Facile Preparation of Polysarcosine-Tethered Inorganic Nanospheres by Using Unimolecular Polypept(o)ides as Template.

Polymer-inorganic nanocomposites that integrate the advantages of both polymers and inorganic nanoparticles (NPs) are broadly exploited for versatile applications. Especially, emerging polymer-templated preparation of inorganic NPs has drawn extensive attention, which is ascribed to simplified synthesis and feasible tunability. However, how to precisely fabricate biocompatible polymer-inorganic NPs remains unsolved. In this article, by mild ring opening polymerization (ROP) of ß-benzyl L-aspartate N-carboxyanhydrides (BLA-NCAs) and sarcosine N-carboxyanhydrides (Sar-NCAs) and subsequent debenzylation, a series of poly(amino acid)-based unimolecular micelles (PAMAM-g-(PLA-b-PSar)) are facilely synthesized. Afterward, by utilization of these star-like polymers as template, the controllable preparation of various PSar-tethered inorganic NPs is investigated and characterized meticulously. This general strategy for the preparation of PSar-tethered inorganic NPs can bring a great chance for future fabrication of biomedical nanoplatforms.

Viral Resistance Analyses From the Remdesivir Phase 3 Adaptive COVID-19 Treatment Trial-1 (ACTT-1).

BACKGROUND: Remdesivir is approved for treatment of coronavirus disease 2019 (COVID-19) in nonhospitalized and hospitalized adult and pediatric patients. Here we present severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) resistance analyses from the phase 3 ACTT-1 randomized placebo-controlled trial conducted in adult participants hospitalized with COVID-19. METHODS: Swab samples were collected at baseline and longitudinally through day 29. SARS-CoV-2 genomes were sequenced using next-generation sequencing. Phenotypic analysis was conducted directly on participant virus isolates and/or using SARS-CoV-2 subgenomic replicons expressing mutations identified in the Nsp12 target gene. RESULTS: Among participants with both baseline and postbaseline sequencing data, emergent Nsp12 substitutions were observed in 12 of 31 (38.7%) and 12 of 30 (40.0%) participants in the remdesivir and placebo arms, respectively. No emergent Nsp12 substitutions in the remdesivir arm were observed in more than 1 participant. Phenotyping showed low to no change in susceptibility to remdesivir relative to wild-type Nsp12 reference for the substitutions tested: A16V (0.8-fold change in EC50), P323L + V792I (2.2-fold), C799F (2.5-fold), K59N (1.0-fold), and K59N + V792I (3.4-fold). CONCLUSIONS: The similar rate of emerging Nsp12 substitutions in the remdesivir and placebo arms and the minimal change in remdesivir susceptibility among tested substitutions support a high barrier to remdesivir resistance development in COVID-19 patients. Clinical Trials Registration. NCT04280705.

Rapid Antigen and Antibody Microfluidic Immunofluorescence Assays Compared to Culture, PCR, and Laboratory Reference Tests: Performance in a Longitudinal Cohort.

We evaluated the performance of rapid antigen (RAg) and antibody (RAb) microfluidic diagnostics with serial sampling of 71 participants at 6 visits over 2 months following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Rapid tests showed strong agreement with laboratory references (κAg = 81.0%; κAb = 87.8%). RAg showed substantial concordance to both virus growth in culture and PCR positivity 0-5 days since symptom onset (κAg-culture = 60.1% and κAg-PCR = 87.1%). PCR concordance to virus growth in culture was similar (κPCR-culture = 70.0%), although agreement between RAg and culture was better overall (κAg-culture = 45.5% vs κPCR-culture = 10.0%). Rapid antigen and antibody testing by microfluidic immunofluorescence platform are highly accurate for characterization of acute infection.

COVID-19 Antigen Results Correlate with the Quantity of Replication-Competent SARS-CoV-2 in a Cross-Sectional Study of Ambulatory Adults during the Delta Wave.

