Imaging, Tracking and Computational Analyses of Virus Entry and Egress with the Cytoskeleton

Viruses have a dual nature: particles are “passive substances” lacking chemical energy transformation, whereas infected cells are “active substances” turning-over energy. How passive viral substances convert to active substances, comprising viral replication and assembly compartments has been of intense interest to virologists, cell and molecular biologists and immunologists. Infection starts with virus entry into a susceptible cell and delivers the viral genome to the replication site. This is a multi-step process, and involves the cytoskeleton and associated motor proteins. Likewise, the egress of progeny virus particles from the replication site to the extracellular space is enhanced by the cytoskeleton and associated motor proteins. This overcomes the limitation of thermal diffusion, and transports virions and virion components, often in association with cellular organelles. This review explores how the analysis of viral trajectories informs about mechanisms of infection. We discuss the methodology enabling researchers to visualize single virions in cells by fluorescence imaging and tracking. Virus visualization and tracking are increasingly enhanced by computational analyses of virus trajectories as well as in silico modeling. Combined approaches reveal previously unrecognized features of virus-infected cells. Using select examples of complementary methodology, we highlight the role of actin filaments and microtubules, and their associated motors in virus infections. In-depth studies of single virion dynamics at high temporal and spatial resolutions thereby provide deep insight into virus infection processes, and are a basis for uncovering underlying mechanisms of how cells function

The nuclear export factor CRM1 controls juxta-nuclear microtubule-dependent virus transport

Transport of large cargo through the cytoplasm requires motor proteins and polarized filaments. Viruses that replicate in the nucleus of post-mitotic cells use microtubules and the dynein–dynactin motor to traffic to the nuclear membrane and deliver their genome through nuclear pore complexes (NPCs) into the nucleus. How virus particles (virions) or cellular cargo are transferred from microtubules to the NPC is unknown. Here, we analyzed trafficking of incoming cytoplasmic adenoviruses by single-particle tracking and superresolution microscopy. We provide evidence for a regulatory role of CRM1 (chromosome-region-maintenance-1; also known as XPO1, exportin-1) in juxta-nuclear microtubule-dependent adenovirus transport. Leptomycin B (LMB) abolishes nuclear targeting of adenovirus. It binds to CRM1, precludes CRM1–cargo binding and blocks signal-dependent nuclear export. LMB-inhibited CRM1 did not compete with adenovirus for binding to the nucleoporin Nup214 at the NPC. Instead, CRM1 inhibition selectively enhanced virion association with microtubules, and boosted virion motions on microtubules less than ∼2 µm from the nuclear membrane. The data show that the nucleus provides positional information for incoming virions to detach from microtubules, engage a slower microtubule-independent motility to the NPC and enhance infectio

Lung macrophage scavenger receptor SR-A6 (MARCO) is an adenovirus type-specific virus entry receptor

<div><p>Macrophages are a diverse group of phagocytic cells acting in host protection against stress, injury, and pathogens. Here, we show that the scavenger receptor SR-A6 is an entry receptor for human adenoviruses in murine alveolar macrophage-like MPI cells, and important for production of type I interferon. Scavenger receptors contribute to the clearance of endogenous proteins, lipoproteins and pathogens. Knockout of SR-A6 in MPI cells, anti-SR-A6 antibody or the soluble extracellular SR-A6 domain reduced adenovirus type-C5 (HAdV-C5) binding and transduction. Expression of murine SR-A6, and to a lower extent human SR-A6 boosted virion binding to human cells and transduction. Virion clustering by soluble SR-A6 and proximity localization with SR-A6 on MPI cells suggested direct adenovirus interaction with SR-A6. Deletion of the negatively charged hypervariable region 1 (HVR1) of hexon reduced HAdV-C5 binding and transduction, implying that the viral ligand for SR-A6 is hexon. SR-A6 facilitated macrophage entry of HAdV-B35 and HAdV-D26, two important vectors for transduction of hematopoietic cells and human vaccination. The study highlights the importance of scavenger receptors in innate immunity against human viruses.</p></div

The Salivary Secretome of the Tsetse Fly Glossina pallidipes (Diptera: Glossinidae) Infected by Salivary Gland Hypertrophy Virus

