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Prune dwarf virus (PDV) is an important viral pathogen of plum, sweet cherry, peach, and many herbaceous test plants. Although PDV has been intensively investigated, mainly in the context of phylogenetic relationship of its genes and proteins, many gaps exist in our knowledge about the mechanism of intercellular transport of this virus. The aim of this work was to investigate alterations in cellular organelles and the cell-to-cell transport of PDV in Cucumis sativus cv. Polan at ultrastructural level. To analyze the role of viral proteins in local transport, double-immunogold assays were applied to localize PDV coat protein (CP) and movement protein (MP). We observe structural changes in chloroplasts, mitochondria, and cellular membranes. We prove that PDV is transported as viral particles via MP-generated tubular structures through plasmodesmata. Moreover, the computer-run 3D modeling reveals structural resemblances between MPs of PDV and of Alfalfa mosaic virus (AMV), implying similarities of transport mechanisms for both viruses.

The mechanical damage of plant tissues leads to the activation of methanol production and its release into the atmosphere. The gaseous methanol or vapors emitted by the damaged plant induce resistance in neighboring intact plants to bacterial pathogens but create favorable conditions for viral infection spread. Among the Nicotiana benthamiana methanol-inducible genes (MIGs), most are associated with plant defense and intercellular transport. Here, we characterize NbMIG21, which encodes a 209 aa protein (NbMIG21p) that does not share any homology with annotated proteins. NbMIG21p was demonstrated to contain a nucleolus localization signal (NoLS). Colocalization studies with fibrillarin and coilin, nucleolus and Cajal body marker proteins, revealed that NbMIG21p is distributed among these subnuclear structures. Our results show that recombinant NbMIG21 possesses DNA-binding properties. Similar to a gaseous methanol effect, an increased NbMIG21 expression leads to downregulation of the nuclear import of proteins with nuclear localization signals (NLSs), as was demonstrated with the GFP-NLS model protein. Moreover, upregulated NbMIG21 expression facilitates tobacco mosaic virus (TMV) intercellular transport and reproduction. We identified an NbMIG21 promoter (PrMIG21) and showed that it is methanol sensitive; thus, the induction of NbMIG21 mRNA accumulation occurs at the level of transcription. Our findings suggest that methanol-activated NbMIG21 might participate in creating favorable conditions for viral reproduction and spread.

Nuclear entry of HIV-1 replication complexes through intact nuclear pore complexes is critical for successful infection. The host protein cleavage-and-polyadenylation-specificity-factor-6 (CPSF6) has been implicated in different stages of early HIV-1 replication. Applying quantitative microscopy of HIV-1 reverse-transcription and pre-integration-complexes (RTC/PIC), we show that CPSF6 is strongly recruited to nuclear replication complexes but absent from cytoplasmic RTC/PIC in primary human macrophages. Depletion of CPSF6 or lack of CPSF6 binding led to accumulation of HIV-1 subviral complexes at the nuclear envelope of macrophages and reduced infectivity. Two-color stimulated-emission-depletion microscopy indicated that under these circumstances HIV-1 complexes are retained inside the nuclear pore and undergo CA-multimer dependent CPSF6 clustering adjacent to the nuclear basket. We propose that nuclear entry of HIV-1 subviral complexes in macrophages is mediated by consecutive binding of Nup153 and CPSF6 to the hexameric CA lattice. Viruses are miniscule parasites that hijack the resources of a cell to make more of themselves. For many, this involves getting inside the nucleus, the fortress that protects the cell’s genetic information. To do so, viruses need to first find a way through a double-layered membrane called the nuclear envelope, which only opens up when a cell divides. Yet, the human immunodeficiency virus type 1 (HIV-1) can infect cells that no longer divide, and in which the nucleus’ walls never come down. The virus cores then head for the nuclear pores, heavily guarded holes in the nuclear envelope that allow the cell's own molecules to go in and out of the nucleus. But HIV-1 is too big to fit through, as its genetic information is encased in a capsid, a coat made of a complex assembly of proteins. However, research shows that these capsid proteins can bind to host proteins at the pore or even inside the nucleus. For example, the capsid protein can recognize the pore protein Nup153, or the nuclear protein CPSF6. These interactions could help the virus make its way in, but how these events unfold is still unclear. To explore this, Bejarano, Peng et al. attached fluorescent labels to HIV-1 and watched as it infected non-dividing cells. Rather than completely get rid of their capsid before they crossed the pores, the virus particles hung on to a large part of their lattice. This remaining coat then attached to CPSF6; when this protein was missing or could not bind to capsid proteins, the viral complexes got stuck in the nuclear pores. This suggests that the capsid lattice could first interact with Nup153 inside the pores: then, CPSF6 would take over, knocking Nup153 away and pulling HIV-1 into the nucleus. Armed with this knowledge, virologists and drug developers could try to block HIV-1 from entering the cell’s nucleus; they could also start to dissect how drugs that target the HIV-1 capsid work. Ultimately, HIV-1 may serve as a model to unravel how large objects can pass the nuclear pore, which may help us understand how molecules are constantly trafficked in and out of the nucleus.

