A newly described coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the causative agent of coronavirus disease 2019 (COVID-19), has infected over 2.3 million people, led to the death of more than 160,000 individuals and caused worldwide social and economic disruption1,2. There are no antiviral drugs with proven clinical efficacy for the treatment of COVID-19, nor are there any vaccines that prevent infection with SARS-CoV-2, and efforts to develop drugs and vaccines are hampered by the limited knowledge of the molecular details of how SARS-CoV-2 infects cells. Here we cloned, tagged and expressed 26 of the 29 SARS-CoV-2 proteins in human cells and identified the human proteins that physically associated with each of the SARS-CoV-2 proteins using affinity-purification mass spectrometry, identifying 332 high-confidence protein–protein interactions between SARS-CoV-2 and human proteins. Among these, we identify 66 druggable human proteins or host factors targeted by 69 compounds (of which, 29 drugs are approved by the US Food and Drug Administration, 12 are in clinical trials and 28 are preclinical compounds). We screened a subset of these in multiple viral assays and found two sets of pharmacological agents that displayed antiviral activity: inhibitors of mRNA translation and predicted regulators of the sigma-1 and sigma-2 receptors. Further studies of these host-factor-targeting agents, including their combination with drugs that directly target viral enzymes, could lead to a therapeutic regimen to treat COVID-19. A human–SARS-CoV-2 protein interaction map highlights cellular processes that are hijacked by the virus and that can be targeted by existing drugs, including inhibitors of mRNA translation and predicted regulators of the sigma receptors.

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A SARS-CoV-2 protein interaction map reveals targets for drug repurposing.

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A User’s Guide

We describe a new reverse-genetics system that allows one to efficiently generate influenza A viruses entirely from cloned cDNAs. Human embryonic kidney cells (293T) were transfected with eight plasmids, each encoding a viral RNA of the A/WSN/33 (H1N1) or A/PR/8/34 (H1N1) virus, flanked by the human RNA polymerase I promoter and the mouse RNA polymerase I terminator—together with plasmids encoding viral nucleoprotein and the PB2, PB1, and PA viral polymerases. This strategy yielded >1 × 103 plaque-forming units (pfu) of virus per ml of supernatant at 48 hr posttransfection. The addition of plasmids expressing all of the remaining viral structural proteins led to a substantial increase in virus production, 3 × 104–5 × 107 pfu/ml. We also used reverse genetics to generate a reassortant virus containing the PB1 gene of the A/PR/8/34 virus, with all other genes representing A/WSN/33. Additional viruses produced by this method had mutations in the PA gene or possessed a foreign epitope in the head of the neuraminidase protein. This efficient system, which does not require helper virus infection, should be useful in viral mutagenesis studies and in the production of vaccines and gene therapy vectors.

https://www.pnas.org/content/pnas/96/16/9345.full.pdf

Generation of influenza A viruses entirely from cloned cDNAs

The extraordinary virulence of the Ebola and Marburg filoviruses has spurred intensive research into the molecular mechanisms by which they multiply and cause disease. Carette et al. use a genome-wide genetic screen in human cells to identify factors required for entry of Ebola virus. The screen uncovered 67 mutations disrupting all six members of the homotypic fusion and vacuole protein-sorting (HOPS) multisubunit tethering complex, which is involved in the fusion of endosomes to lysosomes, and 39 independent mutations that disrupt the endo/lysosomal cholesterol transporter protein Niemann–Pick C1 (NPC1). Cote et al. report the identification of a novel benzylpiperazine adamantane diamide-derived compound that inhibits EboV infection in cell culture, with NPC1 being the target. The unexpected role for the hereditary disease gene NPC1 in Ebola virus infection may facilitate the development of antifilovirus therapeutics. Infections by the Ebola and Marburg filoviruses cause a rapidly fatal haemorrhagic fever in humans for which no approved antivirals are available1. Filovirus entry is mediated by the viral spike glycoprotein (GP), which attaches viral particles to the cell surface, delivers them to endosomes and catalyses fusion between viral and endosomal membranes2. Additional host factors in the endosomal compartment are probably required for viral membrane fusion; however, despite considerable efforts, these critical host factors have defied molecular identification3,4,5. Here we describe a genome-wide haploid genetic screen in human cells to identify host factors required for Ebola virus entry. Our screen uncovered 67 mutations disrupting all six members of the homotypic fusion and vacuole protein-sorting (HOPS) multisubunit tethering complex, which is involved in the fusion of endosomes to lysosomes6, and 39 independent mutations that disrupt the endo/lysosomal cholesterol transporter protein Niemann–Pick C1 (NPC1)7. Cells defective for the HOPS complex or NPC1 function, including primary fibroblasts derived from human Niemann–Pick type C1 disease patients, are resistant to infection by Ebola virus and Marburg virus, but remain fully susceptible to a suite of unrelated viruses. We show that membrane fusion mediated by filovirus glycoproteins and viral escape from the vesicular compartment require the NPC1 protein, independent of its known function in cholesterol transport. Our findings uncover unique features of the entry pathway used by filoviruses and indicate potential antiviral strategies to combat these deadly agents.