Tsetse fly (Diptera; Glossinidae) transmits two devastating diseases to farmers (human African Trypanosomiasis; HAT) and their livestock (Animal African Trypanosomiasis; AAT) in 37 sub-Saharan African countries. During the rainy seasons, vast areas of fertile, arable land remain uncultivated as farmers flee their homes due to the presence of tsetse. Available drugs against trypanosomiasis are ineffective and difficult to administer. Control of the tsetse vector by Sterile Insect Technique (SIT) has been effective. This method involves repeated release of sterilized males into wild tsetse populations, which compete with wild type males for females. Upon mating, there is no offspring, leading to reduction in tsetse populations and thus relief from trypanosomiasis. The SIT method requires large-scale tsetse rearing to produce sterile males. However, tsetse colony productivity is hampered by infections with the salivary gland hypertrophy virus, which is transmitted via saliva as flies take blood meals during membrane feeding and often leads to colony collapse. Here, we investigated the salivary gland secretome proteins of virus-infected tsetse to broaden our understanding of virus infection, transmission and pathology. By this approach, we obtain insight in tsetse-hytrosavirus interactions and identified potential candidate proteins as targets for developing biotechnological strategies to control viral infections in tsetse colonies

Despite WT1 binding sites in the promoter region of human and mouse nucleoporin glycoprotein 210, WT1 does not influence expression of GP210

BACKGROUND: Glycoprotein 210 (GP210) is a transmembrane component of the nuclear pore complex of metazoans, with a short carboxyterminus protruding towards the cytoplasm. Its function is unknown, but it is considered to be a major structural component of metazoan nuclear pores. Yet, our previous findings showed pronounced differences in expression levels in embryonic mouse tissues and cell lines. In order to identify factors regulating GP210, the genomic organization of human GP210 was analyzed in silico. RESULTS: The human gene was mapped to chromosome 3 and consists of 40 exons spread over 102 kb. The deduced 1887 amino acid showed a high degree of alignment homology to previously reported orthologues. Experimentally we defined two transcription initiation sites, 18 and 29 bp upstream of the ATG start codon. The promoter region is characterized by a CpG island and several consensus binding motifs for gene regulatory transcription factors, including clustered sites associated with Sp1 and the Wilms' tumor suppressor gene zinc finger protein (WT1). In addition, distal to the translation start we found a (GT)n repetitive sequence, an element known for its ability to bind WT1. Homologies for these motifs could be identified in the corresponding mouse genomic region. However, experimental tetracycline dependent induction of WT1 in SAOS osteosarcoma cells did not influence GP210 transcription. CONCLUSION: Although mouse GP210 was identified as an early response gene during induced metanephric kidney development, and WT1 binding sites were identified in the promoter region of the human GP210 gene, experimental modulation of WT1 expression did not influence expression of GP210. Therefore, WT1 is probably not regulating GP210 expression. Instead, we suggest that the identified Sp binding sites are involved

Fiber Mediated Receptor Masking in Non-Infected Bystander Cells Restricts Adenovirus Cell Killing Effect but Promotes Adenovirus Host Co-Existence

The basic concept of conditionally replicating adenoviruses (CRAD) as oncolytic agents is that progenies generated from each round of infection will disperse, infect and kill new cancer cells. However, CRAD has only inhibited, but not eradicated tumor growth in xenograft tumor therapy, and CRAD therapy has had only marginal clinical benefit to cancer patients. Here, we found that CRAD propagation and cancer cell survival co-existed for long periods of time when infection was initiated at low multiplicity of infection (MOI), and cancer cell killing was inefficient and slow compared to the assumed cell killing effect upon infection at high MOI. Excessive production of fiber molecules from initial CRAD infection of only 1 to 2% cancer cells and their release prior to the viral particle itself caused a tropism-specific receptor masking in both infected and non-infected bystander cells. Consequently, the non-infected bystander cells were inefficiently bound and infected by CRAD progenies. Further, fiber overproduction with concomitant restriction of adenovirus spread was observed in xenograft cancer therapy models. Besides the CAR-binding Ad4, Ad5, and Ad37, infection with CD46-binding Ad35 and Ad11 also caused receptor masking. Fiber overproduction and its resulting receptor masking thus play a key role in limiting CRAD functionality, but potentially promote adenovirus and host cell co-existence. These findings also give important clues for understanding mechanisms underlying the natural infection course of various adenoviruses