Appropriate interpretation of various diagnostic tests for COVID-19 is critical, yet the association among rapid antigen tests, reverse transcription (RT)-PCR, and viral culture has not been fully defined. To determine whether rapid antigen testing correlates with the presence and quantity of replication-competent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in ambulatory adults, 626 adult participants were enrolled in a cross-sectional diagnostic study. Each participant had two anterior nasal swabs obtained for rapid antigen and RT-PCR testing and SARS-CoV-2 viral culture. The primary outcomes were the presence and quantification of SARS-CoV-2 growth in VeroE6-ACE2-TMPRSS2 cells in asymptomatic and symptomatic ambulatory adults. In this cross-sectional study of 626 adult outpatients, the sensitivity of a single positive antigen test to identify replication-competent SARS-CoV-2 was 63.6% in asymptomatic and 91.0% in symptomatic participants. Viral culture titers were the highest at the onset of symptoms and rapidly declined by 7 days after symptom onset. The positive agreement of the rapid antigen test with RT-PCR at a cycle threshold CT less than 30 was 66.7% in asymptomatic and 90.7% in symptomatic participants. Among symptomatic participants a with a CT less than 30, a single antigen test had a positive agreement of 90.7% (95% confidence interval [CI], 84.8% to 94.8%). There was 100% negative agreement as all 425 RT-PCR-negative participants had a negative antigen test. A positive antigen test in symptomatic adults with COVID-19 has a strong correlation with replication-competent SARS-CoV-2. Rapid antigen test results may be a suitable proxy for infectiousness. IMPORTANCE Do rapid antigen test results correlate with replication-competent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (i.e., infectious) virus? In this cross-sectional diagnostic study of 626 adults, the sensitivity of the antigen test to identify replication-competent SARS-CoV-2 was 63.6% in asymptomatic and 91.0% in symptomatic participants. Viral culture titers were the highest at the onset of symptoms and rapidly declined by 7 days after symptom onset. The positive agreement of the rapid antigen test with reverse transcription (RT)-PCR at a CT of less than 30 was 66.7% in asymptomatic participants and 90.7% in symptomatic participants. A positive antigen test may be an appropriate surrogate for identifying replication-competent virus in symptomatic individuals with COVID-19.

Duration of viral infectiousness and correlation with symptoms and diagnostic testing in non-hospitalized adults during acute SARS-CoV-2 infection: A longitudinal cohort study.

BACKGROUND: Guidelines for SARS-CoV-2 have relied on limited data on duration of viral infectiousness and correlation with COVID-19 symptoms and diagnostic testing. METHODS: We enrolled ambulatory adults with acute SARS-CoV-2 infection and performed serial measurements of COVID-19 symptoms, nasal swab viral RNA, nucleocapsid (N) and spike (S) antigens, and replication-competent SARS-CoV-2 by viral growth in culture. We determined average time from symptom onset to a first negative test result and estimated risk of infectiousness, as defined by positive viral growth in culture. RESULTS: Among 95 adults, median [interquartile range] time from symptom onset to first negative test result was 9 [5] days, 13 [6] days, 11 [4] days, and >19 days for S antigen, N antigen, culture growth, and viral RNA by RT-PCR, respectively. Beyond two weeks, virus growth and N antigen titers were rarely positive, while viral RNA remained detectable among half (26/51) of participants tested 21-30 days after symptom onset. Between 6-10 days from symptom onset, N antigen was strongly associated with culture positivity (relative risk=7.61, 95% CI: 3.01-19.22), whereas neither viral RNA nor symptoms were associated with culture positivity. During the 14 days following symptom onset, the presence of N antigen remained strongly associated (adjusted relative risk=7.66, 95% CI: 3.96-14.82) with culture positivity, regardless of COVID-19 symptoms. CONCLUSIONS: Most adults have replication-competent SARS-CoV-2 for 10-14 after symptom onset. N antigen testing is a strong predictor of viral infectiousness and may be a more suitable biomarker, rather than absence of symptoms or viral RNA, to discontinue isolation within two weeks from symptom onset.

Dynamics of SARS-CoV-2 VOC Neutralization and Novel mAb Reveal Protection against Omicron.

New variants of SARS-CoV-2 continue to emerge and evade immunity. We isolated SARS-CoV-2 temporally across the pandemic starting with the first emergence of the virus in the western hemisphere and evaluated the immune escape among variants. A clinic-to-lab viral isolation and characterization pipeline was established to rapidly isolate, sequence, and characterize SARS-CoV-2 variants. A virus neutralization assay was applied to quantitate humoral immunity from infection and/or vaccination. A panel of novel monoclonal antibodies was evaluated for antiviral efficacy. We directly compared all variants, showing that convalescence greater than 5 months post-symptom onset from ancestral virus provides little protection against SARS-CoV-2 variants. Vaccination enhances immunity against viral variants, except for Omicron BA.1, while a three-dose vaccine regimen provides over 50-fold enhanced protection against Omicron BA.1 compared to a two-dose. A novel Mab neutralizes Omicron BA.1 and BA.2 variants better than the clinically approved Mabs, although neither can neutralize Omicron BA.4 or BA.5. Thus, the need remains for continued vaccination-booster efforts, with innovation for vaccine and Mab improvement for broadly neutralizing activity. The usefulness of specific Mab applications links with the window of clinical opportunity when a cognate viral variant is present in the infected population.

