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International audience; Viruses are a heterogeneous ensemble of entities, all sharing the need for a suitable host to replicate. They are extremely diverse, varying in morphology, size, nature, and complexity of their genomic content. Typically, viruses use host-encoded glycosyltransferases and glycosidases to add and remove sugar residues from their glycoproteins. Thus, the structure of the glycans on the viral proteins have, to date, typically been considered to mimick those of the host. However, the more recently discovered large and giant viruses differ from this paradigm. At least some of these viruses code for an (almost) autonomous glycosylation pathway. These viral genes include those that encode the production of activated sugars, glycosyltransferases, and other enzymes able to manipulate sugars at various levels. This review focuses on large and giant viruses that produce carbohydrate-processing enzymes. A brief description of those harboring these features at the genomic level will be discussed, followed by the achievements reached with regard to the elucidation of the glycan structures, the activity of the proteins able to manipulate sugars, and the organic synthesis of some of these virus-encoded glycans. During this progression, we will also comment on many of the challenging questions on this subject that remain to be addressed. CONTENTS AJ 4.2. Chlorovirus N-Glycans AJ 5. Conclusions and Future Perspectives AN Author Information

Bacteriophages have important roles in the ecology of the human gut microbiome but are under-represented in reference databases. To address this problem, we assembled the Metagenomic Gut Virus catalogue that comprises 189,680 viral genomes from 11,810 publicly available human stool metagenomes. Over 75% of genomes represent double-stranded DNA phages that infect members of the Bacteroidia and Clostridia classes. Based on sequence clustering we identified 54,118 candidate viral species, 92% of which were not found in existing databases. The Metagenomic Gut Virus catalogue improves detection of viruses in stool metagenomes and accounts for nearly 40% of CRISPR spacers found in human gut Bacteria and Archaea. We also produced a catalogue of 459,375 viral protein clusters to explore the functional potential of the gut virome. This revealed tens of thousands of diversity-generating retroelements, which use error-prone reverse transcription to mutate target genes and may be involved in the molecular arms race between phages and their bacterial hosts., Almost 190,000 draft-quality DNA virus genomes are recovered by mining more than 11,000 deposited human stool metagenomes to improve resources for understanding the human gut virome.

Rotavirus is the most common cause of severe diarrhea in infants and children worldwide and is responsible for about 215,000 deaths annually. Over 85% of these deaths originate in low-income/developing countries in Asia and Africa. Therefore, it is necessary to explore the development of vaccines that avoid the use of "living" viruses and furthermore, vaccines that have viral antigens capable of generating powerful heterotypic responses. Our strategy is based on the expression of the fusion of the anti-DEC205 single-chain variable fragment (scFv) coupled by an OLLAS tag to a viral protein (VP6) of Rotavirus in Nicotiana plants. It was possible to express transiently in N. benthamiana and N. sylvestris a recombinant protein consisting of the single chain variable fragment linked by an OLLAS tag to the VP6 protein. The presence of the recombinant protein, which had a molecular weight of approximately 75 kDa, was confirmed by immunodetection, in both plant species and in both cellular compartments (cytoplasm and apoplast) where it was expressed. In addition, the recombinant protein was modeled, and it was observed that some epitopes of interest are exposed on the surface, which could favor their immunogenic response.

The enterovirus genus of the picornavirus family contains many important human pathogens. EV-D68 primarily infects children, and the disease manifestations range from respiratory illnesses to neurological complications such as acute flaccid myelitis (AFM). EV-A71 is a major pathogen for the hand, foot, and mouth disease (HFMD) in children and can also lead to AFM and death in severe cases. CVB3 infection can cause cardiac arrhythmias, acute heart failure, as well as type 1 diabetes. There is currently no FDA-approved antiviral for any of these enteroviruses. In this study, we report our discovery and development of pyrazolopyridine-containing small molecules with potent and broad-spectrum antiviral activity against multiple strains of EV-D68, EV-A71, and CVB3. Serial viral passage experiments, coupled with reverse genetics and thermal shift binding assays, suggested that these molecules target the viral protein 2C. Overall, the pyrazolopyridine inhibitors represent a promising class of candidates for the urgently needed non-polio enterovirus antivirals.

Coxsackievirus B3 (CVB3), is commonly implicated in myocarditis, which can lead to dilated cardiomyopathy, in addition to causing acute pancreatitis and meningitis. Yet, no vaccines are currently available to prevent this infection. Here, we describe the derivation of a live attenuated vaccine virus, termed mutant (Mt) 10, encoding a single amino acid substitution H790A within the viral protein 1, that prevents CVB3 infection in mice and protects from both myocarditis and pancreatitis in challenge studies. We noted that animals vaccinated with Mt 10 developed virus-neutralizing antibodies, predominantly containing IgG2a and IgG2b, and to a lesser extent IgG3 and IgG1. Furthermore, by using major histocompatibility complex class II dextramers and tetramers, we demonstrated that Mt 10 induces antigen-specific T cell responses that preferentially produce interferon-γ. Finally, neither vaccine recipients nor those challenged with the wild-type virus revealed evidence of autoimmunity or cardiac injury as determined by T cell response to cardiac myosin and measurement of circulating cardiac troponin I levels, respectively. Together, our data suggest that Mt 10 is a vaccine candidate that prevents CVB3 infection through the induction of neutralizing antibodies and antigen-specific T cell responses, the two critical components needed for complete protection against virus infections in vaccine studies.

The advent of whole genome sequencing has revealed that common laboratory strains of human cytomegalovirus (HCMV) have major genetic deficiencies resulting from serial passage in fibroblasts. In particular, tropism for epithelial and endothelial cells is lost due to mutations disrupting genes UL128, UL130, or UL131A, which encode subunits of a virion-associated pentameric complex (PC) important for viral entry into these cells but not for entry into fibroblasts. The endothelial cell-adapted strain TB40/E has a relatively intact genome and has emerged as a laboratory strain that closely resembles wild-type virus. However, several heterogeneous TB40/E stocks and cloned variants exist that display a range of sequence and tropism properties. Here, we report the use of PacBio sequencing to elucidate the genetic changes that occurred, both at the consensus level and within subpopulations, upon passaging a TB40/E stock on ARPE-19 epithelial cells. The long-read data also facilitated examination of the linkage between mutations. Consistent with inefficient ARPE-19 cell entry, at least 83% of viral genomes present before adaptation contained changes impacting PC subunits. In contrast, and consistent with the importance of the PC for entry into endothelial and epithelial cells, genomes after adaptation lacked these or additional mutations impacting PC subunits. The sequence data also revealed six single noncoding substitutions in the inverted repeat regions, single nonsynonymous substitutions in genes UL26, UL69, US28, and UL122, and a frameshift truncating gene UL141. Among the changes affecting protein-coding regions, only the one in UL122 was strongly selected. This change, resulting in a D390H substitution in the encoded protein IE2, has been previously implicated in rendering another viral protein, UL84, essential for viral replication in fibroblasts. This finding suggests that IE2, and perhaps its interactions with UL84, have important functions unique to HCMV replication in epithelial cells.