/pdf/ebola-virus-entry-requires-the-cholesterol-transporter-4a0r642kc4.pdf

Ebola virus entry requires the cholesterol transporter Niemann–Pick C1

Crimean-Congo haemorrhagic fever (CCHF) is an often fatal viral infection described in about 30 countries, and it has the most extensive geographic distribution of the medically important tickborne viral diseases, closely approximating the known global distribution of Hyalomma spp ticks. Human beings become infected through tick bites, by crushing infected ticks, after contact with a patient with CCHF during the acute phase of infection, or by contact with blood or tissues from viraemic livestock. Clinical features commonly show a dramatic progression characterised by haemorrhage, myalgia, and fever. The levels of liver enzymes, creatinine phosphokinase, and lactate dehydrogenase are raised, and bleeding markers are prolonged. Infection of the endothelium has a major pathogenic role. Besides direct infection of the endothelium, indirect damage by viral factors or virus-mediated host-derived soluble factors that cause endothelial activations and dysfunction are thought to occur. In diagnosis, enzyme-linked immunoassay and real-time reverse transcriptase PCR are used. Early diagnosis is critical for patient therapy and prevention of potential nosocomial infections. Supportive therapy is the most essential part of case management. Recent studies suggest that ribavirin is effective against CCHF, although definitive studies are not available. Health-care workers have a serious risk of infection, particularly during care of patients with haemorrhages from the nose, mouth, gums, vagina, and injection sites. Simple barrier precautions have been reported to be effective.

/pdf/crimean-congo-haemorrhagic-fever-1mjnt47obh.pdf

Crimean-Congo haemorrhagic fever

Infectious vesicular stomatitis virus (VSV), the prototypic nonsegmented negative-strand RNA virus, was recovered from a full-length cDNA clone of the viral genome. Bacteriophage T7 RNA polymerase expressed from a recombinant vaccinia virus was used to drive the synthesis of a genome-length positive-sense transcript of VSV from a cDNA clone in baby hamster kidney cells that were simultaneously expressing the VSV nucleocapsid protein, phosphoprotein, and polymerase from separate plasmids. Up to 10(5) infectious virus particles were obtained from transfection of 10(6) cells, as determined by plaque assays. This virus was amplified on passage, neutralized by VSV-specific antiserum, and shown to possess specific nucleotide sequence markers characteristic of the cDNA. This achievement renders the biology of VSV fully accessible to genetic manipulation of the viral genome. In contrast to the success with positive-sense RNA, attempts to recover infectious virus from negative-sense T7 transcripts were uniformly unsuccessful, because T7 RNA polymerase terminated transcription at or near the VSV intergenic junctions.

https://www.pnas.org/content/pnas/92/18/8388.full.pdf

Efficient recovery of infectious vesicular stomatitis virus entirely from cDNA clones

