.

American hospitals are increasingly turning to service outsourcing to reduce costs, including laboratory services. Studies of this practice have largely focused on nonacademic medical centers. In contrast, academic medical centers have unique practice environments and unique mission considerations. We sought to elucidate and analyze clinical laboratory outsourcing experiences in US academic medical centers. Seventeen chairs of pathology with relevant experience were willing to participate in in-depth interviews about their experiences. Anticipated financial benefits from joint venture arrangements often eroded after the initial years of the agreement, due to increased test pricing, management fees, duplication of services in support of inpatients, and lack of incentive for utilization control on the part of the for-profit partner. Outsourcing can preclude development of lucrative outreach programs; such programs were successfully launched in several cases after joint ventures were either avoided or terminated. Common complaints included poor test turnaround time and problems with test quality (especially in molecular pathology, microbiology, and flow cytometry), leading to clinician dissatisfaction. Joint ventures adversely affected retention of academically oriented clinical pathology faculty, with adverse effects on research and education, which further exacerbated clinician dissatisfaction due to lack of available consultative expertise. Resident education in pathology and in other disciplines (especially infectious disease) suffered both from lack of on-site laboratory capabilities and from lack of teaching faculty. Most joint ventures were initiated with little or no input from pathology leadership, and input from pathology leadership was seen to have been critical in those cases where such arrangements were declined or terminated.

Benchmarking Subspecialty Practice in Academic Anatomic Pathology: The 2017 Association of Pathology Chairs Survey.

Assessment of physician workloads has become increasingly important in modern academic physician practice, where it is commonly used to allocate resources among departments, to determine staffing, and to set the compensation of individual physicians. The physician work relative value unit system is a frequently used metric in this regard. However, the application of this system to the practice of pathology has proven problematic. One area of uncertainty is the validity of using work relative value unit norms that were derived from general surgical pathology practice to assess the various subspecialties within anatomic pathology. Here, we used data from the 2017 Association of Pathology Chairs practice survey to assess salary and work relative value unit data for single-subspecialty practitioners in US academic pathology departments in the prior year (2016). Five subspecialties were evaluated: dermatopathology, gastrointestinal pathology, hematopathology/hematology, renal pathology, and neuropathology. Data for general surgical pathologists and cytopathologists were included for comparison. For this analysis, survey data were available for 168 practitioners in 43 US academic departments of pathology. Salary ranges varied little among subspecialties, with the exception of dermatopathology, where salaries were higher. In contrast, work relative value unit productivity varied widely among different subspecialties, with median values differing as much as 4- to 7-fold between subspecialties. These results suggest that the use of a single overall work relative value unit standard is not appropriate for specialty- or subspecialty-based anatomic pathology practice, and that either the benchmark norms should be tailored to individual practice patterns, or an alternative system of workload measurement should be developed.

Comprehensive structure-function analysis of the core domain of human telomerase RNA.

Telomerase is a cellular reverse transcriptase that uses part of its integral RNA (called TER) as the template to synthesize telomeric DNA repeats. Vertebrate TERs are thought to share a conserved, highly structured core domain that includes the templating sequence and a pseudoknot, but not all features of the predicted core structure have been verified directly or shown to affect telomerase enzymatic activity. Here, we report a systematic mutational analysis of the core domain (residues 1 to 210) of human telomerase RNA (hTER). Our data confirm that optimal hTER activity requires the integrity of four short helices (P2a.1, P2a, P2b, and P3) which create the proposed pseudoknot and that features of both the primary sequence and secondary structure in P2b and P3 contribute to optimal function. At least part of the long-range P1 pairing is also required, despite the lack of a known P1 counterpart in rodent TERs. Among the predicted single-stranded regions, we found that J2b/3, portions of J2a/3, and residues in and around the template make sequence-specific contributions to telomerase function. Additionally, we provide evidence that naturally occurring hTER sequence polymorphisms found in some patients with aplastic anemia can inhibit telomerase activity by disrupting critical structures within the hTER core domain.

Sustained virologic control in SIV+ macaques after antiretroviral and α4ß7 antibody therapy.

