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Parvovirus B19 (B19V) is a human pathogen with a marked tropism for erythroid progenitor cells (EPCs). The N-terminal of the VP1 unique region (VP1u) contains a receptor-binding domain (RBD), which mediates virus uptake through interaction with an as-yet-unknown receptor (VP1uR). Considering the central role of VP1uR in the virus tropism, we sought to investigate its expression profile in multiple cell types. To this end, we established a PP7 bacteriophage-VP1u bioconjugate, sharing the size and VP1u composition of native B19V capsids. The suitability of the PP7-VP1u construct as a specific and sensitive VP1uR expression marker was validated in competition assays with B19V and recombinant VP1u. VP1uR expression was exclusively detected in erythroid cells and cells reprogrammed towards the erythroid lineage. Sequence alignment and in silico protein structure prediction of the N-terminal of VP1u (N-VP1u) from B19V and other primate erythroparvoviruses (simian, rhesus, and pig-tailed) revealed a similar structure characterized by a fold of three or four α-helices. Functional studies with simian parvovirus confirmed the presence of a conserved RBD in the N-VP1u, mediating virus internalization into human erythroid cells. In summary, this study confirms the exclusive association of VP1uR expression with cells of the erythroid lineage. The presence of an analogous RBD in the VP1u from non-human primate erythroparvoviruses emphasizes their parallel evolutionary trait and zoonotic potential.

As a highly important fish virus, nervous necrosis virus (NNV) has caused severe economic losses to the aquaculture industry worldwide. Autophagy, an evolutionarily conserved intracellular degradation process, is involved in the pathogenesis of several viruses. Although NNV can induce autophagy to facilitate infection in grouper fish spleen cells, how it initiates and mediates autophagy pathways during the initial stage of infection is still unclear. Here, we found that red-spotted grouper NNV (RGNNV) induced autophagosome formation in two fish cell lines at 1.5 and 3 h post infection, indicating that autophagy is activated upon entry of RGNNV. Moreover, autophagic detection showed that RGNNV entry induced incomplete autophagy by impairing the fusion of autophagosomes with lysosomes. Further investigation revealed that binding of the RGNNV capsid protein (CP) to the Lateolabrax japonicus heat shock protein HSP90ab1 (LjHSP90ab1), a cell surface receptor of RGNNV, contributed to RGNNV invasion-induced autophagy. Finally, we found that CP blocked the interaction of L. japonicus protein kinase B (AKT) with LjHSP90ab1 by competitively binding the NM domain of LjHSP90ab1 to inhibit the AKT-mechanistic target of the rapamycin (MTOR) pathway. This study provides novel insight into the relationship between NNV receptors and autophagy, which may help clarify the pathogenesis of NNV.

Papillomavirus L1 and L2, the major and minor capsid proteins, play significant roles in viral assembly, entry, and propagation. In the current study, we investigate the impact of L1 and L2 on viral life cycle and tumor growth with a newly established mouse papillomavirus (MmuPV1) infection model. MmuPV1 L1 knockout, L2 knockout, and L1 plus L2 knockout mutant genomes (designated as L1ATGko-4m, L2ATGko, and L1-L2ATGko respectively) were generated. The mutants were examined for their ability to generate lesions in athymic nude mice. Viral activities were examined by qPCR, immunohistochemistry (IHC), in situ hybridization (ISH), and transmission electron microscopy (TEM) analyses. We demonstrated that viral DNA replication and tumor growth occurred at both cutaneous and mucosal sites infected with each of the mutants. Infections involving L1ATGko-4m, L2ATGko, and L1-L2ATGko mutant genomes generally resulted in smaller tumor sizes compared to infection with the wild type. The L1 protein was absent in L1ATGko-4m and L1-L2ATGko mutant-treated tissues, even though viral transcripts and E4 protein expression were robust. Therefore, L1 is not essential for MmuPV1-induced tumor growth, and this finding parallels our previous observations in the rabbit papillomavirus model. Very few viral particles were detected in L2ATGko mutant-infected tissues. Interestingly, the localization of L1 in lesions induced by L2ATGko was primarily cytoplasmic rather than nuclear. The findings support the hypothesis that the L2 gene influences the expression, location, transport, and assembly of the L1 protein in vivo.

The Open Reading Frame 45 (ORF45) of Kaposi sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus-specific, immediate-early, tegument protein required for efficient viral replication and virion production. We have previously shown that ORF45 interacts with the conserved herpesviral protein ORF33 through the highly conserved C-terminal 19 amino acids (C19) of ORF45. Because the deletion of C19 abolished ORF33 accumulation and viral production, we reasoned that this interaction could be critical for viral production and explored as an antiviral target for gammaherpesviruses. In work described in this article, we characterize this interaction in further detail, first by revealing that this interaction is conserved among gammaherpesviruses, then by identifying residues in C19 critical for its interaction with and stabilization of ORF33. More importantly, we show that disruption of the interaction, either by mutating key residues (W403A or W405A) in C19 or by using competing cell penetration peptide TAT-C19, dramatically reduce the yield of KSHV progeny viruses. Our results not only reveal critical roles of this interaction to viral production but also provide a proof of concept for targeting the ORF33-ORF45 interaction as a novel antiviral strategy against KSHV and other gammaherpesviruses.

The capsid protein of dengue virus strain 2 (DENV2C) promotes nucleic acid structural rearrangements using chaperone activity. However, the role of DENV2C during the interaction of RNA elements in the conserved 5' untranslated region (5'UTR) to the 3' untranslated region (3'UTR) is still unclear. Thus, we investigated the effect of DENV2C on the annealing mechanism of two RNA hairpin elements from the 5'UTR to their complementary sequences during (+)/(-) ds-RNAformation and (+) RNA circularization. DENV2C was found to switch the annealing pathway for RNA elements involved in (+)/(-) ds-RNA formation, but not for RNA elements related to (+) RNA circularization. In addition, we also determined that DENV2C modulates intrinsic dynamics and reduces kinetically trapped unfavourable conformations of the 5'UTR sequence. Thus, our results provide mechanistic insights by which DENV2C chaperones the interactions between RNA elements at the 5' and 3' ends during genome recombination, a prerequisite for DENV replication.

