A Conserved Receptor-Binding Domain in the VP1u of Primate Erythroparvoviruses Determines the Marked Tropism for Erythroid Cells.

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.

Capsid protein from red-spotted grouper nervous necrosis virus induces incomplete autophagy by inactivating the HSP90ab1-AKT-MTOR pathway.

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.

Mouse Papillomavirus L1 and L2 Are Dispensable for Viral Infection and Persistence at Both Cutaneous and Mucosal Tissues.

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.

Disruption of the Interaction between ORF33 and the Conserved Carboxyl-Terminus of ORF45 Abolishes Progeny Virion Production of Kaposi Sarcoma-Associated Herpesvirus.

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.

Dengue virus strain 2 capsid protein switches the annealing pathway and reduces intrinsic dynamics of the conserved 5' untranslated region.

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.

DFT based Computational Methodology of IC50 Prediction.

BACKGROUND: IC50 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 IC50 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 IC50 prediction in the field of in silico drug designing. OBJECTIVE: Recently developed quantum computation based method of IC50 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 IC50, a relative approach of IC50 computation is pursued in the present method. To predict an approximate value of IC50 of a small molecule, only the IC50 of a similar kind of molecule is required for this method. RESULTS: The present method of IC50 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, IC50 based drug search is possible with this method. An algorithm is proposed here for IC50 based drug search.

The Fiber Knob Protein of Human Adenovirus Type 49 Mediates Highly Efficient and Promiscuous Infection of Cancer Cell Lines Using a Novel Cell Entry Mechanism.

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.

The conserved aromatic residue W122 is a determinant of potyviral coat protein stability, replication, and cell-to-cell movement in plants.

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 (W122 ) 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 W122 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 W122 mutant viruses. The substitution of W122 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 W122 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 W122 in the watermelon mosaic virus and potato virus Y CPs. Therefore, we conclude that the aromatic ring in W122 in the core domain of the potyviral CP is critical for cell-to-cell movement through the effects on CP stability and viral replication.

The VP1u of Human Parvovirus B19: A Multifunctional Capsid Protein with Biotechnological Applications.

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 A2 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.

Penaeus stylirostris densovirus proteins CP and NS1 interact with peritrophin of Litopenaeus vannamei.

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.

The Adenovirus Dodecahedron: Beyond the Platonic Story.

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 capsid protein induces autophagy in HEK293T cells by inhibiting phosphorylation of the mammalian target of rapamycin.

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.

Cryo-electron microscopy for the study of virus assembly.

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.

West Nile virus capsid protein inhibits autophagy by AMP-activated protein kinase degradation in neurological disease development.

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.

Bacteriophage P1 does not show spatial preference when infecting Escherichia coli.

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.

Gaussian curvature and the budding kinetics of enveloped viruses.

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.

Maturation of retroviruses.

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.

A New Model System for Exploring Assembly Mechanisms of the HIV-1 Immature Capsid In Vivo.

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.

REM1.3's phospho-status defines its plasma membrane nanodomain organization and activity in restricting PVX cell-to-cell movement.

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.