Epidemiological and clinical characteristics of the largest COVID-19 outbreak along the China-Myanmar border in Ruili City, Yunnan Province, China.

Background: Imported COVID-19 patients posed great challenges to border areas' COVID-19 control. However, research was scarce to reveal epidemiological characteristics of COVID-19 in border areas. This study aimed to explore the detailed transmission chains, and reveal epidemiological and clinical characteristics of the largest COVID-19 outbreak caused by Delta variant of concern (VOC) occurred in the China-Myanmar border area. Methods: During the outbreak from July to September, 2021 in Ruili City, Yunnan Province, China, epidemiological investigation data and clinical-related data pertaining to confirmed COVID-19 patients were collected. Patients' contact history data and viral gene sequencing were used for inference of transmission chains. Sociodemographic and epidemiological characteristics, cycle threshold (Ct) value, and antibodies level were compared between patients who were vaccinated against COVID-19 or not. Results: A total of 117 COVID-19 patients were confirmed during the outbreak, among which 86 (73.5%) were breakthrough infections. These patients evenly split between Chinese and Myanmar people (50.4% vs. 49.6%). Most of these patients were mild (45.3%) or moderate (48.7%) infections with no death reported. Multi-source of infection led to 16 transmission chains with a maximum of 45 patients in one chain. Patients vaccinated against COVID-19 before infection had relatively higher antibodies (IgM and IgG) levels and more rapid response to infection than non-vaccinated patients (p < 0.05). Conclusion: Land border areas have greater risks of imported COVID-19 and more complicated epidemics. It should be cautious in formulating entry and exit requirements for border areas. The immune effect of COVID-19 vaccines and related mechanism should be further explored.

Dynamics of SARS-CoV-2 VOC neutralization and novel mAb reveal protection against Omicron.

To evaluate SARS-CoV-2 variants we isolated SARS-CoV-2 temporally during the pandemic starting with first appearance of virus in the Western hemisphere near Seattle, WA, USA, and isolated each known major variant class, revealing the dynamics of emergence and complete take-over of all new cases by current Omicron variants. We assessed virus neutralization in a first-ever full comparison across variants and evaluated a novel monoclonal antibody (Mab). We found that convalescence greater than 5-months provides little-to-no protection against SARS-CoV-2 variants, vaccination enhances immunity against variants with the exception of Omicron BA.1, and paired testing of vaccine sera against ancestral virus compared to Omicron BA.1 shows that 3-dose vaccine regimen provides over 50-fold enhanced protection against Omicron BA.1 compared to a 2-dose regimen. We also reveal a novel Mab that effectively neutralizes Omicron BA.1 and BA.2 variants over clinically-approved Mabs. Our observations underscore the need for continued vaccination efforts, with innovation for vaccine and Mab improvement, for protection against variants of SARS-CoV-2. Summary: We isolated SARS-CoV-2 temporally starting with emergence of virus in the Western hemisphere. Neutralization analyses across all variant lineages show that vaccine-boost regimen provides protection against Omicron BA.1. We reveal a Mab that protects against Omicron BA.1 and BA.2 variants.

IgM antibodies derived from memory B cells are potent cross-variant neutralizers of SARS-CoV-2.

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.

Efficacy of Parainfluenza Virus 5 (PIV5)-vectored Intranasal COVID-19 Vaccine as a Single Dose Vaccine and as a Booster against SARS-CoV-2 Variants.

Immunization with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines has greatly reduced coronavirus disease 2019 (COVID-19)-related deaths and hospitalizations, but waning immunity and the emergence of variants capable of immune escape indicate the need for novel SARS-CoV-2 vaccines. An intranasal parainfluenza virus 5 (PIV5)-vectored COVID-19 vaccine CVXGA1 has been proven efficacious in animal models and blocks contact transmission of SARS-CoV-2 in ferrets. CVXGA1 vaccine is currently in human clinical trials in the United States. This work investigates the immunogenicity and efficacy of CVXGA1 and other PIV5-vectored vaccines expressing additional antigen SARS-CoV-2 nucleoprotein (N) or SARS-CoV-2 variant spike (S) proteins of beta, delta, gamma, and omicron variants against homologous and heterologous challenges in hamsters. A single intranasal dose of CVXGA1 induces neutralizing antibodies against SARS-CoV-2 WA1 (ancestral), delta variant, and omicron variant and protects against both homologous and heterologous virus challenges. Compared to mRNA COVID-19 vaccine, neutralizing antibody titers induced by CVXGA1 were well-maintained over time. When administered as a boost following two doses of a mRNA COVID-19 vaccine, PIV5-vectored vaccines expressing the S protein from WA1 (CVXGA1), delta, or omicron variants generate higher levels of cross-reactive neutralizing antibodies compared to three doses of a mRNA vaccine. In addition to the S protein, the N protein provides added protection as assessed by the highest body weight gain post-challenge infection. Our data indicates that PIV5-vectored COVID-19 vaccines, such as CVXGA1, can serve as booster vaccines against emerging variants.

