SARS-CoV-2 Antiviral Prescribing Gaps Among Non-Hospitalized High-Risk Adults.

Within a multi-state clinical cohort, SARS-CoV-2 antiviral prescribing patterns were evaluated from April 2022-June 2023 among non-hospitalized SARS-CoV-2-infected patients with risk factors for severe COVID-19. Among 3,247 adults, only 31.9% were prescribed an antiviral agent (87.6% nirmatrelvir/ritonavir, 11.9% molnupiravir, 0.5% remdesivir), highlighting the need to identify and address treatment barriers.

Use of next-generation sequencing to detect mutations associated with antiviral drug resistance in cytomegalovirus.

Cytomegalovirus (CMV) is a significant cause of morbidity and mortality among immunocompromised hosts, including transplant recipients. Antiviral prophylaxis or treatment is used to reduce the incidence of CMV disease in this patient population; however, there is concern about increasing antiviral resistance. Detection of antiviral resistance in CMV was traditionally accomplished using Sanger sequencing of UL54 and UL97 genes, in which specific mutations may result in reduced antiviral activity. In this study, a novel next-generation sequencing (NGS) method was developed and validated to detect mutations in UL54/UL97 associated with antiviral resistance. Plasma samples (n = 27) submitted for antiviral resistance testing by Sanger sequencing were also analyzed using the NGS method. When compared to Sanger sequencing, the NGS assay demonstrated 100% (27/27) overall agreement for determining antiviral resistance/susceptibility and 88% (22/25) agreement at the level of resistance-associated mutations. The limit of detection of the NGS method was determined to be 500 IU/mL, and the lower threshold for detecting mutations associated with resistance was established at 15%. The NGS assay represents a novel laboratory tool that assists healthcare providers in treating patients who are infected with CMV harboring resistance-associated mutations and who may benefit from tailored antiviral therapy.

Standardization of SARS-CoV-2 Cycle Threshold Values: Multisite Investigation Evaluating Viral Quantitation across Multiple Commercial COVID-19 Detection Platforms.

The demand for testing during the coronavirus disease 2019 (COVID-19) pandemic has resulted in the production of several different commercial platforms and laboratory-developed assays for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This has created several challenges, including, but not limited to, the standardization of diagnostic testing, utilization of cycle threshold (CT) values for quantitation and clinical interpretation, and data harmonization. Using reference standards consisting of a linear range of SARS-CoV-2 concentrations quantitated by viral culture-based methods and droplet digital PCR, we investigated the commutability and standardization of SARS-CoV-2 quantitation across different laboratories in the United States. We assessed SARS-CoV-2 CT values generated on multiple reverse transcription-PCR (RT-PCR) platforms and analyzed PCR efficiencies, linearity, gene targets, and CT value agreement. Our results demonstrate the inappropriateness of using SARS-CoV-2 CT values without established standards for viral quantitation. Further, we emphasize the importance of using reference standards and controls validated to independent assays, to compare results across different testing platforms and move toward better harmonization of COVID-19 quantitative test results. IMPORTANCE From the onset of the COVID-19 pandemic, the demand for SARS-CoV-2 testing has resulted in an explosion of analytical tests with very different approaches and designs. The variability in testing modalities, compounded by the lack of available commercial reference materials for standardization early in the pandemic, has led to several challenges regarding data harmonization for viral quantitation. In this study, we assessed multiple commercially available RT-PCR platforms across different laboratories within the United States using standardized reference materials characterized by viral culture methods and droplet digital PCR. We observed variability in the results generated by different instruments and laboratories, further emphasizing the importance of utilizing validated reference standards for quantitation, to better harmonize SARS-CoV-2 test results.

Evaluation of 2 multiplex real-time PCR assays for the detection of HSV-1/2 and Varicella zoster virus directly from clinical samples.

We compared the performance of 2 multiplex assays (Focus Simplexa and Quidel Lyra) to individual real-time PCR for the detection of herpes simplex virus-1 (HSV-1), HSV-2, and Varicella zoster virus (VZV) from clinical specimens. Results were compared to a consensus standard, defined as the result obtained by at least 2 of the 3 molecular methods. The sensitivity of the Quidel assay ranged from 92.0% for HSV-1 to 97.7% for HSV-2, while the specificity for all targets was 100%. The Focus assay demonstrated 100% sensitivity for all targets, and the percent specificity ranged from 96.8% for HSV-1 to 100% for HSV-2 and VZV.

Modulation of B-cell exosome proteins by gamma herpesvirus infection.

The human gamma herpesviruses, Kaposi sarcoma-associated virus (KSHV) and EBV, are associated with multiple cancers. Recent evidence suggests that EBV and possibly other viruses can manipulate the tumor microenvironment through the secretion of specific viral and cellular components into exosomes, small endocytically derived vesicles that are released from cells. Exosomes produced by EBV-infected nasopharyngeal carcinoma cells contain high levels of the viral oncogene latent membrane protein 1 and viral microRNAs that activate critical signaling pathways in recipient cells. In this study, to determine the effects of EBV and KSHV on exosome content, quantitative proteomics techniques were performed on exosomes purified from 11 B-cell lines that are uninfected, infected with EBV or with KSHV, or infected with both viruses. Using mass spectrometry, 871 proteins were identified, of which ∼360 were unique to the viral exosomes. Analysis by 2D difference gel electrophoresis and spectral counting identified multiple significant changes compared with the uninfected control cells and between viral groups. These data predict that both EBV and KSHV exosomes likely modulate cell death and survival, ribosome function, protein synthesis, and mammalian target of rapamycin signaling. Distinct viral-specific effects on exosomes suggest that KSHV exosomes would affect cellular metabolism, whereas EBV exosomes would activate cellular signaling mediated through integrins, actin, IFN, and NFκB. The changes in exosome content identified in this study suggest ways that these oncogenic viruses modulate the tumor microenvironment and may provide diagnostic markers specific for EBV and KSHV associated malignancies.