Coat proteins (CPs) play critical roles in potyvirus cell-to-cell movement. However, the underlying mechanism controlling them remains unclear. Here, we show that substitutions of alanine, glutamic acid, or lysine for the conserved residue tryptophan at position 122 (W 122 ) in tobacco vein banding mosaic virus (TVBMV) CP abolished virus cell-to-cell movement in Nicotiana benthamiana plants. In agroinfiltrated N. benthamiana leaf patches, both the CP and RNA accumulation levels of three W 122 mutant viruses were significantly reduced compared with those of wild-type TVBMV, and CP accumulated to a low level similar to that of a replication-deficient mutant. The results of polyprotein transient expression experiments indicated that CP instability was responsible for the significantly low CP accumulation levels of the three W 122 mutant viruses. The substitution of W 122 did not affect CP plasmodesmata localization or virus particle formation; however, the substitution significantly reduced the number of virus particles. The wild-type TVBMV CP could complement the reduced replication and abolished cell-to-cell movement of the mutant viruses. When the codon for W 122 was mutated to that for a different aromatic residue, phenylalanine or tyrosine, the resultant mutant viruses moved systemically and accumulated up to 80% of the wild-type TVBMV level. Similar results were obtained for the corresponding amino acids of W 122 in the watermelon mosaic virus and potato virus Y CPs. Therefore, we conclude that the aromatic ring in W 122 in the core domain of the potyviral CP is critical for cell-to-cell movement through the effects on CP stability and viral replication., (© 2020 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

The human adenovirus (HAdV) phylogenetic tree is diverse, divided across seven species and comprising over 100 individual types. Species D HAdV are rarely isolated with low rates of preexisting immunity, making them appealing for therapeutic applications. Several species D vectors have been developed as vaccines against infectious diseases, where they induce robust immunity in preclinical models and early phase clinical trials. However, many aspects of the basic virology of species D HAdV, including their basic receptor usage and means of cell entry, remain understudied. Here, we investigated HAdV-D49, which previously has been studied for vaccine and vascular gene transfer applications. We generated a pseudotyped HAdV-C5 presenting the HAdV-D49 fiber knob protein (HAdV-C5/D49K). This pseudotyped vector was efficient at infecting cells devoid of all known HAdV receptors, indicating HAdV-D49 uses an unidentified cellular receptor. Conversely, a pseudotyped vector presenting the fiber knob protein of the closely related HAdV-D30 (HAdV-C5/D30K), differing in four amino acids from HAdV-D49, failed to demonstrate the same tropism. These four amino acid changes resulted in a change in isoelectric point of the knob protein, with HAdV-D49K possessing a basic apical region compared to a more acidic region in HAdV-D30K. Structurally and biologically we demonstrate that HAdV-D49 knob protein is unable to engage CD46, while potential interaction with coxsackievirus and adenovirus receptor (CAR) is extremely limited by extension of the DG loop. HAdV-C5/49K efficiently transduced cancer cell lines of pancreatic, breast, lung, esophageal, and ovarian origin, indicating it may have potential for oncolytic virotherapy applications, especially for difficult to transduce tumor types. IMPORTANCE Adenoviruses are powerful tools experimentally and clinically. To maximize efficacy, the development of serotypes with low preexisting levels of immunity in the population is desirable. Consequently, attention has focused on those derived from species D, which have proven robust vaccine platforms. This widespread usage is despite limited knowledge in their basic biology and cellular tropism. We investigated the tropism of HAdV-D49, demonstrating that it uses a novel cell entry mechanism that bypasses all known HAdV receptors. We demonstrate, biologically, that a pseudotyped HAdV-C5/D49K vector efficiently transduces a wide range of cell lines, including those presenting no known adenovirus receptor. Structural investigation suggests that this broad tropism is the result of a highly basic electrostatic surface potential, since a homologous pseudotyped vector with a more acidic surface potential, HAdV-C5/D30K, does not display a similar pantropism. Therefore, HAdV-C5/D49K may form a powerful vector for therapeutic applications capable of infecting difficult to transduce cells., (Copyright © 2021 Baker et al.)

Background: IC 50 is one of the most important parameters of a drug. But, it is very difficult to predict this value of a new compound without experiment. There are only a few QSAR based methods available for IC 50 prediction, which is also highly dependable on a huge number of known data. Thus, there is an immense demand for a sophisticated computational method of IC 50 prediction in the field of in silico drug designing., Objective: Recently developed quantum computation based method of IC 50 prediction by Bag and Ghorai requires an affordable known data. In present research work, further development of this method is carried out such that the requisite number of known data being minimal., Methods: To retrench the cardinal data span and shrink the effects of variant biological parameters on the computed value of IC 50 , a relative approach of IC 50 computation is pursued in the present method. To predict an approximate value of IC 50 of a small molecule, only the IC 50 of a similar kind of molecule is required for this method., Results: The present method of IC 50 computation is tested for both organic and organometallic compounds as HIV-1 capsid A inhibitor and cancer drugs. Computed results match very well with the experiment., Conclusion: This method is easily applicable to both organic and organometallic compounds with acceptable accuracy. Since this method requires only the dipole moments of an unknown compound and the reference compound, IC 50 based drug search is possible with this method. An algorithm is proposed here for IC 50 based drug search., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The viral protein 1 unique region (VP1u) of human parvovirus B19 (B19V) is a multifunctional capsid protein with essential roles in virus tropism, uptake, and subcellular trafficking. These functions reside on hidden protein domains, which become accessible upon interaction with cell membrane receptors. A receptor-binding domain (RBD) in VP1u is responsible for the specific targeting and uptake of the virus exclusively into cells of the erythroid lineage in the bone marrow. A phospholipase A 2 domain promotes the endosomal escape of the incoming virus. The VP1u is also the immunodominant region of the capsid as it is the target of neutralizing antibodies. For all these reasons, the VP1u has raised great interest in antiviral research and vaccinology. Besides the essential functions in B19V infection, the remarkable erythroid specificity of the VP1u makes it a unique erythroid cell surface biomarker. Moreover, the demonstrated capacity of the VP1u to deliver diverse cargo specifically to cells around the proerythroblast differentiation stage, including erythroleukemic cells, offers novel therapeutic opportunities for erythroid-specific drug delivery. In this review, we focus on the multifunctional role of the VP1u in B19V infection and explore its potential in diagnostics and erythroid-specific therapeutics.

The Penaeus stylirostris densovirus (PstDNV) is a major virus of shrimps that severely harms the shrimp farming industry. Peritrophin is a peritrophic membrane protein with chitin binding activity. To examine the roles of peritrophin in viral infection, we used yeast two-hybrid to analyze the interaction between the Pacific white shrimp (Litopenaeus vannamei) peritrophin and PstDNV proteins (CP, NS1 and NS2). The yeast two-hybrid results showed that NS1 and peritrophin had an interaction, CP and peritrophin had an interaction as well, and NS2 had no interaction with peritrophin. We validated the interactions with GST pull-down assays. We then conducted RNA interference and qRT-PCR. The results showed that when pre-injection of dsRNA-peritrophin, the quantity of PstDNV in the shrimps injected with viruses was significantly lower than in the control group (P < 0.01), indicating the viral infection was decreased when the peritrophin gene expression was inhibited. The results indicated that peritrophin of L. vannamei participated in the PstDNV infection., (Copyright © 2020. Published by Elsevier Ltd.)

Many geometric forms are found in nature, some of them adhering to mathematical laws or amazing aesthetic rules. One of the best-known examples in microbiology is the icosahedral shape of certain viruses with 20 triangular facets and 12 edges. What is less known, however, is that a complementary object displaying 12 faces and 20 edges called a 'dodecahedron' can be produced in huge amounts during certain adenovirus replication cycles. The decahedron was first described more than 50 years ago in the human adenovirus (HAdV3) viral cycle. Later on, the expression of this recombinant scaffold, combined with improvements in cryo-electron microscopy, made it possible to decipher the structural determinants underlying their architecture. Recently, this particle, which mimics viral entry, was used to fish the long elusive adenovirus receptor, desmoglein-2, which serves as a cellular docking for some adenovirus serotypes. This breakthrough enabled the understanding of the physiological role played by the dodecahedral particles, showing that icosahedral and dodecahedral particles live more than a simple platonic story. All these points are developed in this review, and the potential use of the dodecahedron in therapeutic development is discussed.