Antiretroviral drug therapy (ART) effectively suppresses replication of both the immunodeficiency viruses, human (HIV) and simian (SIV); however, virus rebounds soon after ART is withdrawn. SIV-infected monkeys were treated with a 90-day course of ART initiated at 5 weeks post infection followed at 9 weeks post infection by infusions of a primatized monoclonal antibody against the α4ß7 integrin administered every 3 weeks until week 32. These animals subsequently maintained low to undetectable viral loads and normal CD4+ T cell counts in plasma and gastrointestinal tissues for more than 9 months, even after all treatment was withdrawn. This combination therapy allows macaques to effectively control viremia and reconstitute their immune systems without a need for further therapy.

Targeting α4ß7 integrin reduces mucosal transmission of simian immunodeficiency virus and protects gut-associated lymphoid tissue from infection.

α4ß7 integrin-expressing CD4(+) T cells preferentially traffic to gut-associated lymphoid tissue (GALT) and have a key role in HIV and simian immunodeficiency virus (SIV) pathogenesis. We show here that the administration of an anti-α4ß7 monoclonal antibody just prior to and during acute infection protects rhesus macaques from transmission following repeated low-dose intravaginal challenges with SIVmac251. In treated animals that became infected, the GALT was significantly protected from infection and CD4(+) T cell numbers were maintained in both the blood and the GALT. Thus, targeting α4ß7 reduces mucosal transmission of SIV in macaques.

Mutational analyses of the influenza A virus polymerase subunit PA reveal distinct functions related and unrelated to RNA polymerase activity.

Influenza A viral polymerase is a heterotrimeric complex that consists of PA, PB1, and PB2 subunits. We previously reported that a di-codon substitution mutation (G507A-R508A), denoted J10, in the C-terminal half of PA had no apparent effect on viral RNA synthesis but prevented infectious virus production, indicating that PA may have a novel role independent of its polymerase activity. To further examine the roles of PA in the viral life cycle, we have now generated and characterized additional mutations in regions flanking the J10 site from residues 497 to 518. All tested di-codon mutations completely abolished or significantly reduced viral infectivity, but they did so through disparate mechanisms. Several showed effects resembling those of J10, in that the mutant polymerase supported normal levels of viral RNA synthesis but nonetheless failed to generate infectious viral particles. Others eliminated polymerase activity, in most cases by perturbing the normal nuclear localization of PA protein in cells. We also engineered single-codon mutations that were predicted to pack near the J10 site in the crystal structure of PA, and found that altering residues K378 or D478 each produced a J10-like phenotype. In further studies of J10 itself, we found that this mutation does not affect the formation and release of virion-like particles per se, but instead impairs the ability of those particles to incorporate each of the eight essential RNA segments (vRNAs) that make up the viral genome. Taken together, our analysis identifies mutations in the C-terminal region of PA that differentially affect at least three distinct activities: protein nuclear localization, viral RNA synthesis, and a trans-acting function that is required for efficient packaging of all eight vRNAs.

Antibody responses against xenotropic murine leukemia virus-related virus envelope in a murine model.

BACKGROUND: Xenotropic murine leukemia virus-related virus (XMRV) was recently discovered to be the first human gammaretrovirus that is associated with chronic fatigue syndrome and prostate cancer (PC). Although a mechanism for XMRV carcinogenesis is yet to be established, this virus belongs to the family of gammaretroviruses well known for their ability to induce cancer in the infected hosts. Since its original identification XMRV has been detected in several independent investigations; however, at this time significant controversy remains regarding reports of XMRV detection/prevalence in other cohorts and cell type/tissue distribution. The potential risk of human infection, coupled with the lack of knowledge about the basic biology of XMRV, warrants further research, including investigation of adaptive immune responses. To study immunogenicity in vivo, we vaccinated mice with a combination of recombinant vectors expressing codon-optimized sequences of XMRV gag and env genes and virus-like particles (VLP) that had the size and morphology of live infectious XMRV. RESULTS: Immunization elicited Env-specific binding and neutralizing antibodies (NAb) against XMRV in mice. The peak titers for ELISA-binding antibodies and NAb were 1:1024 and 1:464, respectively; however, high ELISA-binding and NAb titers were not sustained and persisted for less than three weeks after immunizations. CONCLUSIONS: Vaccine-induced XMRV Env antibody titers were transiently high, but their duration was short. The relatively rapid diminution in antibody levels may in part explain the differing prevalences reported for XMRV in various prostate cancer and chronic fatigue syndrome cohorts. The low level of immunogenicity observed in the present study may be characteristic of a natural XMRV infection in humans.