The binding mode to TAP (i.e., the peptide transporter associated with antigen processing) from a viral peptide thus far has been unknown in the field of antiviral immunity, but an interfering mode from a virus-encoded TAP inhibitor has been well documented with respect to blocking the TAP function. In the current study, we predicted the structure of the pig TAP transporter and its inhibition complex by the small viral protein ICP47 of the herpes simplex virus (HSV) encoded by the TAP inhibitor to exploit inhibition of the TAP transporter as the host's immune evasion strategy. We found that the hot spots (residues Leu5, Tyr22, and Leu51) on the ICP47 inhibitor interface tended to prevail over the favored Leu and Tyr, which contributed to significant functional binding at the C-termini recognition principle of the TAP. We further characterized the specificity determinants of the peptide transporter from the pig TAP by the ICP47 inhibitor effects and multidrug TmrAB transporter from the Thermus thermophillus and its immunity regarding its structural homolog of the pig TAP. The specialized structure-function relationship from the pig TAP exporter could provide insight into substrate specificity of the unique immunological properties from the host organism. The TAP disarming capacity from all five viral inhibitors (i.e., the five virus-encoded TAP inhibitors of ICP47, UL49.5, U6, BNLF2a, and CPXV012 proteins) was linked to the infiltration of the TAP functional structure in an unstable conformation and the mounting susceptibility caused by the host's TAP polymorphism. It is anticipated that the functional characterization of the pig TAP transporter based on the pig genomic variants will lead to additional insights into the genotype and single nucleotide polymorphism (SNP) in relation to antiviral resistance and disease susceptibility.

Infectious bronchitis virus (IBV) is the only coronavirus known to infect poultry. The replication and pathogenesis of IBV are poorly understood, mainly because of the unavailability of a robust cell culture system. Here, we report that an active ubiquitin proteasome system (UPS) is necessary for efficient replication of IBV in Vero cells. Synthesis of IBV-specific RNA as well as viral protein is hampered in the presence of chemical inhibitors specific for the UPS. Like other coronaviruses, IBV encodes a papain-like protease (PLpro) that exhibits in vitro deubiquitinase activity in addition to proteolytically processing the replicase polyprotein. Our results show that the IBV PLpro enzyme inhibits the synthesis of interferon beta (IFNβ) in infected chicken embryonic fibroblast (DF-1) cells and that this activity is enhanced in the presence of melanoma differentiation-associated protein 5 (MDA5) and TANK binding kinase 1 (TBK1). IBV PLpro, when overexpressed in DF-1 cells, deubiquitinates MDA5 and TBK1. Both of these proteins, along with other adapter molecules such as MAVS, IKKe, and IRF3, form a signaling cascade for the synthesis of IFNβ. Ubiquitination of MDA5 and TBK1 is essential for their activation, and their deubiquitination by IBV PLpro renders them unable to participate in antiviral signaling. This study shows for the first time that there is cross-talk between the UPS and the innate immune response during IBV infection and that the deubiquitinase activity of IBV PLpro is involved in its activity as an IFN antagonist. This insight will be useful for designing better antivirals targeting the catalytic activity of the IBV PLpro enzyme.

The nuclear factor-kappa B (NF-κB) signaling network constitutes a first line of defense against the invading viruses. However, viruses also adopted multiple strategies to interfere with NF-κB activation. Enterovirus 71 (EV71), in the family Picornaviridae, has become the main pathogen responsible for hand, foot, and mouth disease. Recent studies have reported that the nonstructural protein 2C of EV71 inhibits TNF-α induced NF-κB activation by suppressing IKKβ phosphorylation. In our study, we found that 2C can form inclusion bodies (IBs) in infected and transfected cells. Furthermore, 2C was able to sequester IKKβ into IBs through direct interaction with IKKβ. Although 2C did not directly interact with IKKα, viral protein 2C was able to sequester the IKKα into the IBs mediated by IKKβ. Our in vitro data further demonstrated that EV71 2C could suppress IKKα phosphorylation. These all together support a novel mechanism for EV71 to escape from NF-κB response, in which the phosphorylation of IKKα was suppressed by being recruited into viral IBs in the presence of 2C and IKKβ.

Introduction: Dengue Virus (DENV) is the pathogen for human dengue fever and is responsible for 390 million infections per year. The viral genome produces about 10 viral protein products, one of them being NS1. The NS1 protein plays a key role in viral replication and stimulation of humoral immune cells, thus being the perfect candidate to create an effective antiviral drug or vaccine for dengue Methods: Dengue Virus (DENV) is the pathogen for human dengue fever and is responsible for 390 million infections per year. The viral genome produces about 10 viral protein products, one of them being NS1. The NS1 protein plays a key role in viral replication and stimulation of humoral immune cells, thus being the perfect candidate to create an effective antiviral drug or vaccine for dengue Conclusion: The review established promising results of using peptide-based intervention on NS1. Further in vivo and randomized controlled trials are advised to solidify the applicability and biosafety of the intervention

Zika virus has been identified in various body fluids such as semen, urine, saliva, cerebrospinal fluid, and vaginal secretion of an infected individual. The pH of these fluids varies from mildly acidic to mildly alkaline. So it is imperative to understand the impact of these conditions on viral protein functioning. We investigated the NS2B-NS3 protease stability and its activity in different denaturing environments. Finding indicates that NS2B-NS3 protease maintains stability at pH 4.8–8.7. Thus it suggests that the complex remains functionally active to hydrolyze the polyprotein within a diverse environmental condition such as variable pH. Despite a stable structure at a broad pH range, a change in environmental conditions dramatically influence its protease activity. Moreover, it is susceptible to structural transformation leading to increased β-strand or helix content in the presence of alcohol. This study may help further to understand the folding-function relationship of the general flaviviral protease complex.

