Zika virus (ZIKV) establishes persistent infections in multiple human tissues, a phenomenon that likely plays a role in its ability to cause congenital birth defects and neurological disease. Multiple nonstructural proteins encoded by ZIKV, in particular NS5, are known to suppress the interferon (IFN) response by attacking different steps in this critical antiviral pathway. Less well known are the potential roles of structural proteins in affecting the host immune response during ZIKV infection. Capsid proteins of flaviviruses are of particular interest because a pool of these viral proteins is targeted to the nuclei during infection and, as such, they have the potential to affect host cell gene expression. In this study, RNA-seq analyses revealed that capsid proteins from six different flaviviruses suppress expression of type I IFN and IFN-stimulated genes. Subsequent interactome and in vitro ubiquitination assays showed that ZIKV capsid protein binds to and prevents activating ubiquitination of RIG-I CARD domains by TRIM25, a host factor that is important for the induction arm of the IFN response. The other flavivirus capsid proteins also interacted with TRIM25, suggesting that these viral proteins may attenuate antiviral signaling pathways at very early stages of infection, potentially even before nonstructural proteins are produced.

Numerous, diverse plant viruses encode movement proteins (MPs) that aid the virus movement through plasmodesmata, the plant intercellular channels. MPs are essential for virus spread and propagation in distal tissues, and several unrelated MPs have been identified. The 30K superfamily of MPs (named after the molecular mass of tobacco mosaic virus MP, the classical model of plant virology) is the largest and most diverse MP variety, represented in 16 virus families, but its evolutionary origin remained obscure. Here, we show that the core structural domain of the 30K MPs is homologous to the jelly-roll domain of the capsid proteins (CPs) of small RNA and DNA viruses, in particular, those infecting plants. The closest similarity was observed between the 30K MPs and the CPs of the viruses in the families Bromoviridae and Geminiviridae. We hypothesize that the MPs evolved via duplication or horizontal acquisition of the CP gene in a virus that infected an ancestor of vascular plants, followed by neofunctionalization of one of the paralogous CPs, potentially through the acquisition of unique N- and C-terminal regions. During the subsequent coevolution of viruses with diversifying vascular plants, the 30K MP genes underwent explosive horizontal spread among emergent RNA and DNA viruses, likely permitting viruses of insects and fungi that coinfected plants to expand their host ranges, molding the contemporary plant virome.

We have developed a system for analysis of histidine-tagged (His-tagged) retrovirus core (Gag) proteins, assembled in vitro on lipid monolayers consisting of egg phosphatidylcholine (PC) plus the novel lipid DHGN. DHGN was shown to chelate nickel by atomic absorption spectrometry, and DHGN-containing monolayers specifically bound gold conjugates of His-tagged proteins. Using PC + DHGN monolayers, we examined membrane-bound arrays of an N-terminal His-tagged Moloney murine leukemia virus (M-MuLV) capsid (CA) protein, His-MoCA, and in vivo studies suggest that in vitro-derived His-MoCA arrays reflect some of the Gag protein interactions which occur in assembling virus particles. The His-MoCA proteins formed extensive two-dimensional (2D) protein crystals, with reflections out to 9.5 A resolution. The image-analyzed 2D projection of His-MoCA arrays revealed a distinct cage-like network. The asymmetry of the individual building blocks of the network led to the formation of two types of hexamer rings, surrounding protein-free cage holes. These results predict that Gag hexamers constitute a retrovirus core substructure, and that cage hole sizes define an exclusion limit for entry of retrovirus envelope proteins, or other plasma membrane proteins, into virus particles. We believe that the 2D crystallization method will permit the detailed analysis of retroviral Gag proteins and other His-tagged proteins.

Densovirus genome replication and capsid assembly take place in the nucleus of the infected cells. However, the mechanisms underlying such processes as the delivery of virus proteins to the nucleus and the export of progeny virus from the nucleus remain elusive. It is evident that nuclear transport signals should be involved in these processes. We performed an in silico search for the putative nuclear localization signal (NLS) and nuclear export signal (NES) motifs in the capsid proteins of the Blattella germanica Densovirus 1 (BgDV1) densovirus. A high probability NLS motif was found in the common C-terminal of capsid proteins together with a NES motif in the unique N-terminal of VP2. We also performed a global search for the nuclear traffic signals in the densoviruses belonging to five Densovirinae genera, which revealed high diversity in the patterns of NLSs and NESs. Using a heterologous system, the HeLa mammalian cell line expressing GFP-fused BgDV1 capsid proteins, we demonstrated that both signals are functionally active. We suggest that the NLS shared by all three BgDV1 capsid proteins drives the trafficking of the newly-synthesized proteins into the nucleus, while the NES may play a role in the export of the newly-assembled BgDV1 particles into the cytoplasm through nuclear pore complexes.

