Human cytomegalovirus G protein-coupled receptor US28 promotes latency by attenuating c-fos.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that undergoes latency in cells of the hematopoietic compartment, although the mechanisms underlying establishment and maintenance of latency remain elusive. We previously reported that the HCMV-encoded G protein-coupled receptor (GPCR) homolog US28 is required for successful latent infection. We now show that US28 protein (pUS28) provided in trans complements the US28Δ lytic phenotype in myeloid cells, suggesting that sustained US28 expression is necessary for long-term latency. Furthermore, expression of pUS28 at the time of infection represses transcription from the major immediate early promoter (MIEP) within 24 h. However, this repression is only maintained in the presence of continual pUS28 expression provided in trans Our data also reveal that pUS28-mediated signaling attenuates both expression and phosphorylation of cellular fos (c-fos), an AP-1 transcription factor subunit, to repress MIEP-driven transcription. AP-1 binds to the MIEP and promotes lytic replication, and in line with this we find that US28Δ infection results in an increase in AP-1 binding to the MIEP, compared with WT latent infection. Pharmacological inhibition of c-fos represses the MIEP during US28Δ infection to levels similar to those we observe during WT latent infection. Together, our data reveal that US28 is required for both establishment and long-term maintenance of HCMV latency, which is modulated, at least in part, by repressing functional AP-1 binding to the MIEP.

Identification of Zika Virus Inhibitors Using Homology Modeling and Similarity-Based Screening to Target Glycoprotein E.

The World Health Organization has designated Zika virus (ZIKV) as a dangerous, mosquito-borne pathogen that can cause severe developmental defects. The primary goal of this work was identification of small molecules as potential ZIKV inhibitors that target the viral envelope glycoprotein (ZIKV E) involved in membrane fusion and viral entry. A homology model of ZIKV E containing the small molecule ß-octyl glucoside (BOG) was constructed, on the basis of an analogous X-ray structure from dengue virus, and >4 million commercially available compounds were computationally screened using the program DOCK6. A key feature of the screen involved the use of similarity-based scoring to identify inhibitor candidates that make similar interaction energy patterns (molecular footprints) as the BOG reference. Fifty-three prioritized compounds underwent experimental testing using cytotoxicity, cell viability, and tissue culture infectious dose 50% (TCID50) assays. Encouragingly, relative to a known control (NITD008), six compounds were active in both the cell viability assay and the TCID50 infectivity assay, and they showed activity in a third caspase activity assay. In particular, compounds 8 and 15 (tested at 25 µM) and compound 43 (tested at 10 µM) appeared to provide significant protection to infected cells, indicative of anti-ZIKV activity. Overall, the study highlights how similarity-based scoring can be leveraged to computationally identify potential ZIKV E inhibitors that mimic a known reference (in this case BOG), and the experimentally verified hits provide a strong starting point for further refinement and optimization efforts.

Identification of Ebola Virus Inhibitors Targeting GP2 Using Principles of Molecular Mimicry.

A key step in the Ebola virus (EBOV) replication cycle involves conformational changes in viral glycoprotein 2 (GP2) which facilitate host-viral membrane fusion and subsequent release of the viral genome. Ebola GP2 plays a critical role in virus entry and has similarities in mechanism and structure to the HIV gp41 protein for which inhibitors have been successfully developed. In this work, a putative binding pocket for the C-terminal heptad repeat in the N-terminal heptad repeat trimer was targeted for identification of small molecules that arrest EBOV-host membrane fusion. Two computational structure-based virtual screens of ∼1.7 M compounds were performed (DOCK program) against a GP2 five-helix bundle, resulting in 165 commercially available compounds purchased for experimental testing. Based on assessment of inhibitory activity, cytotoxicity, and target specificity, four promising candidates emerged with 50% inhibitory concentration values in the 3 to 26 µM range. Molecular dynamics simulations of the two most potent candidates in their DOCK-predicted binding poses indicate that the majority of favorable interactions involve seven highly conserved residues that can be used to guide further inhibitor development and refinement targeting EBOV.IMPORTANCE The most recent Ebola virus disease outbreak, from 2014 to 2016, resulted in approximately 28,000 individuals becoming infected, which led to over 12,000 causalities worldwide. The particularly high pathogenicity of the virus makes paramount the identification and development of promising lead compounds to serve as inhibitors of Ebola infection. To limit viral load, the virus-host membrane fusion event can be targeted through the inhibition of the class I fusion glycoprotein of Ebolavirus In the current work, several promising small-molecule inhibitors that target the glycoprotein GP2 were identified through systematic application of structure-based computational and experimental drug design procedures.