Viral antagonism of innate immune pathways is a common mechanism by which viruses evade immune surveillance. Infection of host cells with reovirus leads to the blockade of NF-{kappa}B, a key transcriptional regulator of the hosts innate immune response. One mechanism by which reovirus infection results in inhibition of NF-{kappa}B is through a diminishment in levels of upstream activators, IKK{beta} and NEMO. Here, we demonstrate a second, distinct mechanism by which reovirus blocks NF-{kappa}B. We report that expression of a single viral protein, {sigma}3, is sufficient to inhibit expression of NF-{kappa}B target genes. Further, {sigma}3-mediated blockade of NF-{kappa}B occurs without changes to IKK levels or activity. Expression of only a subset of NF-{kappa}B target genes is reduced. Among NF-{kappa}B targets, the expression of type I interferon is significantly diminished by {sigma}3 expression. Correspondingly, ectopic expression of {sigma}3 enhances viral replication. Expression of NF-{kappa}B target genes varies following infection with closely related reovirus strains. Our genetic analysis identifies that these differences are controlled by polymorphisms in the amino acid sequence of {sigma}3. This work identifies a new role for reovirus {sigma}3 as a viral antagonist of the NF-{kappa}B-dependent antiviral pathways. IMPORTANCEHost cells mount a response to curb virus replication in infected cells and prevent spread of virus to neighboring, as yet uninfected cells. The NF-{kappa}B family of proteins is important for the cell to mediate this response. In this study, we show that a single protein, {sigma}3, produced by mammalian reovirus, impairs the function of NF-{kappa}B. We demonstrate that by blocking NF-{kappa}B, {sigma}3 diminishes the hosts response to infection and promotes viral replication. This work identifies a second, previously unknown mechanism by which reovirus blocks this aspect of the host cell response.

Adult T-cell leukemia/lymphoma (ATL) is a T-cell lymphoproliferative neoplasm caused by the human T-cell leukemia virus type 1 (HTLV-1). Two viral proteins, Tax-1 and HBZ play important roles in HTLV-1 infectivity and in HTLV-1-associated pathologies by altering key pathways of cell homeostasis. However, the molecular mechanisms through which the two viral proteins, particularly HBZ, induce and/or sustain the oncogenic process are still largely elusive. Previous results suggested that HBZ interaction with nuclear factors may alter cell cycle and cell proliferation. To have a more complete picture of the HBZ interactions, we investigated in detail the endogenous HBZ interactome in leukemic cells by immunoprecipitating the HBZ-interacting complexes of ATL-2 leukemic cells, followed by tandem mass spectrometry analyses. RNA seq analysis was performed to decipher the differential gene expression and splicing modifications related to HTLV-1. Here we compared ATL-2 with MOLT-4, a non HTLV-1 derived leukemic T cell line and further compared with HBZ-induced modifications in an isogenic system composed by Jurkat T cells and stably HBZ transfected Jurkat derivatives. The endogenous HBZ interactome of ATL-2 cells identified 249 interactors covering three main clusters corresponding to protein families mainly involved in mRNA splicing, nonsense-mediated RNA decay (NMD) and JAK-STAT signaling pathway. Here we analyzed in detail the cluster involved in RNA splicing. RNAseq analysis showed that HBZ specifically altered the transcription of many genes, including crucial oncogenes, by affecting different splicing events. Consistently, the two RNA helicases, members of the RNA splicing family, DDX5 and its paralog DDX17, recently shown to be involved in alternative splicing of cellular genes after NF-κB activation by HTLV-1 Tax-1, interacted and partially co-localized with HBZ. For the first time, a complete picture of the endogenous HBZ interactome was elucidated. The wide interaction of HBZ with molecules involved in RNA splicing and the subsequent transcriptome alteration strongly suggests an unprecedented complex role of the viral oncogene in the establishment of the leukemic state.

The arterivirus porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses to the swine industry worldwide. Here we apply ribosome profiling (RiboSeq) and parallel RNA sequencing (RNASeq) to characterise the transcriptome and translatome of both species of PRRSV and to analyse the host response to infection. We calculated programmed ribosomal frameshift (PRF) efficiency at both sites on the viral genome. This revealed the nsp2 PRF site as the second known example where temporally regulated frameshifting occurs, with increasing -2 PRF efficiency likely facilitated by accumulation of the PRF-stimulatory viral protein, nsp1β. Surprisingly, we find that PRF efficiency at the canonical ORF1ab frameshift site also increases over time, in contradiction of the common assumption that RNA structure-directed frameshift sites operate at a fixed efficiency. This has potential implications for the numerous other viruses with canonical PRF sites. Furthermore, we discovered several highly translated additional viral ORFs, the translation of which may be facilitated by multiple novel viral transcripts. For example, we found a highly expressed 125-codon ORF overlapping nsp12, which is likely translated from novel subgenomic RNA transcripts that overlap the 3' end of ORF1b. Similar transcripts were discovered for both PRRSV-1 and PRRSV-2, suggesting a potential conserved mechanism for temporally regulating expression of the 3'-proximal region of ORF1b. We also identified a highly translated, short upstream ORF in the 5' UTR, the presence of which is highly conserved amongst PRRSV-2 isolates. These findings reveal hidden complexity in the gene expression programmes of these important nidoviruses.Viruses have tiny genomes. Rather than carry all the genetic information they need, they rely on the cells they infect. This makes the few genes they do have all the more important. Many viruses store their genes not in DNA, but in a related molecule called RNA. When the virus infects cells, it uses the cells’ ribosomes – the machines in the cells that make proteins – to build its own proteins. One of the central ideas in biology is that one molecule of RNA carries the instructions for just one type of protein. But many viruses break this rule. The ribosomes in cells read RNA instructions in blocks of three: three RNA letters correspond to one protein building block. But certain sequences in the RNA of viruses act as hidden signals that affect how ribosomes read these molecules. These signals make the ribosomes skip backward by one or two letters on the viral RNA, restarting part way through a three-letter block. Scientists call this a ‘frameshift’, and it is a bit like changing the positions of the spaces in a sentence. The virus causes these frameshifts using proteins or by folding its RNA into a knot-like structure. The frameshifts result in the production of different viral proteins over time. The porcine reproductive and respiratory syndrome virus (PRRSV) uses frameshifts to cause devastating disease in pigs. Besides the sequences in its RNA that allow the ribosomes to skip backwards, the viral enzyme that copies the RNA can also skip forward. This results in shortened copies of its genes, which also changes the proteins they produce. To find out exactly how PRRSV uses these frameshifting techniques, Cook et al. examined infected cells in the laboratory. They monitored the RNA made by the virus and looked closely at the way the cells read it using a technique called ribosome profiling. This revealed that frameshifting increases over the course of an infection. This is partly because the viral protein that causes frameshifts builds up as infection progresses, but it also happened with frameshifts caused by RNA knots. The reason for this is less clear. Cook et al. also discovered several new RNAs made later in infection, which could also change the proteins the virus makes. RNA viruses cause disease in humans as well as pigs. Examples include coronaviruses and HIV. Many of these also have frameshift sites in their genomes. A better understanding of how frameshifts change during infection may aid drug development. Future work could help researchers to understand which proteins viruses make at which stage of infection. This could lead to new treatments for viruses like PRRSV.