Porcine circovirus 3 (PCV3) has been detected in major pig-producing countries around the world since its first report in the US in 2016. Most current studies have focused on epidemiological investigations and detection methods of PCV3 because of lack of live virus strains for research on its pathogenesis in porcine cells or even in pigs. We constructed a recombinant plasmid pCMV-Cap carrying the PCV3 orf2 gene to investigate the effects of capsid (Cap) protein expression on autophagic response in human embryonic kidney cell line 293T (HEK293T). We demonstrate that PCV3 Cap protein induced complete autophagy shown as formation of autophagosomes and autophagosome-like vesicles as well as LC3-II conversion from LC3-I via inhibiting phosphorylation of the mammalian target of rapamycin (mTOR) in HEK293T cells. The ubiquitin-proteasome pathway is also involved in the autophagy process. These findings provide insight for further exploration of PCV3 pathogenetic mechanisms in porcine cells.

Although viruses are extremely diverse in shape and size, evolution has led to a limited number of viral classes or lineages, which is probably linked to the assembly constraints of a viable capsid. Viral assembly mechanisms are restricted to two general pathways, (i) co-assembly of capsid proteins and single-stranded nucleic acids and (ii) a sequential mechanism in which scaffolding-mediated capsid precursor assembly is followed by genome packaging. Cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET), which are revolutionizing structural biology, are central to determining the high-resolution structures of many viral assemblies as well as those of assembly intermediates. This wealth of cryo-EM data has also led to the development and redesign of virus-based platforms for biomedical and biotechnological applications. In this Review, we will discuss recent viral assembly analyses by cryo-EM and cryo-ET showing how natural assembly mechanisms are used to encapsulate heterologous cargos including chemicals, enzymes, and/or nucleic acids for a variety of nanotechnological applications.

To begin its infection, a bacteriophage first needs to adsorb to cells. The adsorption site on the cell surface may influence viral DNA injection, gene expression and cell-fate development. Here, we study the early steps of the infection cycle of coliphage P1, focusing on their correlation with spatial locations at the single-cell level. By fluorescently labeling P1 virions, we found that P1 shows no spatial preference on cell surface adsorption. In addition, live-cell phage DNA imaging revealed that adsorption sites do not affect the success rate for P1 in injecting its DNA into the cell. Furthermore, the lysis-lysogeny decision of P1 does not depend on the adsorption site, based on fluorescence reporters for the lytic and lysogenic pathways. These findings highlight the different infection strategies used by the two paradigmatic coliphages differ from those found in the paradigmatic phage lambda, highlighting that different infection strategies are used by phages., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

West Nile virus (WNV) belongs to the Flaviviridae family and has emerged as a significant cause of viral encephalitis in birds and animals including humans. WNV replication directly induces neuronal injury, followed by neuronal cell death. We previously showed that accumulation of ubiquitinated protein aggregates was involved in neuronal cell death in the WNV-infected mouse brain. In this study, we attempted to elucidate the mechanisms of the accumulation of protein aggregates in the WNV-infected cells. To identify the viral factor inducing the accumulation of ubiquitinated proteins, intracellular accumulation of ubiquitinated proteins was examined in the cells expressing the viral protein. Expression of capsid (C) protein induced the accumulation, while mutations at residues L51 and A52 in C protein abrogated the accumulation. Wild-type (WT) or mutant WNV in which mutations were introduced into the residues was inoculated into human neuroblastoma cells. The expression levels of LC3-II, an autophagy-related protein, and AMP-activated protein kinase (AMPK), an autophagy inducer, were reduced in the cells infected with WT WNV, while the reduction was not observed in the cells infected with WNV with the mutations in C protein. Similarly, ubiquitination and degradation of AMPK were only observed in the cells infected with WT WNV. In the cells expressing C protein, AMPK was co-precipitated with C protein and mutations in L51 and A52 reduced the interaction. Although the viral replication was not affected, the accumulation of ubiquitinated proteins in brain and neurological symptoms were attenuated in the mouse inoculated with WNV with the mutations in C protein as compared with that with WT WNV. Taken together, ubiquitination and degradation of AMPK by C protein resulted in the inhibition of autophagy and the accumulation of protein aggregates, which contributes to the development of neurological disease., Competing Interests: The authors have declared that no competing interests exist.

The formation of a membrane-enveloped virus starts with the assembly of a curved layer of capsid proteins lining the interior of the plasma membrane (PM) of the host cell. This layer develops into a spherical shell (capsid) enveloped by a lipid-rich membrane. In many cases, the budding process stalls prior to the release of the virus. Recently, Brownian dynamics simulations of a coarse-grained model system reproduced protracted pausing and stalling, which suggests that the origin of pausing/stalling is to be found in the physics of the budding process. Here, we propose that the pausing/stalling observed in the simulations can be understood as a purely kinetic phenomenon associated with the neck geometry. A geometrical potential energy barrier develops during the budding that must be overcome by capsid proteins diffusing along the membrane prior to incorporation into the capsid. The barrier is generated by a conflict between the positive Gauss curvature of the assembling capsid and the negative Gauss curvature of the neck region. A continuum theory description is proposed and is compared with the Brownian simulations of the budding of enveloped viruses., Competing Interests: The authors have declared that no competing interests exist.

During retrovirus maturation, cleavage of the precursor structural Gag polyprotein by the viral protease induces architectural rearrangement of the virus particle from an immature into a mature, infectious form. The structural rearrangement encapsidates the viral RNA genome in a fullerene capsid, producing a diffusible viral core that can initiate infection upon entry into the cytoplasm of a host cell. Maturation is an important therapeutic window against HIV-1. In this review, we highlight recent breakthroughs in understanding of the structures of retroviral immature and mature capsid lattices that define the boundary conditions of maturation and provide novel insights on capsid transformation. We also discuss emerging insights on encapsidation of the viral genome in the mature capsid, as well as remaining questions for further study., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The assembly of the HIV-1 immature capsid (HIC) is an essential step in the virus life cycle. In vivo, the HIC is composed of [Formula: see text] hexameric building blocks, and it takes 5-6 min to complete the assembly process. The involvement of numerous building blocks and the rapid timecourse makes it difficult to understand the HIC assembly process. In this work, we study HIC assembly in vivo by using differential equations. We first obtain a full model with 420 differential equations. Then, we reduce six addition reactions for separate building blocks to a single complex reaction. This strategy reduces the full model to 70 equations. Subsequently, the theoretical analysis of the reduced model shows that it might not be an effective way to decrease the HIC concentration at the equilibrium state by decreasing the microscopic on-rate constants. Based on experimental data, we estimate that the nucleating structure is much smaller than the HIC. We also estimate that the microscopic on-rate constant for nucleation reactions is far less than that for elongation reactions. The parametric collinearity investigation testifies the reliability of these two characteristics, which might explain why free building blocks do not readily polymerize into higher-order polymers until their concentration reaches a threshold value. These results can provide further insight into the assembly mechanisms of the HIC in vivo.