Functional characterization of yeast telomerase RNA dimerization.

Telomerase is the cellular RNA-dependent DNA polymerase (i.e. reverse transcriptase) that uses an integral RNA template to synthesize telomeric DNA repeats at the ends of linear chromosomes. Human telomerase RNA (hTERC) is thought to function as a dimeric complex consisting of two RNAs that interact with each other physically as well as genetically. We show here for the first time that the yeast Saccharomyces cerevisiae telomerase RNA TLC1 likewise forms dimers in vitro. TLC1 dimerization depends on a unique 6-base self-complementary sequence, which closely mimics palindromic sequences that mediate functional dimerization of HIV-1 and other retroviral genomes. We found that dissimilar but comparably located TLC1 palindromes from other sensu stricto yeasts can functionally substitute for that of S. cerevisiae. Yeast cells expressing dimerization-defective TLC1 alleles have shorter telomeres than those with wild-type TLC1. This study, therefore, highlights dimerization as a functionally conserved feature of the RNA templates utilized by reverse transcriptases of both viral and cellular origins.

Defective assembly of influenza A virus due to a mutation in the polymerase subunit PA.

The RNA-dependent RNA polymerase of influenza A virus is composed of three subunits that together synthesize all viral mRNAs and also replicate the viral genomic RNA segments (vRNAs) through intermediates known as cRNAs. Here we describe functional characterization of 16 site-directed mutants of one polymerase subunit, termed PA. In accord with earlier studies, these mutants exhibited diverse, mainly quantitative impairments in expressing one or more classes of viral RNA, with associated infectivity defects of varying severity. One PA mutant, however, targeting residues 507 and 508, caused only modest perturbations of RNA expression yet completely eliminated the formation of plaque-forming virus. Polymerases incorporating this mutant, designated J10, proved capable of synthesizing translationally active mRNAs and of replicating diverse cRNA or vRNA templates at levels compatible with viral infectivity. Both the mutant protein and its RNA products were appropriately localized in the cytoplasm, where influenza virus assembly occurs. Nevertheless, J10 failed to generate infectious particles from cells in a plasmid-based influenza virus assembly assay, and hemagglutinating material from the supernatants of such cells contained little or no nuclease-resistant genomic RNA. These findings suggest that PA has a previously unrecognized role in assembly or release of influenza virus virions, perhaps influencing core structure or the packaging of vRNAs or other essential components into nascent influenza virus particles.

NMR structure of the full-length linear dimer of stem-loop-1 RNA in the HIV-1 dimer initiation site.

The packaging signal of HIV-1 RNA contains a stem-loop structure, SL1, which serves as the dimerization initiation site for two identical copies of the genome and is important for packaging of the RNA genome into the budding virion and for overall infectivity. SL1 spontaneously dimerizes via a palindromic hexanucleotide sequence in its apical loop, forming a metastable kissing dimer form. Incubation with nucleocapsid protein causes this form to refold to a thermodynamically stable mature linear dimer. Here, we present an NMR structure of the latter form of the full-length SL1 sequence of the Lai HIV-1 isolate. The structure was refined using nuclear Overhauser effect and residual dipolar coupling data. The structure presents a symmetric homodimer of two RNA strands of 35 nucleotides each; it includes five stems separated by four internal loops. The central palindromic stem is surrounded by two symmetric adenine-rich 1-2 internal loops, A-bulges. All three adenines in each A-bulge are stacked inside the helix, consistent with the solution structures of shorter SL1 constructs determined previously. The outer 4-base pair stems and, proximal to them, purine-rich 1-3 internal loops, or G-bulges, are the least stable parts of the molecule. The G-bulges display high conformational variability in the refined ensemble of structures, despite the availability of many structural restraints for this region. Nevertheless, most conformations share a similar structural motif: a guanine and an adenine from opposite strands form a GA mismatch stacked on the top of the neighboring stem. The two remaining guanines are exposed, one in the minor groove and another in the major groove side of the helix, consistent with secondary structure probing data for SL1. These guanines may be recognized by the nucleocapsid protein, which binds tightly to the G-bulge in vitro.