Saffold Virus, a Human Theiler's-Like Cardiovirus, Is Ubiquitous and Causes Infection Early in Life

The family Picornaviridae contains well-known human pathogens (e.g., poliovirus, coxsackievirus, rhinovirus, and parechovirus). In addition, this family contains a number of viruses that infect animals, including members of the genus Cardiovirus such as Encephalomyocarditis virus (EMCV) and Theiler's murine encephalomyelits virus (TMEV). The latter are important murine pathogens that cause myocarditis, type 1 diabetes and chronic inflammation in the brains, mimicking multiple sclerosis. Recently, a new picornavirus was isolated from humans, named Saffold virus (SAFV). The virus is genetically related to Theiler's virus and classified as a new species in the genus Cardiovirus, which until the discovery of SAFV did not contain human viruses. By analogy with the rodent cardioviruses, SAFV may be a relevant new human pathogen. Thus far, SAFVs have sporadically been detected by molecular techniques in respiratory and fecal specimens, but the epidemiology and clinical significance remained unclear. Here we describe the first cultivated SAFV type 3 (SAFV-3) isolate, its growth characteristics, full-length sequence, and epidemiology. Unlike the previously isolated SAFV-1 and -2 viruses, SAFV-3 showed efficient growth in several cell lines with a clear cytopathic effect. The latter allowed us to conduct a large-scale serological survey by a virus-neutralization assay. This survey showed that infection by SAFV-3 occurs early in life (>75% positive at 24 months) and that the seroprevalence reaches >90% in older children and adults. Neutralizing antibodies were found in serum samples collected in several countries in Europe, Africa, and Asia. In conclusion, this study describes the first cultivated SAFV-3 isolate, its full-length sequence, and epidemiology. SAFV-3 is a highly common and widespread human virus causing infection in early childhood. This finding has important implications for understanding the impact of these ubiquitous viruses and their possible role in acute and/or chronic disease

Tubule-Guided Cell-to-Cell Movement of a Plant Virus Requires Class XI Myosin Motors

Cell-to-cell movement of plant viruses occurs via plasmodesmata (PD), organelles that evolved to facilitate intercellular communications. Viral movement proteins (MP) modify PD to allow passage of the virus particles or nucleoproteins. This passage occurs via several distinct mechanisms one of which is MP-dependent formation of the tubules that traverse PD and provide a conduit for virion translocation. The MP of tubule-forming viruses including Grapevine fanleaf virus (GFLV) recruit the plant PD receptors called Plasmodesmata Located Proteins (PDLP) to mediate tubule assembly and virus movement. Here we show that PDLP1 is transported to PD through a specific route within the secretory pathway in a myosin-dependent manner. This transport relies primarily on the class XI myosins XI-K and XI-2. Inactivation of these myosins using dominant negative inhibition results in mislocalization of PDLP and MP and suppression of GFLV movement. We also found that the proper targeting of specific markers of the Golgi apparatus, the plasma membrane, PD, lipid raft subdomains within the plasma membrane, and the tonoplast was not affected by myosin XI-K inhibition. However, the normal tonoplast dynamics required myosin XI-K activity. These results reveal a new pathway of the myosin-dependent protein trafficking to PD that is hijacked by GFLV to promote tubule-guided transport of this virus between plant cells

Septins Regulate Bacterial Entry into Host Cells

Background: Septins are conserved GTPases that form filaments and are required in many organisms for several processes including cytokinesis. We previously identified SEPT9 associated with phagosomes containing latex beads coated with the Listeria surface protein InlB. Methodology/Principal Findings: Here, we investigated septin function during entry of invasive bacteria in non-phagocytic mammalian cells. We found that SEPT9, and its interacting partners SEPT2 and SEPT11, are recruited as collars next to actin at the site of entry of Listeria and Shigella. SEPT2-depletion by siRNA decreased bacterial invasion, suggesting that septins have roles during particle entry. Incubating cells with InlB-coated beads confirmed an essential role for SEPT2. Moreover, SEPT2-depletion impaired InlB-mediated stimulation of Met-dependent signaling as shown by FRET. Conclusions/Significance: Together these findings highlight novel roles for SEPT2, and distinguish the roles of septin an