Plants respond to pathogens through dynamic regulation of plasma membrane-bound signaling pathways. To date, how the plant plasma membrane is involved in responses to viruses is mostly unknown. Here, we show that plant cells sense the Potato virus X (PVX) COAT PROTEIN and TRIPLE GENE BLOCK 1 proteins and subsequently trigger the activation of a membrane-bound calcium-dependent kinase. We show that the Arabidopsis thaliana CALCIUM-DEPENDENT PROTEIN KINASE 3-interacts with group 1 REMORINs in vivo, phosphorylates the intrinsically disordered N-terminal domain of the Group 1 REMORIN REM1.3, and restricts PVX cell-to-cell movement. REM1.3's phospho-status defines its plasma membrane nanodomain organization and is crucial for REM1.3-dependent restriction of PVX cell-to-cell movement by regulation of callose deposition at plasmodesmata. This study unveils plasma membrane nanodomain-associated molecular events underlying the plant immune response to viruses., Competing Interests: The authors have declared that no competing interests exist.

Detection of viruses by innate immune sensors induces protective antiviral immunity. The viral DNA sensor cyclic GMP-AMP synthase (cGAS) is necessary for detection of HIV by human dendritic cells and macrophages. However, synthesis of HIV DNA during infection is not sufficient for immune activation. The capsid protein, which associates with viral DNA, has a pivotal role in enabling cGAS-mediated immune activation. We now find that NONO is an essential sensor of the HIV capsid in the nucleus. NONO protein directly binds capsid with higher affinity for weakly pathogenic HIV-2 than highly pathogenic HIV-1. Upon infection, NONO is essential for cGAS activation by HIV and cGAS association with HIV DNA in the nucleus. NONO recognizes a conserved region in HIV capsid with limited tolerance for escape mutations. Detection of nuclear viral capsid by NONO to promote DNA sensing by cGAS reveals an innate strategy to achieve distinction of viruses from self in the nucleus., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Viral evolutionary pathways are determined by the fitness landscape, which maps viral genotype to fitness. However, a quantitative description of the landscape and the evolutionary forces on it remain elusive. Here, we apply a biophysical fitness model based on capsid folding stability and antibody binding affinity to predict the evolutionary pathway of norovirus escaping a neutralizing antibody. The model is validated by experimental evolution in bulk culture and in a drop-based microfluidics that propagates millions of independent small viral subpopulations. We demonstrate that along the axis of binding affinity, selection for escape variants and drift due to random mutations have the same direction, an atypical case in evolution. However, along folding stability, selection and drift are opposing forces whose balance is tuned by viral population size. Our results demonstrate that predictable epistatic tradeoffs between molecular traits of viral proteins shape viral evolution.

Background: Disulfiram (DSF), which is used to treat alcohol dependence, has been reported to have anti-cancer effects in various malignant tumors. In this study, we investigated the anti-cancer effects and mechanism of DSF in HNSCC., Methods: Head and neck squamous carcinoma cell lines (FaDu and Hep2) were used to analyze the anti-cancer effects of DSF. The anti-cancer effects of DSF were confirmed in vivo using a xenograft tumor model., Results: The anti-cancer effects of DSF in HNSCC were found to be copper (Cu) dependent. Specifically, DSF/Cu markedly inhibited HNSCC at a concentration of 1 μM. After DSF/Cu administration, production of reactive oxygen species (ROS) was remarkable starting at 0.5 μM, suggesting that the inhibitory effects of DSF/Cu on HNSCC are mediated through the formation of ROS. The levels of phospho-JNK, phospho-cJun and phospho-p38 were increased after DSF/Cu treatment while levels of phospho-Akt were decreased. These results suggested that the inhibitory effects of DSF/Cu on HNSCC cells involve ROS formation and down-regulation of Akt-signaling. Through these molecular mechanisms, DSF ultimately induce the inhibitory effects on HNSCC cell lines mainly through autophagic cell death, not apoptotic cell death. Lastly, we investigated the clinical relevance of DSF/Cu using a HNSCC xenograft animal model, which showed that tumor growth was remarkably decreased by DSF (50 mg/kg injection)., Conclusion: In treating patients with HNSCC, DSF may contribute to improved HNSCC patient's survival. The characteristic anti-cancer effects of DSF on HNSCC may suggest new therapeutic potential for this medication in HNSCC patients., Competing Interests: The authors have no funding sources, financial relationships, or conflicts of interest to disclose.

Disruption of host membranes by nonenveloped viruses, which allows the nucleocapsid or genome to enter the cytosol, is a mechanistically diverse process. Although the membrane-penetrating agents are usually small, hydrophobic or amphipathic peptides deployed from the capsid interior during entry, their manner of membrane interaction varies substantially. In this review, we discuss recent data about the molecular pathways for externalization of viral peptides amidst conformational alterations in the capsid, as well as mechanisms of membrane penetration, which is influenced by structural features of the peptides themselves as well as physicochemical properties of membranes, and other host factors. The membrane-penetrating components of nonenveloped viruses constitute an interesting class of cell-penetrating peptides, and may have potential therapeutic value for gene transfer., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Hepatitis B virus (HBV) core protein (HBc) contains an N-terminal domain (NTD, assembly domain) and a C-terminal domain (CTD), which are linked by a flexible linker region. HBc plays multiple essential roles in viral replication, including capsid assembly, packaging of the viral pregenomic RNA (pgRNA) into nucleocapsids, viral reverse transcription that converts pgRNA to the genomic DNA, and secretion of DNA-containing (complete) virions or genome-free (empty) virions. The HBc linker is generally assumed to act merely as a spacer between NTD and CTD but some results suggest that the linker may affect NTD assembly. To determine its role in viral replication, we have made a number of deletion and substitution mutants in the linker region, in either the presence or absence of CTD, and tested their abilities to support capsid assembly and viral replication in human cells. Our results indicate that the linker could indeed impede NTD assembly in the absence of CTD, which could be partially relieved by partial linker deletion. In contrast, when CTD was present, the linker deletions or substitutions did not affect capsid assembly. Deletion of the entire linker or its C-terminal part resulted in a partial defect in pgRNA packaging and severely impaired viral DNA synthesis. In contrast, deletion of the N-terminal part of the linker, or substitutions of the linker sequence, had little to no effect on RNA packaging or first-strand DNA synthesis. However, the N-terminal linker deletion and two linker substitution mutants were defective in the production of mature double-stranded viral DNA. Secretion of empty virions was blocked by all the linker deletions and substitutions tested. In particular, a conservative linker substitution that allowed mature viral DNA synthesis and secretion of complete virions severely impaired the secretion of empty virions, thus increasing the ratio of complete to empty virions that were secreted. Together, these results demonstrate that the HBc linker region plays critical and complex roles at multiple stages of HBV replication., Competing Interests: The authors have declared that no competing interests exist.