Herpes simplex virus type 1 (HSV-1) can induce in certain individuals with atopic dermatitis (AD) severe cutaneous infections that can spread throughout the entire body, a condition named as AD complicated by eczema herpeticum (ADEH). It has been recently found that ADEH patients can produce specific IgE against HSV-1 proteins, which may contribute to lower protection against HSV-1. However, little is known about the capacity of these HSV-1 proteins to produce an inflammatory response at the skin level. In this study, using a mouse model of AD-like dermatitis, three HSV-1 proteins (glycoprotein D -gD-, glycoprotein B -gB- and VP22) were applied on tape-stripped back skin mice in three exposures periods. Ovalbumin (OVA) and 0.9% NaCl were used as positive and negative controls, respectively. Skin samples were obtained for analysis of specific cell components of skin infiltration. The results showed that the viral protein gD induced a statistically significant increase in the number of dermal infiltrating CD3+, CD4+ cells and mast cells compared with the negative control group. gD was also able to induce epidermal thickening and epidermal infiltration of T cells closely related to the one produced in mice sensitized with OVA. However, VP22 and gB contributed to a lesser extent to skin inflammation. These results showed that proteins from HSV-1, especially gD, can have per se an important T cell and mast cell-driven inflammatory potential at the skin level.

The coronavirus disease-2019 (COVID-19) pandemic, caused by the pathogen severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), started in China during late 2019 and swiftly spread worldwide. Since COVID-19 emergence, many therapeutic regimens have been relentlessly explored, and although two vaccines have just received emergency use authorization by different governmental agencies, antiviral therapeutics based neutralizing antibodies and small-drug inhibitors can still be vital viable options to prevent and treat SARS-CoV-2 infections. The viral spike glycoprotein (S-protein) is the key molecular player that promotes human host cellular invasion via recognition of and binding to the angiotensin-converting enzyme 2 gene (ACE2). In this work, we report the results obtained by mutating in silico the 18 ACE2 residues and the 14 S-protein receptor binding domain (S-RBDCoV-2) residues that contribute to the receptor/viral protein binding interface. Specifically, each wild-type protein-protein interface residue was replaced by a hydrophobic (isoleucine), polar (serine and threonine), charged (aspartic acid/glutamic acid and lysine/arginine), and bulky (tryptophan) residue, respectively, in order to study the different effects exerted by nature, shape, and dimensions of the mutant amino acids on the structure and strength of the resulting binding interface. The computational results were next validated a posteriori against the corresponding experimental data, yielding an overall agreement of 92%. Interestingly, a non-negligible number of mis-sense variations were predicted to enhance ACE2/S-RBDCoV-2 binding, including the variants Q24T, T27D/K/W, D30E, H34S7T/K, E35D, Q42K, L79I/W, R357K, and R393K on ACE2 and L455D/W, F456K/W, Q493K, N501T, and Y505W on S-RBDCoV-2, respectively.

Pathogens usurp host pathways to generate a permissive environment for their propagation. The current spread of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection presents the urgent need to understand the complex pathogen–host interplay for effective control of the virus. SARS-CoV-2 reorganizes the host cytoskeleton for efficient cell entry and controls host transcriptional processes to support viral protein translation. The virus also dysregulates innate cellular defenses using various structural and nonstructural proteins. This results in substantial but delayed hyperinflammation alongside a weakened interferon (IFN) response. We provide an overview of SARS-CoV-2 and its uniquely aggressive life cycle and discuss the interactions of various viral proteins with host signaling pathways. We also address the functional changes in SARS-CoV-2 proteins, relative to SARS-CoV. Our comprehensive assessment of host signaling in SARS-CoV-2 pathogenesis provides some complex yet important strategic clues for the development of novel therapeutics against this rapidly emerging worldwide crisis., Highlights Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) proteins modulate host signaling to generate a permissive environment. SARS-CoV-2 proteins upregulate or downregulate ~100 human kinases involved in cellular physiology, metabolism, and immune activation. Targeting specific proviral cellular signaling could make the host microenvironment resistant to virus replication. Most of the nonstructural proteins of SARS-CoV-2 participate in dysregulating the immune response. Pertaining to the differences in sequence homology, NSP2 may be important in serodiagnosis.

Zika virus (ZIKV), a mosquito-transmitted Flavivirus, emerged in the last decade causing serious diseases and affecting human health globally. Currently, no licensed vaccines or antivirals are available to combat ZIKV, although several vaccine candidates are in the pipeline. In recent years, the presence of non-canonical G-quadruplex (GQ) secondary structures in viral genomes has ignited significant attention as potential targets for antiviral strategy. In this study, we identified several novel conserved potential GQ structures by analyzing published ZIKV genome sequences using an in-house algorithm. Biophysical and biochemical analysis of the RNA sequences containing these potential GQ sequences suggested the existence of such structures in the ZIKV genomes. Studies with known GQ structure-binding and -stabilizing ligands such as Braco-19 and TMPyP4 provided support for this contention. The presence of these ligands in cell culture media led to significant inhibition of infectious ZIKV yield, as well as reduced viral genome replication and viral protein production. Overall, our results, for the first time, show that ZIKV replication can be inhibited by GQ structure-binding and -stabilizing compounds and suggest a new strategy against ZIKV infection mitigation and control., Graphical Abstract, G-quadruplex structures are non-canonical secondary structures present in nucleic acid. In this manuscript, we targeted the Zika GQ structures with GQ-binding ligands like Braco-19 and TMPyP4 to inhibit ZIKV genome replication and transcription. Overall, 100 μM Braco-19 treatment produced >80-fold (96 hpi) reduction in viral titer production in cell culture, having a potential anti-viral effect.

Banana bract mosaic virus (BBrMV), belongs to the genus Potyvirus and it is an important viral pathogen of bananas and plantains. The eukaryotic translation initiation factor, eIF4E, and its isoform play key roles during the virus infection in plants, particularly Potyvirus. The present study was undertaken to determine the role of BBrMV-viral protein genome-linked (VPg) in virus infectivity by analyzing the interaction with the eukaryotic translation initiation factor eIF4E through yeast two-hybrid system. The results suggest that plantain cv. Nendran eIF4E plays an essential role in the initiation of the translation of capped mRNAs and its association with VPg would point to a role of the viral protein in the translation of the virus and may potentially contribute to BBrMV resistance. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13337-021-00672-9) contains supplementary material, which is available to authorized users.