Performance of anterior nares and tongue swabs for nucleic acid, Nucleocapsid, and Spike antigen testing for detecting SARS-CoV-2 against nasopharyngeal PCR and viral culture.

OBJECTIVES: This study assesses and compares the performance of different swab types and specimen collection sites for SARS-CoV-2 testing, to reference standard real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and viral culture. METHODS: Symptomatic adults with COVID-19 who visited routine COVID-19 testing sites used spun polyester and FLOQSwabs to self-collect specimens from the anterior nares and tongue. We evaluated the self-collected specimen from anterior nares and tongue swabs for the nucleocapsid (N) or spike (S) antigen of SARS-CoV-2 by RT-PCR and then compared these results with results from RT-PCR and viral cultures from nurse-collected nasopharyngeal swabs. RESULTS: Diagnostic sensitivity was highest for RT-PCR testing conducted using specimens from the anterior nares collected on FLOQSwabs (84%; 95% CI 68-94%) and spun polyester swabs (82%; 95% CI 66-92%), compared to RT-PCR tests conducted using specimens from nasopharyngeal swabs. Relative to viral culture from nasopharyngeal swabs, diagnostic sensitivities were higher for RT-PCR and antigen testing of anterior nares swabs (91-100%) than that of tongue swabs (18-81%). Antigen testing of anterior nares swabs had higher sensitivities against viral culture (91%) than against nasopharyngeal RT-PCR (38-70%). All investigational tests had high specificity compared with nasopharyngeal RT-PCR. Spun polyester swabs are equally effective as FLOQSwabs for anterior nasal RT-PCR testing. CONCLUSIONS: We found that anterior nares specimens were more sensitive than tongue swab specimens or antigen testing for detecting SARS-CoV-2 by RT-PCR. Thus, self-collected anterior nares specimens may represent an alternative method for diagnostic SARS-CoV-2 testing in some settings.

Exploring the Bridge Cases' Role in the Transmission of the SARS-CoV-2 Delta Variant - Ruili City, Yunnan Province, China, July-September 2021.

WHAT IS ALREADY KNOWN ABOUT THIS TOPIC?: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta variant has proved to have increased transmissibility, and mutations that can cause partial immune escape, which makes its transmission more insidious. WHAT IS ADDED BY THIS REPORT?: This study showed that probable cases who had negative results in nucleic acid testing but had positive IgM test result and/or IgG test value of over 20 S/CO in antibodies testing, might serve as bridges in the Delta variant's transmission chain. WHAT ARE THE IMPLICATIONS FOR PUBLIC HEALTH PRACTICE?: In border inspection and quarantine, tests for SARS-CoV-2 IgM and IgG antibodies should be strengthened alongside nucleic acid tests to prevent probable cases with transmission potential from crossing the land border into China. In contact tracing investigations, the bridging role of probable cases should be considered to reconstruct the transmission chain.

Polymeric Nanoreactors as Emerging Nanoplatforms for Cancer Precise Nanomedicine.

How to precisely detect and effectively cure cancer which is defined as precise nanomedicine has drawn great attention worldwide. Polymeric nanoreactors which can in situ catalyze inert species into activated ones, can greatly increase imaging quality and enhance therapeutic effects along with decreased background interference and reduced serious side effects. After a brief introduction, the design and preparation of polymeric nanoreactors are discussed from the following aspects, that is, solvent-switch, pH-tuning, film rehydration, hard template, electrostatic interaction, and polymerization-induced self-assembly (PISA). Subsequently, the biomedical applications of these nanoreactors in the fields of cancer imaging, cancer therapy, and cancer theranostics are highlighted. The last but not least, conclusions and future perspectives about polymeric nanoreactors are given. It is believed that polymeric nanoreactors can bring a great opportunity for future fabrication and clinical translation of precise nanomedicine.