This communication describes the in vitro assembly of genetically recombinant Cucumber Mosaic Virus (CMV) viral capsid proteins (CPs) into biological nanotubes, several micrometres long yet with a diameter of only approximately 17 nm, triggered by double-stranded DNAs of different lengths.

Enhanced replication of rubella virus (RuV) and replicons by de novo synthesized viral structural proteins has been previously described. Such enhancement can occur by viral capsid proteins (CP) alone in trans. It is not clear whether the CP in the virus particles, i.e., the exogenous CP, modulate viral genome replication. In this study, we found that exogenous RuV CP also enhanced viral genome replication, either when used to package replicons or when mixed with RNA during transfection. We demonstrated that CP does not affect the translation efficiency from genomic (gRNA) or subgenomic RNA (sgRNA), the intracellular distribution of the non-structural proteins (NSP), or sgRNA synthesis. Significantly active RNA replication was observed in transfections supplemented with recombinant CP (rCP), which was supported by accumulated genomic negative-strand RNA. rCP was found to restore replication of a few mutants in NSP but failed to fully restore replicons known to have defects in the positive-strand RNA synthesis. By monitoring the amount of RuV RNA following transfection, we found that all RuV replicon RNAs were well-retained in the presence of rCP within 24 h of post-transfection, compared to non-RuV RNA. These results suggest that the exogenous RuV CP increases efficiency of early viral genome replication by modulating the stage(s) prior to and/or at the initiation of negative-strand RNA synthesis, possibly through a general mechanism such as protecting viral RNA.

Chilo iridescent virus (CIV) is a large (approximately 1850 A diameter) insect virus with an icosahedral, T=147 capsid, a double-stranded DNA (dsDNA) genome, and an internal lipid membrane. The structure of CIV was determined to 13 A resolution by means of cryoelectron microscopy (cryoEM) and three-dimensional image reconstruction. A homology model of P50, the CIV major capsid protein (MCP), was built based on its amino acid sequence and the structure of the homologous Paramecium bursaria chlorella virus 1 Vp54 MCP. This model was fitted into the cryoEM density for each of the 25 trimeric CIV capsomers per icosahedral asymmetric unit. A difference map, in which the fitted CIV MCP capsomers were subtracted from the CIV cryoEM reconstruction, showed that there are at least three different types of minor capsid proteins associated with the capsomers outside the lipid membrane. "Finger" proteins are situated at many, but not all, of the spaces between three adjacent capsomers within each trisymmetron, and "zip" proteins are situated between sets of three adjacent capsomers at the boundary between neighboring trisymmetrons and pentasymmetrons. Based on the results of segmentation and density correlations, there are at least eight finger proteins and three dimeric and two monomeric zip proteins in one asymmetric unit of the CIV capsid. These minor proteins appear to stabilize the virus by acting as intercapsomer cross-links. One transmembrane "anchor" protein per icosahedral asymmetric unit, which extends from beneath one of the capsomers in the pentasymmetron to the internal leaflet of the lipid membrane, may provide additional stabilization for the capsid. These results are consistent with the observations for other large, icosahedral dsDNA viruses that also utilize minor capsid proteins for stabilization and for determining their assembly.

Cellular immune responses against AAV vector capsid represent an obstacle for successful gene therapy. Previous studies have used overlapping peptides spanning the entire capsid sequence to identify T cell epitopes recognized by AAV-specific CD8+ T cells. However, the repertoire of peptides naturally displayed by HLA class I molecules for CD8 T cell recognition is unknown.