Human Cytomegalovirus US28 Is Important for Latent Infection of Hematopoietic Progenitor Cells.

UNLABELLED: Human cytomegalovirus (HCMV) resides latently in hematopoietic progenitor cells (HPCs). During latency, only a subset of HCMV genes is transcribed, including one of the four virus-encoded G protein-coupled receptors (GPCRs), US28. Although US28 is a multifunctional lytic protein, its function during latency has remained undefined. We generated a panel of US28 recombinant viruses in the bacterial artificial chromosome (BAC)-derived clinical HCMV strain TB40/E-mCherry. We deleted the entire US28 open reading frame (ORF), deleted all four of the viral GPCR ORFs, or deleted three of the HCMV GPCRs but not the US28 wild-type protein. Using these recombinant viruses, we assessed the requirement for US28 during latency in the Kasumi-3 in vitro latency model system and in primary ex vivo-cultured CD34(+) HPCs. Our data suggest that US28 is required for latency as infection with viruses lacking the US28 ORF alone or in combination with the remaining HCMV-encoded GPCR results in transcription from the major immediate early promoter, the production of extracellular virions, and the production of infectious virus capable of infecting naive fibroblasts. The other HCMV GPCRs are not required for this phenotype as a virus expressing only US28 but not the remaining virus-encoded GPCRs is phenotypically similar to that of wild-type latent infection. Finally, we found that US28 copurifies with mature virions and is expressed in HPCs upon virus entry although its expression at the time of infection does not complement the US28 deletion latency phenotype. This work suggests that US28 protein functions to promote a latent state within hematopoietic progenitor cells. IMPORTANCE: Human cytomegalovirus (HCMV) is a widespread pathogen that, once acquired, remains with its host for life. HCMV remains latent, or quiescent, in cells of the hematopoietic compartment and upon immune challenge can reactivate to cause disease. HCMV-encoded US28 is one of several genes expressed during latency although its biological function during this phase of infection has remained undefined. Here, we show that US28 aids in promoting experimental latency in tissue culture.

Cryptococcus neoformans Coinfection Dampens the TNF-α Response in HIV-1-Infected Human THP-1 Macrophages.

Cryptococcus neoformans is a devastating opportunistic fungal pathogen. It mostly impacts people in an immunocompromised state, such as people living with HIV/AIDS and following organ transplantation. Macrophages, in addition to being a major cellular reservoir of HIV-1, represent a unique niche in which both C. neoformans and HIV-1 can coinhabit in the course of natural infection. Here, we report the observation that HIV-1 infection of THP-1 macrophages increases the rate at which they phagocytose C. neoformans cells. We investigated the tumor necrosis factor alpha (TNF-α) signaling and nuclear factor kappa B (NF-κB) activation in human monocyte-derived macrophages infected with HIV-1 alone, as well as those coinfected with HIV-1 and C. neoformans Our findings showed that while HIV-1 infection alone upregulates TNF-α production and activates NF-κB signaling, C. neoformans coinfection drastically and rapidly dampens this proinflammatory response. These data suggest an antagonism between two important human pathogens during coinfection of macrophages.IMPORTANCE Fungal infections are one of the leading causes of death for people who live with HIV/AIDS. Even though these pathogens are independently well studied, it is still enigmatic how coinfection with HIV-1 and C. neoformans alters gene expression and cellular processes, especially in clinically relevant cell types. Understanding the interplay between these two pathogens is especially critical because C. neoformans mortality largely depends on the host's immunocompromised state during viral infection. Studying this coinfection is challenging since HIV-1 only infects human cells, and the modified murine HIV-1 virus does not reproduce the clinical landmarks of HIV-1 infection or AIDS in mice. Our observations shed light on how these two pathogens trigger opposing trends in TNF-α and NF-κB signaling in human monocyte-derived macrophages.