The size of viral genomes is limited, thus the majority of encoded proteins possess multiple functions. The main function of tobamoviral movement protein (MP) is to perform plasmodesmata gating and mediate intercellular transport of the viral RNA. MP is a remarkable example of a protein that, in addition to the initially discovered and most obvious function, carries out numerous activities that are important both for the manifestation of its key function and for successful and productive infection in general. Briefly, MP binds the viral genome, delivers it to the plasmodesmata (PD) and mediates its intercellular transfer. To implement the transport function, MP interacts with diverse cellular factors. Each of these cellular proteins has its own function, which could be different under normal conditions and upon viral infection. Here, we summarize the data available at present on the plethora of cellular factors that were identified as tobamoviral MP partners and analyze the role of these interactions in infection development.

Gram-negative bacteria naturally secrete nano-sized outer membrane vesicles (OMVs), which are important mediators of communication and pathogenesis. OMV uptake by host cells activates TLR signalling via transported PAMPs. As important resident immune cells, alveolar macrophages are located at the air-tissue interface where they comprise the first line of defence against inhaled microorganisms and particles. To date, little is known about the interplay between alveolar macrophages and OMVs from pathogenic bacteria. The immune response to OMVs and underlying mechanisms are still elusive. Here, we investigated the response of primary human macrophages to bacterial vesicles ( Legionella   pneumophila , Klebsiella   pneumoniae , Escherichia coli , Salmonella   enterica , Streptococcus   pneumoniae ) and observed comparable NF-κB activation across all tested vesicles. In contrast, we describe differential type I IFN signalling with prolonged STAT1 phosphorylation and strong Mx1 induction, blocking influenza A virus replication only for Klebsiella , E.coli and Salmonella OMVs. OMV-induced antiviral effects were less pronounced for endotoxin-free Clear coli OMVs and Polymyxin-treated OMVs. LPS stimulation could not mimic this antiviral status, while TRIF knockout abrogated it. Importantly, supernatant from OMV-treated macrophages induced an antiviral response in alveolar epithelial cells (AEC), suggesting OMV-induced intercellular communication. Finally, results were validated in an ex vivo infection model with primary human lung tissue. In conclusion, Klebsiella , E.coli and Salmonella OMVs induce antiviral immunity in macrophages via TLR4-TRIF-signaling to reduce viral replication in macrophages, AECs and lung tissue. These gram-negative bacteria induce antiviral immunity in the lung through OMVs, with a potential decisive and tremendous impact on bacterial and viral coinfection outcome. 7FMb6rvPxRFWumGrBQKEMq Video Abstract

The complex retrovirus, human T-cell leukemia virus type 1 (HTLV-1), primarily infects CD4 + T-cells in vivo . Infectious spread within this cell population requires direct contact between virally-infected and target cells. The HTLV-1 accessory protein, HBZ, was recently shown to enhance HTLV-1 infection by activating intracellular adhesion molecule 1 (ICAM-1) expression, which promotes binding of infected cells to target cells and facilitates formation of a virological synapse. In this study we show that HBZ additionally enhances HTLV-1 infection by activating expression of myoferlin (MyoF), which functions in membrane fusion and repair and vesicle transport. Results from ChIP assays and quantitative reverse transcriptase PCR indicate that HBZ forms a complex with c-Jun or JunB at two enhancer sites within the MYOF gene and activates transcription through recruitment of the coactivator p300/CBP. In HTLV-1-infected T-cells, specific inhibition of MyoF using the drug, WJ460, or shRNA-mediated knockdown of MyoF reduced infection efficiency. This effect was associated with a decrease in cell adhesion and an intracellular reduction in the abundance of HTLV-1 envelope (Env) surface unit (SU) and transmembrane domain (TM). Lysosomal protease inhibitors partially restored SU levels in WJ460-treated cells, and SU localization to LAMP-2 sites was increased by MyoF knockdown, suggesting that MyoF restricts SU trafficking to lysosomes for degradation. Consistent with these effects, less SU was associated with cell-free virus particles. Together, these data suggest that MyoF contributes to HTLV-1 infection through modulation of Env trafficking and cell adhesion.