Analysis of global sumoylation changes occurring during keratinocyte differentiation.

Sumoylation is a highly dynamic process that plays a role in a multitude of processes ranging from cell cycle progression to mRNA processing and cancer. A previous study from our lab demonstrated that SUMO plays an important role in keratinocyte differentiation. Here we present a new method of tracking the sumoylation state of proteins by creating a stably transfected HaCaT keratinocyte cell line expressing an inducible SNAP-SUMO3 protein. The SNAP-tag allows covalent fluorescent labeling that is denaturation resistant. When combined with two-dimensional gel electrophoresis, the SNAP-tag technology provides direct visualization of sumoylated targets and can be used to follow temporal changes in the global cohort of sumoylated proteins during dynamic processes such as differentiation. HaCaT keratinocyte cells expressing SNAP-SUMO3 displayed normal morphological and biochemical features that are consistent with typical keratinocyte differentiation. SNAP-SUMO3 also localized normally in these cells with a predominantly nuclear signal and some minor cytoplasmic staining, consistent with previous reports for untagged SUMO2/3. During keratinocyte differentiation the total number of proteins modified by SNAP-SUMO3 was highest in basal cells, decreased abruptly after induction of differentiation, and slowly rebounded beginning between 48 and 72 hours as differentiation progressed. However, within this overall trend the pattern of change for individual sumoylated proteins was highly variable with both increases and decreases in amount over time. From these results we conclude that sumoylation of proteins during keratinocyte differentiation is a complex process which likely reflects and contributes to the biochemical changes that drive differentiation.

HPV E6 proteins target Ubc9, the SUMO conjugating enzyme.

The human papillomavirus oncogenic protein, E6, interacts with a number of cellular proteins, and for some targets, E6 directs their degradation through the ubiquitin-proteasome pathway. Post-translational modification with ubiquitin-like modifiers, such as SUMO, also influences protein activities, protein-protein interactions, and protein stability. We report that the high risk HPVE6 proteins reduce the intracellular quantity of the sole SUMO conjugation enzyme, Ubc9, concomitant with decreased host sumoylation. E6 did not significantly influence transcription of Ubc9, indicating that the effects were likely at the protein level. Consistent with typical E6-mediated proteasomal degradation, E6 bound to Ubc9 in vitro, and required E6AP for reduction of Ubc9 levels. Under stable E6 expression conditions in differentiating keratinocytes there was a decrease in Ubc9 and a loss of numerous sumoylated targets indicating a significant perturbation of the normal sumoylation profile. While E6 is known to inhibit PIASy, a SUMO ligase, our results suggest that HPV E6 also targets the Ubc9 protein to modulate host cell sumoylation, suggesting that the sumoylation system may be an important target during viral reproduction and possibly the subsequent development of cervical cancer.

Host cell sumoylation level influences papillomavirus E2 protein stability.

The stability of papillomavirus E2 proteins is regulated by proteasomal degradation, and regulation of degradation could contribute to the higher expression levels of E2 proteins observed in suprabasal layers of differentiated skin. We have recently shown that the E2 proteins are modified by sumoylation [Wu Y-C, Roark AA, Bian X-L, Wilson, VG (2008) Virol 378:329-338], and that sumoylation levels are up-regulated during keratinocyte differentiation [Deyrieux AF, Rosas-Acosta G, Ozbun MA, Wilson VG (2007) J Cell Sci 120:125-136]. These observations, coupled with the known ability of sumoylation to prevent proteasomal degradation of certain proteins, suggested that this modification might contribute to stabilizing E2 proteins in suprabasal keratinocytes. Conditions that increased overall sumoylation were found to increase the intracellular amounts of the HPV11, 16, and 18 E2 proteins. No effect of sumoylation was seen on E2 transcripts, and the increased levels of E2 proteins resulted from a greatly increased half-life for the E2 proteins. In vitro studies confirmed that sumoylation could block the proteasomal degradation of the 16E2 protein. Interestingly, this stabilization effect was indirect as it did not require sumoylation of 16E2 itself and must be acting through sumoylation of a cellular target(s). This sumoylation-dependent, indirect stabilization of E2 proteins is a novel process that may couple E2 levels to changes in the cellular environment. Specifically, our results suggest that the levels of papillomavirus E2 protein could be up-regulated in differentiating keratinocytes in response to the increased overall sumoylation that accompanies differentiation.

Ubiquitin proteolytic system: focus on SUMO.

The small ubiquitin-like modifier proteins (Smt3 in yeast and SUMOs 1-4 in vertebrates) are members of the ubiquitin super family. Like ubiquitin, the SUMOs are protein modifiers that are covalently attached to the epsilon-amino group of lysine residues in the substrates. The application of proteomics to the SUMO field has greatly expanded both the number of known targets and the number of identified target lysines. As new refinements of proteomic techniques are developed and applied to sumoylation, an explosion of novel data is likely in the next 5 years. This ability to examine sumoylated proteins globally, rather than individually, will lead to new insights into both the functions of the individual SUMO types, and how dynamic changes in overall sumoylation occur in response to alterations in cellular environment. In addition, there is a growing appreciation for the existence of cross-talk mechanisms between the sumoylation and ubiquitinylation processes. Rather than being strictly parallel, these two systems have many points of intersection, and it is likely that the coordination of these two systems is a critical contributor to the regulation of many fundamental cellular events.