Many viruses, including adenovirus, exhibit bidirectional transport along microtubules following cell entry. Cytoplasmic dynein is responsible for microtubule minus end transport of adenovirus capsids after endosomal escape. However, the identity and roles of the opposing plus end-directed motor(s) remain unknown. We performed an RNAi screen of 38 kinesins, which implicated Kif5B (kinesin-1 family) and additional minor kinesins in adenovirus 5 (Ad5) capsid translocation. Kif5B RNAi markedly increased centrosome accumulation of incoming Ad5 capsids in human A549 pulmonary epithelial cells within the first 30 min post infection, an effect dramatically enhanced by blocking Ad5 nuclear pore targeting using leptomycin B. The Kif5B RNAi phenotype was rescued by expression of RNAi-resistant Kif5A, B, or C, and Kif4A. Kif5B RNAi also inhibited a novel form of microtubule-based "assisted-diffusion" behavior which was apparent between 30 and 60 min p.i. We found the major capsid protein penton base (PB) to recruit kinesin-1, distinct from the hexon role we previously identified for cytoplasmic dynein binding. We propose that adenovirus uses independently recruited kinesin and dynein for directed transport and for a more random microtubule-based assisted diffusion behavior to fully explore the cytoplasm before docking at the nucleus, a mechanism of potential importance for physiological cargoes as well., Competing Interests: The authors have declared that no competing interests exist.

Competing Interests: The authors have declared that no competing interests exist.

Capsid (CA) protein is a major virion-constituent of all retroviruses including human immunodeficiency virus type 1 (HIV-1), and is essential for early and late phases in viral replication cycle through interaction with numerous cellular factors. In particular, N-terminal domain (NTD) of HIV-1 CA has been frequently and well reported to bind to various host cell proteins that considerably affect viral replication potential. In this study, in order to better define biological bases of the CA-NTD for HIV-1 replication, we performed an extensive mutational analysis in an unprecedented manner. By aligning CA-NTD sequences derived from representative infectious molecular clones of HIV-1, HIV-2, and simian immunodeficiency virus isolated from the rhesus macaque (SIVmac), a number of amino acids specific to HIV-1 were selected, and were replaced with those from SIVmac at the corresponding sites. Mutant viruses thus generated were then examined for multi-cycle infectivity, single-cycle infectivity, and ability to produce progeny virions. While some CA-NTD mutations affected viral replication ability to varying degrees, those in helix 7 abolished viral growth potential without exception. These results highlight functional importance of non-conserved amino acids in helix 7, and give new insights into functionality of HIV-1 CA-NTD. J. Med. Invest. 65:110-115, February, 2018.

Protein tyrosine sulfation (PTS) is a key modulator of extracellular protein-protein interaction (PPI), which regulates principal biological processes. For example, the capsid protein VP1 of enterovirus 71 (EV71) specifically interacts with sulfated P-selectin glycoprotein ligand-1 (PSGL-1) to facilitate virus invasion. Currently available methods cannot be used to directly observe PTS-induced PPI. In this study, atomic force microscopy was used to measure the interaction between sulfated or mutated PSGL-1 and VP1. We found that the binding strength increased by 6.7-fold following PTS treatment on PSGL-1 with a specific antisulfotyrosine antibody. Similar results were obtained when the antisulfotyrosine antibody was replaced with the VP1 protein of EV71; however, the interaction forces of VP1 were only approximately one-third of those of the antisulfotyrosine antibody. We also found that PTS on the tyrosine-51 residue of glutathione S-transferases fusion-PSGL-1 was mainly responsible for the PTS-induced PPI. Our results contribute to the fundamental understanding of PPI regulated through PTS.

Almost a century has passed since the discovery of papillomaviruses. A few decades of research have given a wealth of information on the molecular biology of papillomaviruses. Several excellent studies have been performed looking at the long- and short-term evolution of these viruses. However, when and how papillomaviruses originate is still a mystery. In this study, we systematically searched the (sequenced) biosphere to find distant homologs of papillomaviral protein domains. Our data show that, even including structural information, which allows us to find deeper evolutionary relationships compared to sequence-only based methods, only half of the protein domains in papillomaviruses have relatives in the rest of the biosphere. We show that the major capsid protein L1 and the replication protein E1 have relatives in several viral families, sharing three protein domains with Polyomaviridae and Parvoviridae . However, only the E1 replication protein has connections with cellular organisms. Most likely, the papillomavirus ancestor is of marine origin, a biotope that is not very well sequenced at the present time. Nevertheless, there is no evidence as to how papillomaviruses originated and how they became vertebrate and epithelium specific., Competing Interests: The authors declare no conflicts of interest.

The long distance movement of potyviruses is a poorly understood step of the viral cycle. Only factors inhibiting this process, referred to as "Restricted TEV Movement" (RTM), have been identified in Arabidopsis thaliana. On the virus side, the potyvirus coat protein (CP) displays determinants required for long-distance movement and for RTM-based resistance breaking. However, the potyvirus CP was previously shown not to interact with the RTM proteins. We undertook the identification of Arabidopsis factors which directly interact with either the RTM proteins or the CP of lettuce mosaic virus (LMV). An Arabidopsis cDNA library generated from companion cells was screened with LMV CP and RTM proteins using the yeast two-hybrid system. Fourteen interacting proteins were identified. Two of them were shown to interact with CP and the RTM proteins suggesting that a multiprotein complex could be formed between the RTM proteins and virions or viral ribonucleoprotein complexes. Co-localization experiments in Nicotiana benthamiana showed that most of the viral and cellular protein pairs co-localized at the periphery of chloroplasts which suggests a putative role for plastids in this process.

Bovine adenovirus type 3 (BAdV3) is being used in the development of potential vehicles for gene therapy and vectored vaccine. To that end, a more comprehensive description of BAdV3 biology is essential. In this study, we focused on the role of pIX in BAdV3 virion rescue after full-length BAdV3 genome transfection. Initially, pIX deletion or initiation codon mutation abolished the production of progeny virions, which suggested that pIX was essential for the rescue of BAdV3 containing a full-length genome. Moreover, through transfection of a panel of pIX mutant BAdV3 genomes, we observed that the conserved N-terminus and the putative leucine zipper element (PLZP) were essential for virion rescue, whereas the C-terminus following the coiled-coil domain was non-essential. In addition, swap of the PLZP element and its following region of BAdV3 pIX to corresponding domains of human adenovirus type 5 (HAdV5) did not affect virion production, whereas swap of the entire pIX abolished production of progeny virions. We suggest that failure of the full-length BAdV3 pIX swap might be due to species specificity of its N-terminus region before the PLZP element.