Zaire ebolavirus, commonly called Ebola virus (EBOV), is an RNA virus that causes severe hemorrhagic fever with high mortality. Viral protein 35 (VP35) is a virulence factor encoded in the EBOV genome. VP35 inhibits host innate immune responses and functions as a critical cofactor for viral RNA replication. EBOV VP35 contains a short conserved motif that interacts with dynein light chain 8 (LC8), which serves as a regulatory hub protein by associating with various LC8-binding proteins. Herein, we present the crystal structure of human LC8 bound to the peptide comprising residues 67-76 of EBOV VP35. Two VP35 peptides were found to interact with homodimeric LC8 by extending the central β-sheets, constituting a 2:2 complex. Structural analysis demonstrated that the intermolecular binding between LC8 and VP35 is mainly sustained by a network of hydrogen bonds and supported by hydrophobic interactions in which Thr73 and Thr75 of VP35 are involved. These findings were verified by binding measurements using isothermal titration calorimetry. Biochemical analyses also verified that residues 67-76 of EBOV VP35 constitute a core region for interaction with LC8. In addition, corresponding motifs from other members of the genus Ebolavirus commonly bound to LC8 but with different binding affinities. Particularly, VP35 peptides originating from pathogenic species interacted with LC8 with higher affinity than those from noninfectious species, suggesting that the binding of VP35 to LC8 is associated with the pathogenicity of the Ebolavirus species.

Aim. Production of recombinant viral antigens of the main immunodominant proteins: glycoprotein (GPΔMLD), nucleoprotein (NP) and matrix protein (VP40) of the Marburg virus, as well as the study of their antigenic and immunogenic properties.Materials and methods. To create recombinant proteins GPΔMLD, NP and VP40 of the Marburg virus, synthesized nucleotide sequences encoding these proteins cloned into the pET21a expression vector were used. The immunogenic and antigenic properties of the obtained recombinant proteins were tested using a number of biomodels (mice, chickens, and guinea pigs).Results and discussion. Recombinant plasmids containing genes encoding proteins GPΔMLD, NP, VP40 of the Marburg virus, as well as Escherichia coli producing strains, with the yield of purified preparations of recombinant proteins GPΔMLD, NP, VP40 from one liter of culture fluid – 5, 10, and 10 μg were obtained, respectively. When mice are immunized, recombinant proteins GP, NP, and VP40 MARV induce the synthesis of high titer antibodies (recombinant proteins NP and VP40 – more than 409600, and recombinant protein GPΔMLD – 12800). Mouse antibodies specific to recombinant proteins interact in an enzyme-linked immunosorbent assay (ELISA) with the antigen of inactivated MARV. Antibodies of chickens immunized with virus-like particles containing the surface glycoprotein of the Marburg virus and antibodies of guinea pigs immunized with an experimental DNA vaccine containing the GPΔMLD MARV gene recognize the recombinant GPΔMLD protein and the viral protein in the inactivated MARV. The resulting recombinant proteins are immunogenic/antigenic and can be used for the development of enzymelinked immunosorbent assay systems.

Viral infections are the cause of serious infirmities in humans and kill millions of people every year. Management of viral diseases is one of the challenges faced by the whole world which needs improvement in prevention and treatment options. Complete understanding of the consequences of viral proteins interaction network on host physiology is essential. Towards this goal, deciphering viral protein-protein interactions is one of the perspective which can help in our understanding about the basis of viral pathogenesis and the development of new antivirals. Indeed, viral infection network based on viral-viral proteins will provide an elusive and investigative framework to articulate rationalize drug discovery based on proteomics scale of viruses. In this study, proteomics a collection of viral-viral protein interactions reporting different studies of Hepatitis C virus, Influenza A virus, Dengue virus and SARS Coronavirus. Our effort of protein-interactions was focused on different studies reporting interactions between viral proteins encoded by the viruses under study. The study is integrated with a broad and original literature-curated data of viral-viral protein (197 non-redundant) interactions.

Plants protect against viruses through passive and active resistance mechanisms, and in most cases characterized thus far, natural recessive resistance to potyviruses has been mapped to mutations in the eukaryotic initiation factor eIF4E or eIF(iso)4E genes. Five eIF4E copies and three eIF(iso)4E copies were detected in Brassica rapa. The eIF4E and eIF(iso)4E genes could interact with turnip mosaic virus (TuMV) viral protein linked to the genome (VPg) to initiate virus translation. From the yeast two-hybrid system (Y2H) and bimolecular fluorescence complementation (BiFC) assays, the TuMV-CHN2/CHN3 VPgs could not interact with BraA.eIF4E.a/c or BraA.eIF(iso)4E.c, but they could interact with BraA.eIF(iso)4E.a in B. rapa. Further analysis indicated that the amino acid substitution L186F (nt T556C) in TuMV-UK1 VPg was important for the interaction networks between the TuMV VPg and eIF(iso)4E proteins. An interaction model of the BraA. eIF(iso)4E protein with TuMV VPg was constructed to infer the effect of the significant amino acids on the interaction of TuMV VPgs-eIF(iso)4Es, particularly whether the L186F in TuMV-UK1 VPg could change the structure of the TuMV-UK1 VPg protein, which may terminate the interaction of the BraA.eIF(iso)4E and TuMV VPg protein. This study provides new insights into the interactions between plant viruses and translation initiation factors to reveal the working of key amino acids.

The current pandemic resulted from SARS-CoV-2 still remains as the major public health concern globally. The precise mechanism of viral pathogenesis is not fully understood, which remains a major hurdle for medical intervention. Here we generated an interactome profile of protein-protein interactions based on host and viral protein structural similarities information. Further computational biological study combined with Gene enrichment analysis predicted key enriched pathways associated with viral pathogenesis. The results show that axon guidance, membrane trafficking, vesicle-mediated transport, apoptosis, clathrin-mediated endocytosis, Vpu mediated degradation of CD4 T cell, and interferon-gamma signaling are key events associated in SARS-CoV-2 life cycle. Further, degree centrality analysis reveals that IRF1/9/7, TP53, and CASP3, UBA52, and UBC are vital proteins for IFN-γ-mediated signaling, apoptosis, and proteasomal degradation of CD4, respectively. We crafted chronological events of the virus life cycle. The SARS-CoV-2 enters through clathrin-mediated endocytosis, and the genome is trafficked to the early endosomes in a RAB5-dependent manner. It is predicted to replicate in a double-membrane vesicle (DMV) composed of the endoplasmic reticulum, autophagosome, and ERAD machinery. The SARS-CoV-2 down-regulates host translational machinery by interacting with protein kinase R, PKR-like endoplasmic reticulum kinase, and heme-regulated inhibitor and can phosphorylate eIF2a. The virion assembly occurs in the ER-Golgi intermediate compartment (ERGIC) organized by the spike and matrix protein. Collectively, we have established a spatial link between viral entry, RNA synthesis, assembly, pathogenesis, and their associated diverse host factors, those could pave the way for therapeutic intervention., Graphical Abstract Communicated by Ramaswamy H. Sarma