Key Points Glycans that are present on glycoproteins often have a pivotal role in protein folding, trafficking and protection against degradation, the efficient operation of the innate immune system and, as in the case of pathogens such as HIV, escape by the immune system. There exists a wide range of carbohydrate-binding agents (CBAs), including lectins and synthetic small-size non-peptidic agents, and their molecular interactions with carbohydrates are well described. A number of CBAs, with a preference for mannose and/or N -acetylglucosamine (GlcNAc) recognition have been shown to inhibit HIV infections by blocking the viral entry process. They have also been shown to be active against other viruses such as human hepatitis C virus (HCV), coronavirus and influenza virus. When HIV is exposed to CBAs, virus escape eventually occurs predominantly by deleting its glycans on the viral envelope glycoprotein gp120. It has been shown that such mutant virus strains have an increased sensitivity to neutralizing antibodies. Numerous other pathogens, including HCV, bacteria such as Mycobacterium tuberculosis and Helicobacter pylori , parasites such as Leishmania spp. or fungi such as Aspergillus spp. and Candida spp., efficiently use the C-type lectin dendritic-cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) present on DCs for transmission and/or immune suppression. These pathogens may represent prime candidate compounds that can be exposed to CBAs in an attempt to interrupt the infection or transmission process in the host. CBA therapy of HIV (and possibly other pathogens) may represent a therapeutic approach with a dual mechanism of action: the direct inhibition of virus capture, transmission and entry into its target cells; and the recruitment of the immune system after glycan deletions occur under CBA pressure, uncovering previously hidden immunogenic epitopes on the viral envelope. Carbohydrate-binding agents (CBAs) are a family of diverse molecules that can bind to specific glycan structures on viruses or target cells. Jan Balzarini describes a new antiviral mechanism that is based on the specific interaction of CBAs with the glycans that are present on viral-envelope glycoproteins. Several chronic viral infections (such as HIV and hepatitis C virus) are highly prevalent and are a serious health risk. The adaptation of animal viruses to the human host, as recently exemplified by influenza viruses and the severe acute respiratory syndrome coronavirus, is also a continuous threat. There is a high demand, therefore, for new antiviral lead compounds and novel therapeutic concepts. In this Review, an original therapeutic concept for suppressing enveloped viruses is presented that is based on a specific interaction of carbohydrate-binding agents (CBAs) with the glycans present on viral-envelope glycoproteins. This approach may also be extended to other pathogens, including parasites, bacteria and fungi.

Viral infection, which entails synthesis of viral proteins and active reproduction of the viral genome, effects significant changes in the functions of many intracellular systems in plants. Along with these processes, a virus has to suppress cellular defense to create favorable conditions for its successful systemic spread in a plant. The virus exploits various cellular factors of a permissive host modulating its metabolism as well as local and systemic transport of macromolecules and photoassimilates. The Nicotiana benthamiana stress-induced gene encoding Kunitz peptidase inhibitor-like protein (KPILP) has recently been shown to be involved in chloroplast retrograde signaling regulation and stimulation of intercellular transport of macromolecules. In this paper we demonstrate the key role of KPILP in the development of tobamovius infection. Systemic infection of N. benthamiana plants with tobacco mosaic virus (TMV) or the closely related crucifer-infecting tobamovirus (crTMV) induces a drastic increase in KPILP mRNA accumulation. KPILP knockdown significantly reduces the efficiency of TMV and crTMV intercellular transport and reproduction. Plants with KPILP silencing become partially resistant to tobamovirus infection. Therefore, KPILP could be regarded as a novel proviral factor in the development of TMV and crTMV infection in N. benthamiana plants.

Intercellular bridges (ICBs) connecting germ cells are essential for spermatogenesis, and their deletion causes male infertility. However, the functions and component factors of ICBs are still unknown. We previously identified novel ICB-associated proteins by proteomics analysis using ICB enrichment. Here, we performed immunoprecipitation–proteomics analyses using antibodies specific to known ICB proteins MKLP1, RBM44, and ectoplasmic specialization-associated protein KIAA1210 and predicted protein complexes in the ICB cores. KIAA1210, its binding protein topoisomerase2B (TOP2B), and tight junction protein ZO1 were identified as novel ICB proteins. On the other hand, as well as KIAA1210 and TOP2B, MKLP1 and RBM44, but not TEX14, were localized at the XY body of spermatocytes, suggesting that there is a relationship between ICB proteins and meiotic chromosomes. Moreover, small RNAs interacted with an ICB protein complex that included KIAA1210, RBM44, and MKLP1. These results indicate dynamic movements of ICB proteins and suggest that ICB proteins could be involved not only in the communication between germ cells but also in their epigenetic regulation. Our results provide a novel perspective on the function of ICBs and could be helpful in revealing the biological function of the ICB. Summary Sentence Through the analyses of the dynamic movement of ICB-related proteins and their association with RNA, it was suggested that ICB may have other unknown functions to regulate spermatogenesis but not to connect germ cells.

The ability of cells to receive, process, and respond to information is essential for a variety of biological processes. This is true for the simplest single cell entity as it is for the highly specialized cells of multicellular organisms. In the latter, most cells do not exist as independent units, but are organized into specialized tissues. Within these functional assemblies, cells communicate with each other in different ways to coordinate physiological processes. Recently, a new type of cell-to-cell communication was discovered, based on de novo formation of membranous nanotubes between cells. These F-actin-rich structures, referred to as tunneling nanotubes (TNT), were shown to mediate membrane continuity between connected cells and facilitate the intercellular transport of various cellular components. The subsequent identification of TNT-like structures in numerous cell types revealed some structural diversity. At the same time it emerged that the direct transfer of cargo between cells is a common functional property, suggesting a general role of TNT-like structures in selective, long-range cell-to-cell communication. Due to the growing number of documented thin and long cell protrusions in tissue implicated in cell-to-cell signaling, it is intriguing to speculate that TNT-like structures also exist in vivo and participate in important physiological processes.