Inferring host gene subnetworks involved in viral replication.

Systematic, genome-wide loss-of-function experiments can be used to identify host factors that directly or indirectly facilitate or inhibit the replication of a virus in a host cell. We present an approach that combines an integer linear program and a diffusion kernel method to infer the pathways through which those host factors modulate viral replication. The inputs to the method are a set of viral phenotypes observed in single-host-gene mutants and a background network consisting of a variety of host intracellular interactions. The output is an ensemble of subnetworks that provides a consistent explanation for the measured phenotypes, predicts which unassayed host factors modulate the virus, and predicts which host factors are the most direct interfaces with the virus. We infer host-virus interaction subnetworks using data from experiments screening the yeast genome for genes modulating the replication of two RNA viruses. Because a gold-standard network is unavailable, we assess the predicted subnetworks using both computational and qualitative analyses. We conduct a cross-validation experiment in which we predict whether held-aside test genes have an effect on viral replication. Our approach is able to make high-confidence predictions more accurately than several baselines, and about as well as the best baseline, which does not infer mechanistic pathways. We also examine two kinds of predictions made by our method: which host factors are nearest to a direct interaction with a viral component, and which unassayed host genes are likely to be involved in viral replication. Multiple predictions are supported by recent independent experimental data, or are components or functional partners of confirmed relevant complexes or pathways. Integer program code, background network data, and inferred host-virus subnetworks are available at http://www.biostat.wisc.edu/~craven/chasman_host_virus/.

Genome-wide analysis of host factors in nodavirus RNA replication.

Flock House virus (FHV), the best studied of the animal nodaviruses, has been used as a model for positive-strand RNA virus research. As one approach to identify host genes that affect FHV RNA replication, we performed a genome-wide analysis using a yeast single gene deletion library and a modified, reporter gene-expressing FHV derivative. A total of 4,491 yeast deletion mutants were tested for their ability to support FHV replication. Candidates for host genes modulating FHV replication were selected based on the initial genome-wide reporter gene assay and validated in repeated Northern blot assays for their ability to support wild type FHV RNA1 replication. Overall, 65 deletion strains were confirmed to show significant changes in the replication of both FHV genomic RNA1 and sub-genomic RNA3 with a false discovery rate of 5%. Among them, eight genes support FHV replication, since their deletion significantly reduced viral RNA accumulation, while 57 genes limit FHV replication, since their deletion increased FHV RNA accumulation. Of the gene products implicated in affecting FHV replication, three are localized to mitochondria, where FHV RNA replication occurs, 16 normally reside in the nucleus and may have indirect roles in FHV replication, and the remaining 46 are in the cytoplasm, with functions enriched in translation, RNA processing and trafficking.

Limited agreement of independent RNAi screens for virus-required host genes owes more to false-negative than false-positive factors.

Systematic, genome-wide RNA interference (RNAi) analysis is a powerful approach to identify gene functions that support or modulate selected biological processes. An emerging challenge shared with some other genome-wide approaches is that independent RNAi studies often show limited agreement in their lists of implicated genes. To better understand this, we analyzed four genome-wide RNAi studies that identified host genes involved in influenza virus replication. These studies collectively identified and validated the roles of 614 cell genes, but pair-wise overlap among the four gene lists was only 3% to 15% (average 6.7%). However, a number of functional categories were overrepresented in multiple studies. The pair-wise overlap of these enriched-category lists was high, ∼19%, implying more agreement among studies than apparent at the gene level. Probing this further, we found that the gene lists implicated by independent studies were highly connected in interacting networks by independent functional measures such as protein-protein interactions, at rates significantly higher than predicted by chance. We also developed a general, model-based approach to gauge the effects of false-positive and false-negative factors and to estimate, from a limited number of studies, the total number of genes involved in a process. For influenza virus replication, this novel statistical approach estimates the total number of cell genes involved to be ∼2,800. This and multiple other aspects of our experimental and computational results imply that, when following good quality control practices, the low overlap between studies is primarily due to false negatives rather than false-positive gene identifications. These results and methods have implications for and applications to multiple forms of genome-wide analysis.

Systematic identification of novel, essential host genes affecting bromovirus RNA replication.