The capsid precursor P1 constitutes the N-terminal part of the enterovirus polyprotein. It is processed into VP0, VP3, and VP1 by the viral proteases, and VP0 is cleaved autocatalytically into VP4 and VP2. We observed that poliovirus VP0 is recognized by an antibody against a cellular autophagy protein, LC3A. The LC3A-like epitope overlapped the VP4/VP2 cleavage site. Individually expressed VP0-EGFP and P1 strongly colocalized with a marker of selective autophagy, p62/SQSTM1. To assess the role of capsid proteins in autophagy development we infected different cells with poliovirus or encapsidated polio replicon coding for only the replication proteins. We analyzed the processing of LC3B and p62/SQSTM1, markers of the initiation and completion of the autophagy pathway and investigated the association of the viral antigens with these autophagy proteins in infected cells. We observed cell-type-specific development of autophagy upon infection and found that only the virion signal strongly colocalized with p62/SQSTM1 early in infection. Collectively, our data suggest that activation of autophagy is not required for replication, and that capsid proteins contain determinants targeting them to p62/SQSTM1-dependent sequestration. Such a strategy may control the level of capsid proteins so that viral RNAs are not removed from the replication/translation pool prematurely.

Viruses are a major cause of production losses in the world shrimp-farming industry. Despite this, little is known about viral-host interactions in shrimp due in part to the lack of continuous shrimp cell lines. Here, the yeast two-hybrid assay system was employed to study interactions between three Taura syndrome viral capsid proteins (VP1-VP3) and proteins from a cDNA library of the black tiger shrimp Penaeus monodon. VP1 interacted with beta-actin, elongation factor 1alpha (EF1alpha), lysozyme (Lys) and laminin receptor/ribosomal protein p40 (Lamr/p40) containing a putative palindromic laminin binding region LMWWML. VP2 interacted with beta-actin and EF1alpha, while VP3 bound to the same proteins as VP1 except for Lamr/p40. In vitro pull-down assays confirmed these interactions. The most interesting interaction was specific binding between VP1 and Lamr/p40 since Lamr/p40 has been identified as the mammalian cell receptor for several arthropod-borne viruses (arboviruses). A search of mosquito vector and Drosophila sequences at available databases revealed the presence of putative Lamr/p40 proteins with high homology to the Lamr/p40 from shrimp.

Previously, we demonstrated that input SV40 particles undergo a partial disassembly in the endoplasmic reticulum, which exposes internal capsid proteins VP2 and VP3 to immunostaining. Then, in the cytoplasm, disassembly progresses further to also make the genomic DNA accessible to immune detection, as well as to detection by an ethynyl-2-deoxyuridine (EdU)-based chemical reaction. The cytoplasmic partially disassembled SV40 particles retain some of the SV40 capsid proteins, VP1, VP2, and VP3, in addition to the viral genome.

The HIV-1 capsid is a shell that encapsulates viral RNA, and forms a conical structure by assembling oligomers of capsid (CA) proteins. Since the CA proteins are highly conserved among many strains of HIV-1, the inhibition of the CA function could be an appropriate goal for suppression of HIV-1 replication, but to date, no drug targeting CA has been developed. Hydrophobic interactions between two CA molecules through Trp184 and Met185 in the protein are known to be indispensable for conformational stabilization of the CA multimer. In our previous study, a small molecule designed by in silico screening as a dipeptide mimic of Trp184 and Met185 in the interaction site was synthesized and found to have significant anti-HIV-1 activity. In the present study, molecules with different scaffolds based on a dipeptide mimic of Trp184 and Met185 have been designed and synthesized. Their significant anti-HIV activity and their advantages compared to the previous compounds were examined. The present results should be useful in the design of novel CA-targeting anti-HIV agents.

Porcine circovirus-like virus P1 is a relatively new kind of virus that is closely related to the post-weaning multisystemic wasting syndrome, congenital tremors, and abortions in swine. The molecular mechanisms of P1 virus infection and pathogenesis are fully unknown. To analyze P1 and its host interactions, we used a yeast two-hybrid (Y2H) assay to identify cellular proteins interacting with the Cap of the P1 virus. In this study, the Cap of the P1 virus exhibited no self-activation and toxicity to yeast cells and was used as bait to screen the Y2H library prepared from the pancreas tissue.