Using atomic force microscopy imaging and nanoindentation measurements, we investigated the effect of the minor capsid proteins pUL17 and pUL25 on the structural stability of icosahedral herpes simplex virus capsids. pUL17 and pUL25, which form the capsid vertex-specific component (CVSC), particularly contributed to capsid resilience along the 5-fold and 2-fold but not along the 3-fold icosahedral axes. Our detailed analyses, including quantitative mass spectrometry of the protein composition of the capsids, revealed that both pUL17 and pUL25 are required to stabilize the capsid shells at the vertices. This indicates that herpesviruses withstand the internal pressure that is generated during DNA genome packaging by locally reinforcing the mechanical sturdiness of the vertices, the most stressed part of the capsids. IMPORTANCE In this study, the structural, material properties of herpes simplex virus 1 were investigated. The capsid of herpes simplex virus is built up of a variety of proteins, and we scrutinized the influence of two of these proteins on the stability of the capsid. For this, we used a scanning force microscope that makes detailed, topographic images of the particles and that is able to perform mechanical deformation measurements. Using this approach, we revealed that both studied proteins play an essential role in viral stability. These new insights support us in forming a complete view on viral structure and furthermore could possibly help not only to develop specific antivirals but also to build protein shells with improved stability for drug delivery purposes., (Copyright © 2017 American Society for Microbiology.)

Simian virus 40 capsid protein (VP1) is a unique system for studying substrate-dependent assembly of a nanoparticle. Here, we investigate a simplest case of this system where 12 VP1 pentamers and a single polyanion, e.g., RNA, form a T = 1 particle. To test the roles of polyanion substrate length and structure during assembly, we characterized the assembly products with size exclusion chromatography, transmission electron microscopy, and single-particle resistive-pulse sensing. We found that 500 and 600 nt RNAs had the optimal length and structure for assembly of uniform T = 1 particles. Longer 800 nt RNA, shorter 300 nt RNA, and a linear 600 unit poly(styrene sulfonate) (PSS) polyelectrolyte produced heterogeneous populations of products. This result was surprising as the 600mer PSS and 500-600 nt RNA have similar mass and charge. Like ssRNA, PSS also has a short 4 nm persistence length, but unlike RNA, PSS lacks a compact tertiary structure. These data indicate that even for flexible substrates, shape as well as size affect assembly and are consistent with the hypothesis that work, derived from protein-protein and protein-substrate interactions, is used to compact the substrate.

Porcine circovirus type 2 (PCV2) has recently been reported to elicit the unfolded protein response (UPR) via activation of the PERK/eIF2α (RNA-activated protein kinase-like endoplasmic reticulum (ER) kinase/eukaryotic initiation factor 2α) pathway. This study attempted to examine which viral protein might be involved in inducing UPR and whether this cellular event would lead to apoptosis of the cells expressing the viral protein. By transient expression, we found that both replicase (Rep) and capsid (Cap) proteins of PCV2 could induce ER stress as shown by increased phosphorylation of PERK with subsequent activation of the eIF2α-ATF4 (activating transcription factor 4)-CHOP (CCAAT/enhancer-binding protein homologous protein) axis. Cap expression, but not Rep, significantly reduced anti-apoptotic B-cell lymphoma-2 (Bcl-2) and increased caspase-3 cleavage, possibly due to increased expression of CHOP. Since knockdown of PERK by RNA interference clearly reduced Cap-induced CHOP expression, caspase-3 cleavage, and apoptotic cell death possibly by partially rescuing Bcl-2 expression, we propose that there is connection between Cap-induced UPR and apoptosis via the PERK/eIF2α/ATF4/CHOP/Bcl-2 pathway. This study, together with our earlier studies, provides insight into the mechanisms underlying PCV2 pathogenesis.

Baculovirus occlusion-derived virus (ODV) initiates infection of lepidopteran larval hosts by binding to the midgut epithelia, which is mediated by per os infectivity factors (PIFs). Autographa californica multiple nucleopolyhedrovirus (AcMNPV) encodes seven PIF proteins, of which PIF1 to PIF4 form a core complex in ODV envelopes to which PIF0 and PIF6 loosely associate. Deletion of any pif gene results in ODV being unable to bind or enter midgut cells. AC83 also associates with the PIF complex, and this study further analyzed its role in oral infectivity to determine if it is a PIF protein. It had been proposed that AC83 possesses a chitin binding domain that enables transit through the peritrophic matrix; however, no chitin binding activity has ever been demonstrated. AC83 has been reported to be found only in the ODV envelopes, but in contrast, the Orgyia pseudotsugata MNPV AC83 homolog is associated with both ODV nucleocapsids and envelopes. In addition, unlike known pif genes, deletion of ac83 eliminates nucleocapsid formation. We propose a new model for AC83 function and show AC83 is associated with both ODV nucleocapsids and envelopes. We also further define the domain required for nucleocapsid assembly. The cysteine-rich region of AC83 is also shown not to be a chitin binding domain but a zinc finger domain required for the recruitment or assembly of the PIF complex to ODV envelopes. As such, AC83 has all the properties of a PIF protein and should be considered PIF8. In addition, pif7 ( ac110 ) is reported as the 38th baculovirus core gene. IMPORTANCE ODV is essential for the per os infectivity of the baculovirus AcMNPV. To initiate infection, ODV binds to microvilli of lepidopteran midgut cells, a process which requires a group of seven virion envelope proteins called PIFs. In this study, we reexamined the function of AC83, a protein that copurifies with the ODV PIFs, to determine its role in the oral infection process. A zinc finger domain was identified and a new model for AC83 function was proposed. In contrast to previous studies, AC83 was found to be physically located in both the envelope and nucleocapsid of ODV. By deletion analysis, the AC83 domain required for nucleocapsid assembly was more finely delineated. We show that AC83 is required for PIF complex formation and conclude that it is a true per os infectivity factor and should be called PIF8., (© Crown copyright 2017.)

Tailed bacteriophages and herpesviruses assemble infectious particles via an empty precursor capsid (or 'procapsid') built by multiple copies of coat and scaffolding protein and by one dodecameric portal protein. Genome packaging triggers rearrangement of the coat protein and release of scaffolding protein, resulting in dramatic procapsid lattice expansion. Here, we provide structural evidence that the portal protein of the bacteriophage P22 exists in two distinct dodecameric conformations: an asymmetric assembly in the procapsid (PC-portal) that is competent for high affinity binding to the large terminase packaging protein, and a symmetric ring in the mature virion (MV-portal) that has negligible affinity for the packaging motor. Modelling studies indicate the structure of PC-portal is incompatible with DNA coaxially spooled around the portal vertex, suggesting that newly packaged DNA triggers the switch from PC- to MV-conformation. Thus, we propose the signal for termination of 'Headful Packaging' is a DNA-dependent symmetrization of portal protein., Competing Interests: The authors declare no competing financial interests.