Positive-strand RNA virus replication involves viral proteins and cellular proteins at nearly every replication step. Brome mosaic virus (BMV) is a well-established model for dissecting virus-host interactions and is one of very few viruses whose RNA replication, gene expression and encapsidation have been reproduced in the yeast Saccharomyces cerevisiae. Previously, our laboratory identified ∼100 non-essential host genes whose loss inhibited or enhanced BMV replication at least 3-fold. However, our isolation of additional BMV-modulating host genes by classical genetics and other results underscore that genes essential for cell growth also contribute to BMV RNA replication at a frequency that may be greater than that of non-essential genes. To systematically identify novel, essential host genes affecting BMV RNA replication, we tested a collection of ∼900 yeast strains, each with a single essential gene promoter replaced by a doxycycline-repressible promoter, allowing repression of gene expression by adding doxycycline to the growth medium. Using this strain array of ∼81% of essential yeast genes, we identified 24 essential host genes whose depleted expression reproducibly inhibited or enhanced BMV RNA replication. Relevant host genes are involved in ribosome biosynthesis, cell cycle regulation and protein homeostasis, among other cellular processes. BMV 2a(Pol) levels were significantly increased in strains depleted for a heat shock protein (HSF1) or proteasome components (PRE1 and RPT6), suggesting these genes may affect BMV RNA replication by directly or indirectly modulating 2a(Pol) localization, post-translational modification or interacting partners. Investigating the diverse functions of these newly identified essential host genes should advance our understanding of BMV-host interactions and normal cellular pathways, and suggest new modes of virus control.

Intersection of the multivesicular body pathway and lipid homeostasis in RNA replication by a positive-strand RNA virus.

Like many positive-strand RNA viruses, brome mosaic virus (BMV) RNA replication occurs in membrane-invaginated vesicular compartments. BMV RNA replication compartments show parallels with membrane-enveloped, budding retrovirus virions, whose release depends on the cellular multivesicular body (MVB) sorting pathway. BMV RNA replication compartments are not released from their parent membranes, but might depend on MVB functions for membrane invagination. Prior results show that BMV RNA replication is severely inhibited by deletion of the crucial MVB gene DOA4 or BRO1. We report here that involvement of DOA4 and BRO1 in BMV RNA replication is not dependent on the MVB pathway's membrane-shaping functions but rather is due to their roles in recycling ubiquitin from MVB cargos. We show that deleting DOA4 or BRO1 inhibits the ubiquitination- and proteasome-dependent activation of homologous transcription factors Mga2p and Spt23p, which regulate many lipid metabolism genes, including the fatty acid desaturase gene OLE1, which is essential for BMV RNA replication. However, Mga2p processing and BMV RNA replication are restored by supplementing free ubiquitin, which is depleted in doa4Δ and bro1Δ cells. The results identify Mga2p and Spt23p processing and lipid regulation as sensitive targets of ubiquitin depletion and correctly predict multiple effects of modulating additional host genes RFU1, UBP6, and UFD3. Our results also show that BMV RNA replication depends on additional Mga2p-regulated genes likely involved in lipid metabolism beyond OLE1. Among other points, these findings show the potential for blocking viral RNA replication by modulating lipid synthesis at multiple levels.

Drosophila RNAi screen identifies host genes important for influenza virus replication.

All viruses rely on host cell proteins and their associated mechanisms to complete the viral life cycle. Identifying the host molecules that participate in each step of virus replication could provide valuable new targets for antiviral therapy, but this goal may take several decades to achieve with conventional forward genetic screening methods and mammalian cell cultures. Here we describe a novel genome-wide RNA interference (RNAi) screen in Drosophila that can be used to identify host genes important for influenza virus replication. After modifying influenza virus to allow infection of Drosophila cells and detection of influenza virus gene expression, we tested an RNAi library against 13,071 genes (90% of the Drosophila genome), identifying over 100 for which suppression in Drosophila cells significantly inhibited or stimulated reporter gene (Renilla luciferase) expression from an influenza-virus-derived vector. The relevance of these findings to influenza virus infection of mammalian cells is illustrated for a subset of the Drosophila genes identified; that is, for three implicated Drosophila genes, the corresponding human homologues ATP6V0D1, COX6A1 and NXF1 are shown to have key functions in the replication of H5N1 and H1N1 influenza A viruses, but not vesicular stomatitis virus or vaccinia virus, in human HEK 293 cells. Thus, we have demonstrated the feasibility of using genome-wide RNAi screens in Drosophila to identify previously unrecognized host proteins that are required for influenza virus replication. This could accelerate the development of new classes of antiviral drugs for chemoprophylaxis and treatment, which are urgently needed given the obstacles to rapid development of an effective vaccine against pandemic influenza and the probable emergence of strains resistant to available drugs.