Flaviviruses such as dengue virus (DENV) and Zika virus (ZIKV) have evolved sophisticated mechanisms to suppress the host immune system. For instance, flavivirus infections were found to sabotage peroxisomes, organelles with an important role in innate immunity. The current model suggests that the capsid (C) proteins of DENV and ZIKV downregulate peroxisomes, ultimately resulting in reduced production of interferons by interacting with the host protein PEX19, a crucial chaperone in peroxisomal biogenesis. Here, we aimed to explore the importance of peroxisomes and the role of C interaction with PEX19 in the flavivirus life cycle. By infecting cells lacking peroxisomes we show that this organelle is required for optimal DENV replication. Moreover, we demonstrate that DENV and ZIKV C bind PEX19 through a conserved PEX19-binding motif, which is also commonly found in cellular peroxisomal membrane proteins (PMPs). However, in contrast to PMPs, this interaction does not result in the targeting of C to peroxisomes. Furthermore, we show that the presence of C results in peroxisome loss due to impaired peroxisomal biogenesis, which appears to occur by a PEX19-independent mechanism. Hence, these findings challenge the current model of how flavivirus C might downregulate peroxisomal abundance and suggest a yet unknown role of peroxisomes in flavivirus biology.

Our previous studies have demonstrated that haploid AAV vectors made from capsids of two different serotypes induced high transduction and prevented serotype-specific antibody binding. In this study, we explored the transduction efficiency of several haploid viruses, which were made from the VP1/VP2 of one serotype and VP3 of another compatible serotype. After systemic injection of 2 × 1010 vg of AAV vectors into mice, the haploid AAV vectors, composed of VP1/VP2 from serotypes 8 or 9, and VP3 from AAV2, displayed a two to seven-fold increase in liver transduction compared with those of parental AAV2 vectors. Furthermore, a chimeric AAV2/8 VP1/VP2 with N-terminus of VP1/VP2 from AAV2 and C-terminus (VP3 domain) from AAV8 was constructed, and produced the haploid vector 28m-2VP3 with AAV2 VP3. The haploid 28m-2VP3 vector showed a five-fold higher transduction than that of the vectors composed solely of AAV2 VPs. Remarkably, the 28m-2VP3 vectors also induced a significant increase in transgene expression compared to the vectors composed of AAV8 VP1/VP2 with AAV2 VP3. The results suggest that the difference in the VP1/VP2 N-terminal region between AAV2 and AAV8 may allow better "communication" between the VP1/VP2 N-terminus of AAV2 with its cognate VP3. Similarly, the haploid vectors, VP1/VP2 from serotypes 8 or 9 and VP3 from AAV3, achieved higher transductions in multiple tissue types beyond typical tropism compared with those of AAV3 vectors. Consistently, higher vector genome copy numbers were detected in these tissues, indicating that an incorporation of non-cognate VP1/VP2 might influence the cellular tropism of the haploid vectors. However, there was no significant difference or even decreased transductions when compared with those of parental AAV8 or AAV9 vectors. In summary, these studies provide insight into current development strategies of AAV vectors that can increase AAV transduction across multiple tissues.

Virus-host interaction is a tug of war between pathogenesis and immunity, followed by either activating the host immune defense system to eliminate virus or manipulating host immune control mechanisms to survive and facilitate virus propagation. Comprehensive knowledge of interactions between host and viral proteins might provide hints for developing novel antiviral strategies. To gain a more detailed knowledge of the interactions with porcine circovirus type 2 capsid protein, we employed a coimmunoprecipitation combined with liquid chromatography mass spectrometry (LC-MS) approach and 222 putative PCV2 Cap-interacting host proteins were identified in the infected porcine kidney (PK-15) cells. Further, a protein-protein interactions (PPIs) network was plotted, and the PCV2 Cap-interacting host proteins were potentially involved in protein binding, DNA transcription, metabolism and innate immune response based on the gene ontology annotation and Kyoto Encyclopedia of Genes and Genomes database enrichment. Verification in vitro assay demonstrated that eight cellular proteins, namely heterogeneous nuclear ribonucleoprotein C, nucleophosmin-1, DEAD-box RNA helicase 21, importin β3, eukaryotic translation initiation factor 4A2, snail family transcriptional repressor 2, MX dynamin like GTPase 2, and intermediate chain 1 interacted with PCV2 Cap. Thus, this work effectively provides useful protein-related information to facilitate further investigation of the underlying mechanism of PCV2 infection and pathogenesis.