The nonsegmented negative-strand (NNS) RNA viruses of the order Mononegavirales include a wide variety of human, animal, and plant pathogens. The NNS RNA genomes of these viruses are templates for two distinct RNA synthetic processes: transcription to generate mRNAs and replication of the genome via production of a positive-sense antigenome that acts as template to generate progeny negative-strand genomes. The four virus families within the Mononegavirales all express the information encoded in their genomes by transcription of discrete subgenomic mRNAs. The key feature of transcriptional control in the NNS RNA viruses is entry of the virus-encoded RNA-dependent RNA polymerase at a single 3′ proximal site followed by obligatory sequential transcription of the linear array of genes. Levels of gene expression are primarily regulated by position of each gene relative to the single promoter and also by cis-acting sequences located at the beginning and end of each gene and at the intergenic junctions. Obligatory sequential transcription dictates that termination of each upstream gene is required for initiation of downstream genes. Therefore, termination is a means to regulate expression of individual genes within the framework of a single transcriptional promoter. By engineering either whole virus genomes or subgenomic replicon derivatives, elements important for signaling transcript initiation, 5′ end modification, 3′ end polyadenylation, and transcription termination have been identified. Although the diverse families of NNS RNA virus use different sequences to control these processes, transcriptional termination is a common theme in controlling gene expression and overall transcriptional regulation is key in controlling the outcome of viral infection. The latest models for control of replication and transcription are discussed.

Transcription and Replication of Nonsegmented Negative-Strand RNA Viruses

The family Bunyaviridae of segmented, negative-stranded RNA viruses includes over 350 members that infect a bewildering variety of animals and plants. Many of these bunyaviruses are the causative agents of serious disease in their respective hosts, and are classified as emerging viruses because of their increased incidence in new populations and geographical locations throughout the world. Emerging bunyaviruses, such as Crimean–Congo hemorrhagic fever virus, tomato spotted wilt virus and Rift Valley fever virus, are currently attracting great interest due to migration of their arthropod vectors, a situation possibly linked to climate change. These and other examples of continued emergence suggest that bunyaviruses will probably continue to pose a sustained global threat to agricultural productivity, animal welfare and human health. The threat of emergence is particularly acute in light of the lack of effective preventative or therapeutic treatments for any of these viruses, making their study an important priority. This review presents recent advances in the understanding of the bunyavirus life cycle, including aspects of their molecular, cellular and structural biology. Whilst special emphasis is placed upon the emerging bunyaviruses, we also describe the extensive body of work involving model bunyaviruses, which have been the subject of major contributions to our overall understanding of this important group of viruses.

Recent advances in the molecular and cellular biology of bunyaviruses

We investigated the cis-acting sequences involved in termination of vesicular stomatis virus mRNA synthesis by using bicistronic genomic analogs. All of the cis-acting signals necessary for termination reside within the first 13 nucleotides of the 23-nucleotide conserved gene junction. This 13-nucleotide termination sequence at the end of the upstream gene comprises the tetranucleotide AUAC, the tract containing seven uridines (U7 tract), and the intergenic dinucleotide (GA), but it does not include the downstream gene start sequence. Data presented here show that upstream mRNA termination is independent of downstream mRNA initiation. Alteration of any nucleotide in the 13-nucleotide sequence decreased the termination activity of the gene junction and resulted in increased synthesis of a bicistronic readthrough RNA. This finding indicated that the wild-type gene junction has evolved to achieve the maximum termination efficiency. The most critical position of the AUAC sequence was the C, which could not be altered without complete loss of mRNA termination. Reducing the length of the wild-type U7 tract to zero, five, or six U residues also totally abolished mRNA termination, resulting in exclusive synthesis of the bicistronic readthrough mRNA. Shortening the wild-type U7 tract to either five or six U residues abolished VSV polymerase slippage during readthrough RNA synthesis. Since neither the U5 nor U6 template was able to direct mRNA termination, these data imply that polymerase slippage is a prerequisite for termination. Evidence is also presented to show that in addition to causing polymerase slippage, the U7 tract itself or its poly(A) product constitutes an essential signal for mRNA termination.

cis-Acting signals involved in termination of vesicular stomatitis virus mRNA synthesis include the conserved AUAC and the U7 signal for polyadenylation.

John N. Barr

Papers

Efficient recovery of infectious vesicular stomatitis virus entirely from cDNA clones

Transcription and Replication of Nonsegmented Negative-Strand RNA Viruses

Recent advances in the molecular and cellular biology of bunyaviruses

cis-Acting signals involved in termination of vesicular stomatitis virus mRNA synthesis include the conserved AUAC and the U7 signal for polyadenylation.

Transcriptional control of the RNA-dependent RNA polymerase of vesicular stomatitis virus