Superinfection exclusion (SIE) is an antagonistic virus-virus interaction whereby initial infection by one virus prevents subsequent infection by closely related viruses. Although SIE has been described in diverse viruses infecting plants, humans, and animals, its mechanisms, including involvement of specific viral determinants, are just beginning to be elucidated. In this study, SIE determinants encoded by two economically important wheat viruses, Wheat streak mosaic virus (WSMV; genus Tritimovirus, family Potyviridae) and Triticum mosaic virus (TriMV; genus Poacevirus, family Potyviridae), were identified in gain-of-function experiments that used heterologous viruses to express individual virus-encoded proteins in wheat. Wheat plants infected with TriMV expressing WSMV P1, HC-Pro, P3, 6K1, CI, 6K2, NIa-VPg, or NIb cistrons permitted efficient superinfection by WSMV expressing green fluorescent protein (WSMV-GFP). In contrast, wheat infected with TriMV expressing WSMV NIa-Pro or coat protein (CP) substantially excluded superinfection by WSMV-GFP, suggesting that both of these cistrons are SIE effectors encoded by WSMV. Importantly, SIE is due to functional WSMV NIa-Pro or CP rather than their encoding RNAs, as altering the coded protein products by minimally changing RNA sequences led to abolishment of SIE. Deletion mutagenesis further revealed that elicitation of SIE by NIa-Pro requires the entire protein while CP requires only a 200-amino-acid (aa) middle fragment (aa 101 to 300) of the 349 aa. Strikingly, reciprocal experiments with WSMV-mediated expression of TriMV proteins showed that TriMV CP, and TriMV NIa-Pro to a lesser extent, likewise excluded superinfection by TriMV-GFP. Collectively, these data demonstrate that WSMV- and TriMV-encoded CP and NIa-Pro proteins are effectors of SIE and that these two proteins trigger SIE independently of each other., Importance: Superinfection exclusion (SIE) is an antagonistic virus-virus interaction that prevents secondary invasions by identical or closely related viruses in the same host cells. Although known to occur in diverse viruses, SIE remains an enigma in terms of key molecular determinants and action mechanisms. In this study, we found that Wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV) encode two independently functioning cistrons that serve as effectors of SIE at the protein but not the RNA level. The coat protein and NIa-Pro encoded by these two viruses, when expressed from a heterologous virus, exerted SIE to the cognate viruses. The identification of virus-encoded effectors of SIE and their transgenic expression could potentially facilitate the development of virus-resistant crop plants. Additionally, functional conservation of SIE in diverse virus groups suggests that a better understanding of the underlying mechanisms of SIE could facilitate the development of novel antiviral therapies against viral diseases., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

The human papillomavirus (HPV) capsid protein L2 is essential for viral entry. To gain a deeper understanding of the role of L2, we searched for novel cellular L2-interacting proteins. A yeast two-hybrid analysis uncovered the actin-depolymerizing factor gelsolin, the membrane glycoprotein dysadherin, the centrosomal protein 68 (Cep68), and the cytoskeletal adaptor protein obscurin-like 1 protein (OBSL1) as putative L2 binding molecules. Pseudovirus (PsV) infection assays identified OBSL1 as a host factor required for gene transduction by three oncogenic human papillomavirus types, HPV16, HPV18, and HPV31. In addition, we detected OBSL1 expression in cervical tissue sections and noted the involvement of OBSL1 during gene transduction of primary keratinocytes by HPV16 PsV. Complex formation of HPV16 L2 with OBSL1 was demonstrated in coimmunofluorescence and coimmunoprecipitation studies after overexpression of L2 or after PsV exposure. We observed a strong colocalization of OBSL1 with HPV16 PsV and tetraspanin CD151 at the plasma membrane, suggesting a role for OBSL1 in viral endocytosis. Indeed, viral entry assays exhibited a reduction of viral endocytosis in OBSL1-depleted cells. Our results suggest OBSL1 as a novel L2-interacting protein and endocytosis factor in HPV infection., Importance: Human papillomaviruses infect mucosal and cutaneous epithelia, and the high-risk HPV types account for 5% of cancer cases worldwide. As recently discovered, HPV entry occurs by a clathrin-, caveolin-, and dynamin-independent endocytosis via tetraspanin-enriched microdomains. At present, the cellular proteins involved in the underlying mechanism of this type of endocytosis are under investigation. In this study, the cytoskeletal adaptor OBSL1 was discovered as a previously unrecognized interaction partner of the minor capsid protein L2 and was identified as a proviral host factor required for HPV16 endocytosis into target cells. The findings of this study advance the understanding of a so far less well-characterized endocytic pathway that is used by oncogenic HPV subtypes., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Proteins that form the reovirus outer capsid play an active role in the entry of reovirus into host cells. Among these, the σ1 protein mediates attachment of reovirus particles to host cells via interaction with cell surface glycans or the proteinaceous receptor junctional adhesion molecule A (JAM-A). The μ1 protein functions to penetrate the host cell membrane to allow delivery of the genome-containing viral core particle into the cytoplasm to initiate viral replication. We demonstrate that a reassortant virus that expresses the M2 gene-encoded μ1 protein derived from prototype strain T3D in an otherwise prototype T1L background (T1L/T3DM2) infects cells more efficiently than parental T1L. Unexpectedly, the enhancement in infectivity of T1L/T3DM2 is due to its capacity to attach to cells more efficiently. We present genetic data implicating the central region of μ1 in altering the cell attachment property of reovirus. Our data indicate that the T3D μ1-mediated enhancement in infectivity of T1L is dependent on the function of σ1 and requires the expression of JAM-A. We also demonstrate that T1L/T3DM2 utilizes JAM-A more efficiently than T1L. These studies revealed a previously unknown relationship between two nonadjacent reovirus outer capsid proteins, σ1 and μ1., Importance: How reovirus attaches to host cells has been extensively characterized. Attachment of reovirus to host cells is mediated by the σ1 protein, and properties of σ1 influence the capacity of reovirus to target specific host tissues and produce disease. Here, we present new evidence indicating that the cell attachment properties of σ1 are influenced by the nature of μ1, a capsid protein that does not physically interact with σ1. These studies could explain the previously described role for μ1 in influencing reovirus pathogenesis. These studies are also of broader significance because they highlight an example of how genetic reassortment between virus strains could produce phenotypes that are distinct from those of either parent., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Staphylococcus aureus pathogenicity islands (SaPIs) are phage satellites that exploit the life cycle of their helper phages for their own benefit. Most SaPIs are packaged by their helper phages using a headful (pac) packaging mechanism. These SaPIs interfere with pac phage reproduction through a variety of strategies, including the redirection of phage capsid assembly to form small capsids, a process that depends on the expression of the SaPI-encoded cpmA and cpmB genes. Another SaPI subfamily is induced and packaged by cos-type phages, and although these cos SaPIs also block the life cycle of their inducing phages, the basis for this mechanism of interference remains to be deciphered. Here we have identified and characterized one mechanism by which the SaPIs interfere with cos phage reproduction. This mechanism depends on a SaPI-encoded gene, ccm, which encodes a protein involved in the production of small isometric capsids, compared with the prolate helper phage capsids. As the Ccm and CpmAB proteins are completely unrelated in sequence, this strategy represents a fascinating example of convergent evolution. Moreover, this result also indicates that the production of SaPI-sized particles is a widespread strategy of phage interference conserved during SaPI evolution.This article is part of the themed issue 'The new bacteriology'., (© 2016 The Authors.)