The capsid of human immunodeficiency virus type 1 (HIV-1) is a shell that encloses viral RNA and is highly conserved among many strains of the virus. It forms a conical structure by assembling oligomers of capsid (CA) proteins. CA dysfunction is expected to be an important target of suppression of HIV-1 replication, and it is important to understand a new mechanism that could lead to the CA dysfunction. A drug targeting CA however, has not been developed to date. Hydrophobic interactions between two CA molecules via Trp184/Met185 in CA were recently reported to be important for stabilization of the multimeric structure of CA. In the present study, a small molecule designed by in silico screening as a dipeptide mimic of Trp184 and Met185 in the interaction site, was synthesized and its significant anti-HIV-1 activity was confirmed. Structure activity relationship (SAR) studies of its derivatives were performed and provided results that are expected to be useful in the future design and development of novel anti-HIV agents targeting CA.

Human parechoviruses (HPeV) are picornaviruses with a highly-ordered RNA genome contained within icosahedrally-symmetric capsids. Ordered RNA structures have recently been shown to interact with capsid proteins VP1 and VP3 and facilitate virus assembly in HPeV1. Using an assay that combines reversible cross-linking, RNA affinity purification and peptide mass fingerprinting (RCAP), we mapped the RNA-interacting regions of the capsid proteins from the whole HPeV1 virion in solution. The intrinsically-disordered N-termini of capsid proteins VP1 and VP3, and unexpectedly, VP0, were identified to interact with RNA. Comparing these results to those obtained using recombinantly-expressed VP0 and VP1 confirmed the virion binding regions, and revealed unique RNA binding regions in the isolated VP0 not previously observed in the crystal structure of HPeV1. We used RNA fluorescence anisotropy to confirm the RNA-binding competency of each of the capsid proteins' N-termini. These findings suggests that dynamic interactions between the viral RNA and the capsid proteins modulate virus assembly, and suggest a novel role for VP0.

Rhinoviruses (RVs) are major causes of the common cold, but they can also trigger exacerbations of asthma. More than 160 different RV strains exist and can be classified into three genetic species (RV-A, RV-B and RV-C) which bind to different receptors on human cells including intracellular adhesion molecule 1 (ICAM-1), the low-density lipoprotein receptor (LDLR) or the cadherin-related family member 3 (CDHR3). Epitopes located in the RV capsid have mainly been determined for RV2, a minor-group RV-A strain binding to LDLR, and for RV14, a major-group RV-B strain binding to ICAM-1. In order to study epitopes involved in the neutralization of RV89, an ICAM-1-binding RV-A strain which is highly different from RV2 and RV14 in terms of receptor specificity and sequence, respectively, we analyzed the specificity and epitopes of a highly neutralizing antiserum using recombinantly produced RV89 capsid proteins (VP1, VP2, VP3 and VP4), recombinant fragments and synthetic overlapping peptides thereof. We found that the antiserum which neutralized in vitro RV89 infection up to a dilution of 1:24,000 reacted with the capsid proteins VP1 and VP2 but not with VP3 and VP4. The neutralizing antibodies recognized recombinant fragments comprising approximately 100 amino acids of the N- and C-terminus of VP1 and the middle part of VP2, in particular, three peptides which, according to molecular modeling based on the three-dimensional structure of RV16, were surface-exposed on the viral capsid. Two recombinant fusion proteins containing the identified peptides fused to hepatitis B (HBV)-derived preS as a carrier protein induced upon immunization of rabbits antibodies capable of neutralizing in vitro RV89 infections. Interestingly, the virus-neutralizing epitopes determined for RV89 corresponded to those determined for minor-group RV2 binding to LDL and major-group RV14 belonging to the RV-B species, which are highly different from RV89. Our results indicate that highly different RV strains, even when reacting with different receptors, seem to engage similar parts of their capsid in the infection process. These results may be important for the design of active and passive immunization strategies for RV.

Although citrus leprosis disease has been known for more than a hundred years, one of its causal agents, citrus leprosis virus C2 (CiLV-C2), is poorly characterized. This study described the association of CiLV-C2 movement protein (MP) and capsid protein (p29) with biological membranes. Our findings obtained by computer predictions, chemical treatments after membrane fractionation, and biomolecular fluorescence complementation assays revealed that p29 is peripherally associated, while the MP is integrally bound to the cell membranes. Topological analyses revealed that both the p29 and MP expose their N- and C-termini to the cell cytoplasmic compartment. The implications of these results in the intracellular movement of the virus were discussed.