Prospective assessment of catheter-associated bacteriuria clinical presentation, epidemiology, and colonization dynamics in nursing home residents.

BACKGROUNDCatheterization facilitates continuous bacteriuria, for which the clinical significance remains unclear. This study aimed to determine the clinical presentation, epidemiology, and dynamics of bacteriuria in a cohort of long-term catheterized nursing home residents.METHODSProspective urine culture, urinalysis, chart review, and assessment of signs and symptoms of infection were performed weekly for 19 study participants over 7 months. All bacteria ≥ 1 × 103 cfu/mL were cultured, isolated, identified, and tested for susceptibility to select antimicrobials.RESULTSIn total, 226 of the 234 urine samples were polymicrobial (97%), with an average of 4.7 isolates per weekly specimen. A total of 228 urine samples (97%) exhibited ≥ 1 × 106 CFU/mL, 220 (94%) exhibited abnormal urinalysis, 126 (54%) were associated with at least 1 possible sign or symptom of infection, and 82 (35%) would potentially meet a standardized definition of catheter-associated urinary tract infection (CAUTI), but only 3 had a caregiver diagnosis of CAUTI. Bacterial isolates (286; 30%) were resistant to a tested antimicrobial agent, and bacteriuria composition was remarkably stable despite a combined total of 54 catheter changes and 23 weeks of antimicrobial use.CONCLUSIONBacteriuria composition was largely polymicrobial, including persistent colonization by organisms previously considered to be urine culture contaminants. Neither antimicrobial use nor catheter changes sterilized the urine, at most resulting in transient reductions in bacterial burden followed by new acquisition of resistant isolates. Thus, this patient population exhibits a high prevalence of bacteriuria coupled with potential indicators of infection, necessitating further exploration to identify sensitive markers of true infection.FUNDINGThis work was supported by the NIH (R00 DK105205, R01 DK123158, UL1 TR001412).

Activin receptor signaling regulates cocaine-primed behavioral and morphological plasticity.

Activin receptor signaling, including the transcription factor Smad3, was upregulated in the rat nucleus accumbens (NAc) shell following withdrawal from cocaine. Direct genetic and pharmacological manipulations of this pathway bidirectionally altered cocaine seeking while governing morphological plasticity in NAc neurons. Thus, Activin/Smad3 signaling is induced following withdrawal from cocaine, and such regulation may be a key molecular mechanism underlying behavioral and cellular plasticity in the brain following cocaine self-administration.

Transforming growth factor beta receptor 1 is increased following abstinence from cocaine self-administration, but not cocaine sensitization.

The addicted phenotype is characterized as a long-lasting, chronically relapsing disorder that persists following long periods of abstinence, suggesting that the underlying molecular changes are stable and endure for long periods even in the absence of drug. Here, we investigated Transforming Growth Factor-Beta Type I receptor (TGF-ß R1) expression in the nucleus accumbens (NAc) following periods of withdrawal from cocaine self-administration (SA) and a sensitizing regimen of non-contingent cocaine. Rats were exposed to either (i) repeated systemic injections (cocaine or saline), or (ii) self-administration (cocaine or saline) and underwent a period of forced abstinence (either 1 or 7 days of drug cessation). Withdrawal from cocaine self-administration resulted in an increase in TGF-ß R1 protein expression in the NAc compared to saline controls. This increase was specific for volitional cocaine intake as no change in expression was observed following a sensitizing regimen of experimenter-administered cocaine. These findings implicate TGF-ß signaling as a novel potential therapeutic target for treating drug addiction.