Epizootic hemorrhagic disease virus (EHDV) is a distinct species within the genus Orbivirus, within the family Reoviridae. The epizootic hemorrhagic disease virus genome comprises ten segments of linear, double stranded (ds) RNA, which are packaged within each virus particle. The EHDV virion has a three layered capsid-structure, generated by four major viral proteins: VP2 and VP5 (outer capsid layer); VP7 (intermediate, core-surface layer) and VP3 (innermost, sub-core layer). Although EHDV infects cattle sporadically, several outbreaks have recently occurred in this species in five Mediterranean countries, indicating a potential threat to the European cattle industry. EHDV is transmitted by biting midges of the genus Culicoides, which can travel long distances through wind-born movements (particularly over water), increasing the potential for viral spread in new areas/countries. Expression systems to generate self-assembled virus like particles (VLPs) by simultaneous expression of the major capsid-proteins, have been established for several viruses (including bluetongue virus). This study has developed expression systems for production of EHDV VLPs, for use as non-infectious antigens in both vaccinology and serology studies, avoiding the risk of genetic reassortment between vaccine and field strains and facilitating large scale antigen production. Genes encoding the four major-capsid proteins of a field strain of EHDV-6, were isolated and cloned into transfer vectors, to generate two recombinant baculoviruses. The expression of these viral genes was assessed in insect cells by monitoring the presence of specific viral mRNAs and by western blotting. Electron microscopy studies confirmed the formation and purification of assembled VLPs., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Sri Lankan cassava mosaic virus (SLCMV) is bipartite begomovirus infecting cassava in India and Sri Lanka. Interestingly, the DNA-A component of the SLCMV alone is able to infect Nicotiana benthamiana causing symptoms of upward leaf rolling and stunting. One of the differences between monopartite and bipartite begomoviruses is the requirement of Coat Protein (CP) for infectivity; CP being essential for the former, but dispensable in the latter. This investigation was aimed to determine the importance of CP in the infectivity of the bipartite SLCMV, behaving as a monopartite virus in N. benthamiana. We tested CP-null mutants, single amino acid replacement mutants and double, triple and quadruple combinations of the above in SLCMV DNA-A, for infectivity, symptom development and viral DNA accumulation in N. benthamiana. While CP-null mutants were non-infectious, a majority of the single amino acid replacement mutants and their combinations retained infectivity, some with attenuated symptoms and reduced viral titers. Some of the combined mutations restored the attenuated symptoms to wild type levels. Some of the mutations were predicted to cause changes in the secondary structure of the CP, which roughly correlated with the attenuation of symptoms and the reduction in viral titers., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Clinically available drugs active against Epstein-Barr virus (EBV) and other human herpesviruses are limited to those targeting viral DNA replication. To identify compounds directed against other steps in the viral life cycle, we searched for drugs active against the EBV SM protein, which is essential for infectious virus production. SM has a highly gene-specific mode of action and preferentially enhances expression of several late lytic cycle EBV genes. Here we demonstrate that spironolactone, a mineralocorticoid receptor antagonist approved for clinical use, inhibits SM function and infectious EBV production. Expression of EBV viral capsid antigen is highly SM dependent, and spironolactone inhibits viral capsid antigen synthesis and capsid formation, blocking EBV virion production at a step subsequent to viral DNA replication. In addition, spironolactone inhibits expression of other SM-dependent genes necessary for infectious virion formation. We further demonstrate that molecules structurally related to spironolactone with similar antimineralocorticoid blocking activity do not inhibit EBV production. These findings pave the way for development of antiherpesvirus drugs with new mechanisms of action directed against SM and homologous essential proteins in other herpesviruses.

The mechanical properties of virus capsids correlate with local conformational dynamics in the capsid structure. They also reflect the required stability needed to withstand high internal pressures generated upon genome loading and contribute to the success of important events in viral infectivity, such as capsid maturation, genome uncoating and receptor binding. The mechanical properties of biological nanoparticles are often determined from monitoring their dynamic deformations in Atomic Force Microscopy nanoindentation experiments; but a comprehensive theory describing the full range of observed deformation behaviors has not previously been described. We present a new theory for modeling dynamic deformations of biological nanoparticles, which considers the non-linear Hertzian deformation, resulting from an indenter-particle physical contact, and the bending of curved elements (beams) modeling the particle structure. The beams' deformation beyond the critical point triggers a dynamic transition of the particle to the collapsed state. This extreme event is accompanied by a catastrophic force drop as observed in the experimental or simulated force (F)-deformation (X) spectra. The theory interprets fine features of the spectra, including the nonlinear components of the FX-curves, in terms of the Young's moduli for Hertzian and bending deformations, and the structural damage dependent beams' survival probability, in terms of the maximum strength and the cooperativity parameter. The theory is exemplified by successfully describing the deformation dynamics of natural nanoparticles through comparing theoretical curves with experimental force-deformation spectra for several virus particles. This approach provides a comprehensive description of the dynamic structural transitions in biological and artificial nanoparticles, which is essential for their optimal use in nanotechnology and nanomedicine applications.

The main function of virus coat protein is formation of the capsid that protects the virus genome against degradation. However, besides the structural function, coat proteins have many additional important activities in the infection cycle of the virus and in the defense response of host plants to viral infection. This review focuses on noncanonical functions of coat proteins of helical RNA-containing plant viruses with positive genome polarity. Analysis of data on the structural organization of coat proteins of helical viruses has demonstrated that the presence of intrinsically disordered regions within the protein structure plays an important role in implementation of nonstructural functions and largely determines the multifunctionality of coat proteins.

There are two stages generally recognized in the viral capsid assembly: nucleation and elongation. This paper focuses on the nucleation stage and develops mathematical models for HIV-1 viral capsid nucleation based on six-species dynamical systems. The Particle Swarm Optimization (PSO) algorithm is used for parameter fitting to estimate the association and dissociation rates from biological experiment data. Numerical simulations of capsid protein (CA) multimer concentrations demonstrate a good agreement with experimental data. Sensitivity and elasticity analysis of CA multimer concentrations with respect to the association and dissociation rates further reveals the importance of CA trimer-of- dimers in the nucleation stage of viral capsid self- assembly., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Many viruses are transmitted by arthropod vectors. An important mode of transmission is the noncirculative or mechanical transmission where viruses attach to the vector mouthparts for transport to a new host. It has long been assumed that noncirculative transmission is an unsophisticated mode of viral spread, and in the simplest case mere contamination of the vector mouthparts. However, emerging evidence strongly suggests that noncirculative transmission, like other transmission strategies, results from specific interactions between pathogens, hosts, and vectors. Recently, new insights into this concept have been obtained, by demonstrating that a plant virus responds instantly to the presence of its aphid vector on the host by forming transmission morphs. This novel concept, named Transmission Activation (TA), where viruses respond directly or via the host to the outside world, opens new research horizons., (Copyright © 2015 Elsevier B.V. All rights reserved.)