The Human Immunodeficiency Virus (HIV-1) type 1 viral protein is a life threatening virus causing HIV/AIDS in infected humans. The HIV-1 envelope (ENV) trimer glycoprotein GP160 (GP120-GP41) is gaining attention in recent years as a potential vaccine candidate for HIV-1/AIDS. However, the sequence variation and charge polarity at the interacting sites across clades is a shortcoming faced in the development of an effective HIV-1 vaccine. We analyzed the interfaces in terms of its interface area, interface size, and interface energies (van der Waals, hydrogen bonds, and electrostatics). The interfaces were divided as dominant (≥60%) and subdominant (

Vaccinia virus (VACV) belonging to the poxvirus family enters the host cell via two different entry pathways; either endocytosis or virus/host cell membrane fusion. With respect to the virus/host cell membrane fusion, there are eleven viral membrane proteins forming a complicated entry-fusion complex (EFC), including A28, A21, A16, F9, G9, G3, H2, J5, L5, L1 and O3, to conduct the fusion function. These EFC components are highly conserved in all poxviruses and each of them is essential and necessary for the fusion activity. So far, with the exceptions of L1 and F9 whose crystal structures were reported, the structural information about other EFC components remains largely unclear. We aim to conduct a structural and functional investigation of VACV virus-entry membrane protein A28. In this work, we expressed and purified a truncated form of A28 (14 kDa; residues 38–146, abbreviated as tA28 hereinafter), with deletion of its transmembrane domain (residues 1–22) and a hydrophobic segment (residues 23–37). And the assignments of its backbone and side chain 1H, 13C and 15N chemical shifts of tA28 are reported. The secondary structure propensity from TALOS+ indicates that tA28 does contain three α-helices, six β-strands and connecting loops. Aside from this, we demonstrated that tA28 does interact with fusion suppressor viral protein A26 (residues 351–500) by the 1H–15N HSQC spectrum. We interpret that A28 binding to A26 deactivates EFC fusion activity. The current study provides a valuable framework towards further structural analyses of this protein and for better understanding virus/host cell membrane fusion mechanism in association with virus entry.

Coronavirus diseases 2019 (COVID-19) are seriously affecting human health all over the world. Nucleotide inhibitors have promising results in terms of its efficacy against different viral polymerases. In this study, detailed molecular docking and dynamics simulations are used to evaluate the binding affinity of a clinically approved drug, sofosbuvir, with the solved structure of the viral protein RNA-dependent RNA polymerase (RdRp) and compare it to the clinically approved drug, Remdesivir. These drugs are docked onto the three-dimensional structure of the nsp12 protein of SARS-CoV-2, which controls the polymerization process. Hence, it is considered one of the primary therapeutic targets for coronaviruses. Sofosbuvir is a drug that is currently used for HCV treatment; therefore, HCV RdRp is used as a positive control protein target. The protein dynamics are simulated for 100 ns, while the binding is tested during different dynamics states of the SARS-CoV-2 RdRp. Additionally, the drug-protein complexes are further simulated for 20 ns to explore the binding mechanism. The interaction of SARS-CoV-2 RdRp as a target with the active form of sofosbuvir as a ligand demonstrates binding effectiveness. One of the FDA-approved antiviral drugs, such as sofosbuvir, can help us in this mission, aiming to limit the danger of COVID-19. Sofosbuvir was found to bind nsp12 with comparable binding energies to that of Remdesivir, which has been reported for its potential against COVID-19 RdRp and is currently approved by the FDA. Graphic abstract

We present in this work a first X‐ray Absorption Spectroscopy study of the interactions of Zn with human BST2/tetherin and SARS‐CoV‐2 orf7a proteins as well as with some of their complexes. The analysis of the XANES region of the measured spectra shows that Zn binds to BST2, as well as to orf7a, thus resulting in the formation of BST2‐orf7a complexes. This structural information confirms the the conjecture, recently put forward by some of the present Authors, according to which the accessory orf7a (and possibly also orf8) viral protein are capable of interfering with the BST2 antiviral activity. Our explanation for this behavior is that, when BST2 gets in contact with Zn bound to the orf7a Cys15 ligand, it has the ability of displacing the metal owing to the creation of a new disulfide bridge across the two proteins. The formation of this BST2‐orf7a complex destabilizes BST2 dimerization, thus impairing the antiviral activity of the latter., Think zinc! We conjecture that in the case of SARS‐CoV‐2, the orf7a accessory protein acts as a BST2 antagonist. We provide evidence, based on sequence analysis, Molecular Dynamics simulations as well as XAS experiments, that Zn ions play a key role in the SARS‐CoV‐2 virus strategy to escape the immune response mediated by the BST2 host protein.

Investigation of the role of animals that have recovered and survived from African swine fever (ASF) in carrying the ASF virus is currently intense and ongoing. However, no clear definition of the carrier stage has been established. The aim of the present study was to establish criteria to elucidate a clear status of survival in naturally ASF-infected domestic pigs in Vietnam. Seroconversion from previous infection was confirmed by serological assay, and the absence of the viral genome in various organs was also assured by molecular analysis of a partial p72 gene. We recognized that histopathological evidence could benefit from further insights into the status and role of the surviving animals; therefore, we performed a histopathological study on four pigs from farms with a history of ASF outbreak. We found fibrotic changes in the reparative process as the main finding in all four pigs. Immunohistochemical detection of viral protein revealed an interesting result. Despite the negative result from viral genome detection, the p30 protein gave a positive signal in the tonsils, lung, and stomach. This raises the possibility of stress-induced viral reactivation in long-term survivors and the risk of further outbreaks from human handling of contaminated carcasses.

Nigella sativa (N. sativa) is extensively used as medicinal plant all over the world. It has the potential properties as the antiviral and antibacterial application. Its seed contain thymoquinone (TQ), thymohydroquinone (THQ), thymol (THY), p-cymene as major and other minor components. TQ and THQ exhibit broad spectrum of antimicrobial properties against the activity of bacteria, viruses, parasites, schistosoma and fungi. This work provides credence to the fabrication of antibacterial nanofibrous membrane by electrospinning machine from N. sativa extract with polyvinyl alcohol (PVA) solution for wound dressing. The morphology of the developed membrane is also characterized using scanning electron microscope. Fourier transform infrared spectroscopy (FTIR) data has been showed that the functional groups of N. sativa are present in the prepared PVA-N. sativa nanofibrous membrane and its antibacterial activity was investigated. The disk diffusion method has been used to evaluate the antibacterial activity of PVA-N. sativa nanofibrous membrane against Staphylococcus aureus (S. aureus) bacteria and the inhibition zone with a value of 10 mm is formed. Considering the inherent properties of N. sativa, a conceptual reaction mechanism has been proposed to deactivate the viral proteins by the action of TQ and THQ.

