Pokeweed antiviral protein (PAP), a single chain ribosome-inactivating protein (RIP) isolated from pokeweed plants (Phytolacca americana), removes specific adenine and guanine residues from the highly conserved, ␣-sarcin/ ricin loop in the large rRNA, resulting in inhibition of protein synthesis. We recently demonstrated that PAP could also inhibit translation of mRNAs and viral RNAs that are capped by binding to the cap structure and depurinating the RNAs downstream of the cap. Cell growth is inhibited when PAP cDNA is expressed in the yeast Saccharomyces cerevisiae under the control of the galactose-inducible GAL1 promoter. Here, we show that overexpression of wild type PAP in yeast leads to a decrease in PAP mRNA abundance. The decrease in mRNA levels is not observed with an active site mutant, indicating that it is due to the N-glycosidase activity of the protein. PAP expression had no effect on steady state levels of mRNA from four different endogenous yeast genes examined, indicating specificity. We demonstrate that PAP can depurinate the rRNA in trans in a translation-independent manner. When rRNA is depurinated and translation is inhibited, the steady state levels of PAP mRNA increase dramatically relative to the U3 snoRNA. Using a PAP variant which depurinates rRNA, inhibits translation but does not destabilize its mRNA, we demonstrate that PAP mRNA is destabilized after its levels are up-regulated by a mechanism that occurs independently of rRNA depurination and translation. We quantify the extent of rRNA depurination in vivo using a novel primer extension assay and show that the temporal pattern of rRNA depurination is similar to the pattern of PAP mRNA destabilization, suggesting that they may occur by a common mechanism. These results provide the first in vivo evidence that a single chain RIP targets not only the large rRNA but also its own mRNA. These findings have implications for understanding the biological function of RIPs.Pokeweed antiviral protein (PAP), 1 a single chain ribosomeinactivating protein (RIP) isolated from the leaves of pokeweed plants (Phytolacca americana), removes specific adenine and guanine residues from the highly conserved, ␣-sarcin/ricin (S/R) loop in the large rRNA (1-3). The enzymatic removal of specific purines from the S/R loop has been reported to interfere with the binding of eEF-2 (elongation factor 2) and inhibit protein synthesis at the translocation step (4, 5). RIPs are protein toxins produced by organisms ranging from bacteria to plants. Because of their selective toxicity, they have been used as biological weapons, to protect plants against pathogens, and as therapies against cancer. Their biological function in the organisms that produce them is unknown. PAP is thought to be a defense protein because it is a potent inhibitor of animal and plant viral pathogens, including human immunodeficiency virus, poliovirus, herpes simplex virus, influenza, potato virus X, and brome mosaic virus (6 -10). Because of its cytotoxicity to dividing cells, PAP is currently und...

The distribution of upper respiratory viral loads (VL) in asymptomatic children infected with SARS-CoV-2 is unknown. We assessed PCR cycle threshold (Ct) values and estimated VL in infected asymptomatic children diagnosed in nine pediatric hospital testing programs. Records for asymptomatic and symptomatic patients with positive clinical SARS-CoV-2 tests were reviewed. Ct values were adjusted by centering each value around the institutional median Ct value from symptomatic children tested with that assay, and converted to estimated VL (copies/mL) using internal or manufacturer data. Adjusted Ct values and estimated VL for asymptomatic versus symptomatic children (118 vs. 197 ages 0-4; 79 vs 97 ages 5-9; 69 vs 75 ages 10-13; 73 vs 109 ages 14-17) were compared. The median adjusted Ct value in asymptomatic children was 10.3 cycles higher than for symptomatic children (p< 0.0001), and VL 3-4 logs lower (p<0.0001); differences were consistent (p<0.0001) across all four age brackets. These differences were consistent across all institutions and by sex, ethnicity, and race. Asymptomatic children with diabetes (OR 6.5, p = 0.01), recent contact (OR 2.3, p = 0.02), and testing for surveillance (OR 2.7, p = 0.005) had higher estimated risk of having a Ct value in the lowest quartile than children without, while immunocompromise had no effect. Children with asymptomatic SARS-CoV-2 infection had lower levels of virus in the nasopharynx/oropharynx than symptomatic children, but timing of infection relative to diagnosis likely impacted levels in asymptomatic children. Caution is recommended when choosing diagnostic tests for screening of asymptomatic children.

Natural killer (NK) cells play a critical role in controlling murine cytomegalovirus (MCMV) and can mediate both cytokine production and direct cytotoxicity. The NK cell activation receptor, Ly49H, is responsible for genetic resistance to MCMV in C57BL/6 mice. Recognition of the viral m157 protein by Ly49H is sufficient for effective control of MCMV infection. Additionally, during the host response to infection, distinct immune and non-immune cells elaborate a variety of pleiotropic cytokines which have the potential to impact viral pathogenesis, NK cells, and other immune functions, both directly and indirectly. While the effects of various immune deficiencies have been examined for general antiviral phenotypes, their direct effects on Ly49H-dependent MCMV control are poorly understood. To specifically interrogate Ly49H-dependent functions, herein we employed an in vivo viral competition approach to show Ly49H-dependent MCMV control is specifically mediated through cytotoxicity but not IFNγ production. Whereas m157 induced Ly49H-dependent degranulation, efficient cytotoxicity also required either IL-12 or type I interferon (IFN-I) which acted directly on NK cells to produce granzyme B. These studies demonstrate that both of these distinct NK cell-intrinsic mechanisms are integrated for optimal viral control by NK cells.

Pokeweed antiviral protein and several other ribosome inactivating proteins are effective against a broad range of viruses. Recent results have shown that their enzymatic activity is not limited to depurination of the large rRNA, they can depurinate other nucleic acids, including viral RNAs. Antiviral activity of RIPs is summarized here in light of their novel activities and recent developments in the field.

Lateral-flow assays (LFAs) are rapid and inexpensive, yet they are nearly 1,000-fold less sensitive than laboratory-based tests. Here we show that plasmonically active antibody-conjugated fluorescent gold nanorods can make conventional LFAs ultrasensitive. With sample-to-answer times within 20 min, plasmonically enhanced LFAs read out via a standard benchtop fluorescence scanner attained about 30-fold improvements in dynamic range and in detection limits over 4-h-long gold-standard enzyme-linked immunosorbent assays, and achieved 95% clinical sensitivity and 100% specificity for antibodies in plasma and for antigens in nasopharyngeal swabs from individuals with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Comparable improvements in the assay's performance can also be achieved via an inexpensive portable scanner, as we show for the detection of interleukin-6 in human serum samples and of the nucleocapsid protein of SARS-CoV-2 in nasopharyngeal samples. Plasmonically enhanced LFAs outperform standard laboratory tests in sensitivity, speed, dynamic range, ease of use and cost, and may provide advantages in point-of-care diagnostics.Lateral-flow assays (LFAs) are among the simplest, fastest and cheapest point-of-care (POC) diagnostic methods, and offer broad potential for population-level screening for disease 1,2 . Although numerous LFAs for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies 3-5 and antigens 6,7 have been introduced, none has sensitivity and quantitation comparable to laboratory-based diagnostics such as real-time PCR with reverse transcription (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) [8][9][10] . In general, conventional colorimetric LFAs are ~1,000-fold less sensitive than these standard laboratory tests 11,12 , and diagnosis using LFAs requires an additional confirmatory laboratory-based test to correctly establish negative results. Colorimetric LFAs are often inadequate for quantitative read-outs, owing to limited changes in colour with respect to the variation of the concentration of the target analyte 13 .