cis-Acting packaging signals in the influenza virus PB1, PB2, and PA genomic RNA segments.

The influenza A virus genome consists of eight negative-sense RNA segments. The cis-acting signals that allow these viral RNA segments (vRNAs) to be packaged into influenza virus particles have not been fully elucidated, although the 5' and 3' untranslated regions (UTRs) of each vRNA are known to be required. Efficient packaging of the NA, HA, and NS segments also requires coding sequences immediately adjacent to the UTRs, but it is not yet known whether the same is true of other vRNAs. By assaying packaging of genetically tagged vRNA reporters during plasmid-directed influenza virus assembly in cells, we have now mapped cis-acting sequences that are sufficient for packaging of the PA, PB1, and PB2 segments. We find that each involves portions of the distal coding regions. Efficient packaging of the PA or PB1 vRNAs requires at least 40 bases of 5' and 66 bases of 3' coding sequences, whereas packaging of the PB2 segment requires at least 80 bases of 5' coding region but is independent of coding sequences at the 3' end. Interestingly, artificial reporter vRNAs carrying mismatched ends (i.e., whose 5' and 3' ends are derived from different vRNA segments) were poorly packaged, implying that the two ends of any given vRNA may collaborate in forming specific structures to be recognized by the viral packaging machinery.

Functional characterization of telomerase RNA variants found in patients with hematologic disorders.

Human telomerase uses a specific cellular RNA, called hTERC, as the template to synthesize telomere repeats at chromosome ends. Approximately 10% to 15% of patients with aplastic anemia or other bone marrow failure syndromes are carriers of hTERC sequence variants whose functional significance, in most cases, is unknown. We screened 10 reported and 2 newly discovered hTERC variants from such patients and found that 10 of these negatively affected telomerase enzymatic function when they were used to reconstitute telomerase enzymatic function in human cells. Most functional deficits were due to perturbations of hTERC secondary structure and correlated well with the degrees of telomere shortening and reduced telomerase activity observed in peripheral blood lymphocytes of the representative patients. We also found no evidence of dominant-negative activity in any of the mutants. Therefore, loss of telomerase activity and of telomere maintenance resulting from inherited hTERC mutations may limit marrow stem cell renewal and predispose some patients to bone marrow failure.

Identification and functional characterization of 2 variant alleles of the telomerase RNA template gene (TERC) in a patient with dyskeratosis congenita.

Heterozygous mutations of the human telomerase RNA template gene (TERC) have been described in patients with acquired aplastic anemia and the autosomal dominant form of dyskeratosis congenita (DKC). Patients with mutations in both TERC alleles have not yet been reported. Here, we report a patient with DKC who inherited 2 distinct TERC sequence variants from her parents; a deletion (216_229del) in one and a point mutation (37A>G) in the other allele of the TERC gene. Her marrow was hypocellular and showed an abnormal clone [46, XX t(7;21)(q34;q22)]. The telomere lengths in leukocytes of the patient and her relatives were shorter than those of the age-matched controls and were progressively shorter in subsequent generations of family members with the 216_229del allele. Telomerase enzymatic levels in lymphocytes from the patient were approximately half of those measured in healthy controls. The 216_229del mutation failed to reconstitute telomerase activity in transfected cells, but, when coexpressed with the 37A>G variant, telomerase activity was only modestly suppressed. These clinical and laboratory findings support the concept that telomerase levels in human hematopoietic stem cells are tightly controlled as even moderately reduced levels result in accelerated telomere shortening and eventual marrow failure.