Severe acute respiratory syndrome (SARS) is a newly emerged infectious disease that caused pandemic spread in 2003. The etiological agent of SARS is a novel coronavirus (SARS-CoV). The coronaviral surface spike protein S is a type I transmembrane glycoprotein that mediates initial host binding via the cell surface receptor angiotensin-converting enzyme 2 (ACE2), as well as the subsequent membrane fusion events required for cell entry. Here we report the crystal structure of the S1 receptor binding domain (RBD) in complex with a neutralizing antibody, 80R, at 2.3 A resolution, as well as the structure of the uncomplexed S1 RBD at 2.2 A resolution. We show that the 80R-binding epitope on the S1 RBD overlaps very closely with the ACE2-binding site, providing a rationale for the strong binding and broad neutralizing ability of the antibody. We provide a structural basis for the differential effects of certain mutations in the spike protein on 80R versus ACE2 binding, including escape mutants, which should facilitate the design of immunotherapeutics to treat a future SARS outbreak. We further show that the RBD of S1 forms dimers via an extensive interface that is disrupted in receptor- and antibody-bound crystal structures, and we propose a role for the dimer in virus stability and infectivity.

SARS-CoV-2 is a novel pathogen causing pneumonia named COVID-19 and leading to a severe pandemic since the end of 2019. The genome of SARS-CoV-2 contains a macro domain that may play an important role in regulating ADP-ribosylation in host cells and initiating viral replication. Here, we report the 1H, 13C, and 15N resonance assignments of the SARS-CoV-2 macro domain. This work provides the ground for further structural deciphering and biophysical investigation in protein function and antiviral agent design. Supplementary Information The online version of this article (10.1007/s12104-020-09996-x) contains supplementary material, which is available to authorized users.

The novel coronavirus pandemic has spread over in 213 countries as of July 2020. Approximately 12 million people have been infected so far according to the reports from World Health Organization (WHO). Preventive measures are being taken globally to avoid the rapid spread of virus. In the current study, an in silico approach is carried out as a means of inhibiting the spike protein of the novel coronavirus by flavonoids from natural sources that possess both antiviral and anti-inflammatory properties. The methodology is focused on molecular docking of 10 flavonoid compounds that are docked with the spike protein of SARS-CoV-2, to determine the highest binding affinity at the binding site. Molecular dynamics simulation was carried out with the flavonoid-protein complex showing the highest binding affinity and highest interactions. The flavonoid naringin showed the least binding energy of −9.8 Kcal/mol with the spike protein which was compared with the standard drug, dexamethasone which is being repurposed to treat critically ill patients. MD simulation was carried out on naringin-spike protein complex for their conformational stability in the active site of the novel coronavirus spike protein. The RMSD of the complex appeared to be more stable when compared to that of the protein from 0.2 nm to 0.4 nm. With the aid of this in silico approach further in vitro studies can be carried out on these flavonoids against the novel coronavirus as a means of viral protein inhibitors.

The binding ability of lectins has gained attention owing to the carbohydrate-specific interactions of these proteins. Such interactions can be applied to diverse fields of biotechnology, including the detection, isolation, and concentration of biological target molecules. The physiological aspects of the lectin concanavalin A (ConA) have been intensively studied through structural and functional investigations. X-ray crystallography studies have proven that ConA has two β-sheets and a short α-helix and that it exists in the form of a metalloprotein containing Mn2+ and Ca2+. These heterometals are coordinated with side chains located in a metal-coordinated domain (MCD), and they affect the structural environment in the carbohydrate-binding domain (CBD), which interacts with carbohydrates through hydrogen bonds. Recent studies have shown that ConA can regulate biophysical interactions with glycoproteins in virus envelopes because it specifically interacts with diverse polysaccharides through its CBD (Tyr, Asn, Asp, and Arg residues positioned next to the MCD). Owing to their protein–protein interaction abilities, ConA can form diverse self-assembled complexes including monomers, dimers, trimers, and tetramers, thus affording unique results in different applications. In this regard, herein, we present a review of the structural modifications in ConA through metal-ion coordination and their effect on complex formation. In recent approaches, ConA has been applied for viral protein detection, on the basis of the interactions of ConA. These aspects indicate that lectins should be thoroughly investigated with respect to their biophysical interactions, for avoiding unexpected changes in their interaction abilities.

Purpose Acyclovir is most commonly used for treating ocular Herpes Keratitis, a leading cause of infectious blindness. However, emerging resistance to Acyclovir resulting from mutations in the thymidine kinase gene of Herpes Simplex Virus −1 (HSV-1), has prompted the need for new therapeutics directed against a different viral protein. One novel target is the HSV-1 Processivity Factor which is essential for tethering HSV-1 Polymerase to the viral genome to enable long-chain DNA synthesis. Methods A series of peptides, based on the crystal structure of the C-terminus of HSV-1 Polymerase, were constructed with hydrocarbon staples to retain their alpha-helical conformation. The stapled peptides were tested for blocking both HSV-1 DNA synthesis and infection. The most effective peptide was further optimized by replacing its negative N-terminus with two hydrophobic valine residues. This di-valine stapled peptide was tested for inhibiting HSV-1 infection of human primary corneal epithelial cells. Results The stapled peptides blocked HSV-1 DNA synthesis and HSV-1 infection. The unstapled control peptide had no inhibitory effects. Specificity of the stapled peptides was confirmed by their inabilities to block infection by an unrelated virus. Significantly, the optimized di-valine stapled peptide effectively blocked HSV-1 infection in human primary corneal epithelial cells with selectivity index of 11.6. Conclusions Hydrocarbon stapled peptides that simulate the α-helix from the C-terminus of HSV-1 DNA polymerase can specifically block DNA synthesis and infection of HSV-1 in human primary corneal epithelial cells. These stapled peptides provide a foundation for developing a topical therapeutic for treating human ocular Herpes Keratitis.

Given the on-going SARS-CoV-2 pandemic, identification of immunogenic targets against the viral protein will provide crucial advances towards the development of sensitive diagnostic tools and vaccination strategies. Our previous study has found that ORF8 protein of SARS-CoV-2 is highly immunogenic and shows high sensitivity in identifying COVID-19 disease. In this study, by employing overlapping linear peptides, we characterized the IgG immunodominant regions on SARS-CoV-2 ORF8 protein that are seropositive in the sera from SARS-CoV-2-infected patients. The major immunogenic epitopes are localized at (1) N-termini alpha helix, (2) the resides spanning beta 2 and 3 sheets, and (3) the loop between beta 4 and 5 sheets. Additionally, hamster model infected by SARS-CoV-2 further validates the seropositivity of the linear epitopes in vivo, demonstrating a potential application of the linear peptide-based immunization strategy. Taken together, identification and validation of these B-cell linear epitopes will provide insights into the design of serological diagnostics and peptide-based vaccination approach against this pandemic virus of high priority.

The envelope glycoprotein, termed the spike protein, of severe acute respiratory syndrome coronavirus (SARS-CoV) is known to mediate viral entry. Similar to other class 1 viral fusion proteins, the heptad repeat regions of SARS-CoV spike are thought to undergo conformational changes from a prefusion form to a subsequent post-fusion form that enables fusion of the viral and host membranes. Recently, the structure of a post-fusion form of SARS-CoV spike, which consists of isolated domains of heptad repeats 1 and 2 (HR1 and HR2), has been determined by x-ray crystallography. To date there is no structural information for the prefusion conformations of SARS-CoV HR1 and HR2. In this work we present the NMR structure of the HR2 domain (residues 1141-1193) from SARS-CoV (termed S2-HR2) in the presence of the co-solvent trifluoroethanol. We find that in the absence of HR1, S2-HR2 forms a coiled coil symmetric trimer with a complex molecular mass of 18 kDa. The S2-HR2 structure, which is the first example of the prefusion form of coronavirus envelope, supports the current model of viral membrane fusion and gives insight into the design of structure-based antagonists of SARS.

Kaposi’s sarcoma-associated herpesvirus (KSHV) causes the inflammatory and angiogenic endothelial cell neoplasm, Kaposi’s sarcoma (KS). We previously demonstrated that expression of the KSHV protein, Kaposin B (KapB), promoted inflammation via the disassembly of cytoplasmic ribonucleoprotein granules called processing bodies (PBs). Processing bodies (PBs) modify gene expression by silencing or degrading labile messenger RNAs (mRNAs) including many transcripts that encode inflammatory or angiogenic proteins that are associated with KS disease. Although our work implicated PB disassembly as one of the causes of inflammation during KSHV infection, the precise mechanism used by KapB to elicit PB disassembly was unclear. Here we reveal a new connection between the degradative process of autophagy and PB disassembly. We show that both latent KSHV infection and KapB expression enhanced autophagic flux via the phosphorylation of the autophagy regulatory protein, Beclin 1. KapB was necessary for this effect, as infection with a recombinant virus that does not express the KapB protein did not induce Beclin 1 phosphorylation or autophagic flux. Moreover, we reveal that PB disassembly mediated by KSHV or KapB depended on canonical autophagy genes and the selective autophagy receptor NDP52/CALCOCO2 and that the PB scaffolding protein, Pat1b, co-immunoprecipitated with NDP52. These studies reveal a new role for autophagy and the selective autophagy receptor NDP52 in promoting PB turnover and the concomitant synthesis of inflammatory molecules during KSHV infection.Author SummaryKaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent of the inflammatory, endothelial cell cancer, Kaposi’s Sarcoma (KS). KSHV induces a pro-tumourigenic inflammatory environment which aids in the establishment and maintenance of the KS lesion. Processing bodies (PBs) are cellular structures that dampen inflammatory gene expression by suppression or decay of their cognate RNA molecules. We previously showed that the viral protein KapB caused PB disappearance during KSHV infection, identifying a new pathway used by KSHV to elicit inflammation. Now, we show that KSHV and KapB hijack the cellular degradative process of autophagy to promote PB disassembly and increase inflammatory gene expression. This places autophagy as central to the regulation of inflammation by KSHV and illustrates another remarkable strategy used by these viruses to create a tumourigenic microenvironment.

HIV Type 1 (HIV-1) and simian immunodeficiency virus (SIV) display differential replication kinetics in macrophages. This is because high expression levels of the active host deoxynucleotide triphosphohydrolase sterile α motif domain and histidine-aspartate domain–containing protein 1 (SAMHD1) deplete intracellular dNTPs, which restrict HIV-1 reverse transcription, and result in a restrictive infection in this myeloid cell type. Some SIVs overcome SAMHD1 restriction using viral protein X (Vpx), a viral accessory protein that induces proteasomal degradation of SAMHD1, increasing cellular dNTP concentrations and enabling efficient proviral DNA synthesis. We previously reported that SAMHD1-noncounteracting lentiviruses may have evolved to harbor RT proteins that efficiently polymerize DNA, even at low dNTP concentrations, to circumvent SAMHD1 restriction. Here we investigated whether RTs from SIVmac239 virus lacking a Vpx protein evolve during in vivo infection to more efficiently synthesize DNA at the low dNTP concentrations found in macrophages. Sequence analysis of RTs cloned from Vpx (+) and Vpx (−) SIVmac239–infected animals revealed that Vpx (−) RTs contained more extensive mutations than Vpx (+) RTs. Although the amino acid substitutions were dispersed indiscriminately across the protein, steady-state and pre-steady-state analysis demonstrated that selected SIVmac239 Vpx (−) RTs are characterized by higher catalytic efficiency and incorporation efficiency values than RTs cloned from SIVmac239 Vpx (+) infections. Overall, this study supports the possibility that the loss of Vpx may generate in vivo SIVmac239 RT variants that can counteract the limited availability of dNTP substrate in macrophages.

The purpose of this study was to examine the deltoid skin biopsy in twenty-three patients with coronavirus disease 2019 (COVID-19), most severely ill, for vascular complement deposition and correlate this with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral RNA and protein localization and ACE2 expression. Deltoid skin microvascular complement screening has been applied to patients with various systemic complement-mediated microvascular syndromes, best exemplified by atypical hemolytic uremic syndrome. In 21 of 23 cases, substantial microvascular deposition of complement components was identified. The two patients without significant complement deposition included one patient with moderate disease and a severely ill patient who although on a ventilator for a day was discharged after 3 days. The dominant microvascular complement immunoreactant identified was the terminal membranolytic attack complex C5b-9. Microvascular complement deposition strongly colocalized in situ with the SARS-CoV-2 viral proteins including spike glycoproteins in the endothelial cells as well as the viral receptor ACE2 in lesional and nonlesional skin; viral RNA was not evident. Microvascular SARS-CoV-2 viral protein, complement, and ACE2 expression was most conspicuous in the subcutaneous fat. Although the samples from severely ill patients with COVID-19 were from grossly normal skin, light microscopically focal microvascular abnormalities were evident that included endothelial cell denudement, basement membrane zone reduplication, and small thrombi. It is concluded that complement activation is common in grossly normal skin, especially in the subcutaneous fat which may provide a link between severe disease and obesity, in people with severe COVID-19, and the strong colocalization with the ACE2 receptor and viral capsid proteins without viral RNA suggests that circulating viral proteins (ie, pseudovirions) may dock onto the endothelial of these microvessels and induce complement activation.