Varicella-zoster virus: molecular virology and virus-host interactions

Cosmid-based mutagenesis and methods to examine varicella-zoster virus (VZV) tropism for differentiated human cells in vivo provide new information about molecular mechanisms of VZV infection. How specific VZV gene products contribute to viral replication has been further defined, and effects of VZV on expression of cellular genes have been demonstrated.     

微小RNA在病毒与宿主相互作用中的功能

微小RNA(microRNA,miRNA)是一类长度为22个核苷酸左右的内源性非编码小RNA分子。自1993年最先从秀丽隐杆线虫体内发现miRNA以来,目前为止已有35 000多条miRNA在植物、动物及病毒中被发现。它们作为重要的转录调控因子,参与细胞分化、凋亡、代谢、信号转导、免疫等多种生物学过程。病毒和宿主细胞均可编码miRNA,病毒编码的miRNA可改变宿主内环境,而宿主编码的miRNA则可影响病毒生存。本文就miRNA对病毒与宿主相互作用的调控进行综述。     

Virus genomes and virus-host interactions in aquaculture animals

Over the last 30 years,aquaculture has become the fastest growing form of agriculture production in the world,but its development has been hampered by a diverse range of pathogenic viruses.During the last decade,a large number of viruses from aquatic animals have been identified,and more than 100 viral genomes have been sequenced and genetically characterized.These advances are leading to better understanding about antiviral mechanisms and the types of interaction occurring between aquatic viruses and their hosts.Here,based on our research experience of more than 20 years,we review the wealth of genetic and genomic information from studies on a diverse range of aquatic viruses,including iridoviruses,herpesviruses,reoviruses,and rhabdoviruses,and outline some major advances in our understanding of virus–host interactions in animals used in aquaculture.     

ZIKV viral proteins and their roles in virus-host interactions

The re-emergence of Zika virus(ZIKV) and its associated neonatal microcephaly and Guillain-Barré syndrome have led the World Health Organization to declare a global health emergency. Until today, many related studies have successively reported the role of various viral proteins of ZIKVin the process of ZIKVinfection and pathogenicity. These studies have provided significant insights for the treatment and prevention of ZIKV infection. Here we review the current research advances in the functional characterization of the interactions between each ZIKV viral protein and its host factors.     

Sulfolobus tengchongensis spindle-shaped virus STSV1: virus-host interactions and genomic features

A virus infecting the hyperthermophilic archaeon Sulfolobus tengchongensis has been isolated from a field sample from Tengchong, China, and characterized. The virus, denoted STSV1 (Sulfolobus tengchongensis spindle-shaped virus 1), has the morphology of a spindle (230 by 107 nm) with a tail of variable length (68 nm on average) at one end and is the largest of the known spindle-shaped viruses. After infecting its host, the virus multiplied rapidly to high titers (>10(10) PFU/ml). Replication of the virus retarded host growth but did not cause lysis of the host cells. STSV1 did not integrate into the host chromosome and existed in a carrier state. The STSV1 DNA was modified in an unusual fashion, presumably by virally encoded modification systems. STSV1 harbors a double-stranded DNA genome of 75,294 bp, which shares no significant sequence similarity to those of fuselloviruses. The viral genome contains a total of 74 open reading frames (ORFs), among which 14 have a putative function. Five ORFs encode viral structural proteins, including a putative coat protein of high abundance. The products of the other nine ORFs are probably involved in polysaccharide biosynthesis, nucleotide metabolism, and DNA modification. The viral genome divides into two nearly equal halves of opposite gene orientation. This observation as well as a GC-skew analysis point to the presence of a putative viral origin of replication in the 1.4-kb intergenic region between ORF1 and ORF74. Both morphological and genomic features identify STSV1 as a novel virus infecting the genus Sulfolobus.     

Global network of specific virus-host interactions in hypersaline environments

Hypersaline environments are dominated by archaea and bacteria and are almost entirely devoid of eukaryotic organisms. In addition, hypersaline environments contain considerable numbers of viruses. Currently, there is only a limited amount of information about these haloviruses. The ones described in detail mostly resemble head-tail bacteriophages, whereas observations based on direct microscopy of the hypersaline environmental samples highlight the abundance of non-tailed virus-like particles. Here we studied nine spatially distant hypersaline environments for the isolation of new halophilic archaea (61 isolates), halophilic bacteria (24 isolates) and their viruses (49 isolates) using a culture-dependent approach. The obtained virus isolates approximately double the number of currently described archaeal viruses. The new isolates could be divided into three tailed and two non-tailed virus morphotypes, suggesting that both types of viruses are widely distributed and characteristic for haloarchaeal viruses. We determined the sensitivity of the hosts against all isolated viruses. It appeared that the host ranges of numerous viruses extend to hosts in distant locations, supporting the idea that there is a global exchange of microbes and their viruses. It suggests that hypersaline environments worldwide function like a single habitat.     

NK cells stimulate proliferation of T and NK cells through 2B4/CD48 interactions

Few studies have addressed the consequences of physical interactions between NK and T cells, as well as physical interactions among NK cells themselves. We show in this study that NK cells can enhance T cell activation and proliferation in response to CD3 cross-linking and specific Ag through interactions between 2B4 (CD244) on NK cells and CD48 on T cells. Furthermore, 2B4/CD48 interactions between NK cells also enhanced proliferation of NK cells in response to IL-2. Overall, these results suggest that NK cells augment the proliferation of neighboring T and NK cells through direct cell-cell contact. These results provide new insights into NK cell-mediated control of innate and adaptive immunity and demonstrate that receptor/ligand-specific cross talk between lymphocytes may occur in settings other than T-B cell or T-T cell interactions.     

Coordinate expression of cytokines and chemokines by NK cells during murine cytomegalovirus infection

Cytokines and chemokines activate and direct effector cells during infection. We previously identified a functional group of five cytokines and chemokines, namely, IFN-gamma, activation-induced T cell-derived and chemokine-related cytokine/lymphotactin, macrophage-inflammatory protein 1alpha, macrophage-inflammatory protein 1beta, and RANTES, coexpressed in individual activated NK cells, CD8(+) T cells, and CD4(+) Th1 cells in vitro and during in vivo infections. However, the stimuli during infection were not known. In murine CMV (MCMV) infection, the DAP12/KARAP-associated Ly49H NK cell activation receptor is crucial for resistance through recognition of MCMV-encoded m157 but NK cells also undergo in vivo nonspecific responses to uncharacterized stimuli. In this study, we show that Ly49H ligation by m157 resulted in a coordinated release of all five cytokines/chemokines from Ly49H(+) NK cells. Whereas other cytokines also triggered the release of these cytokines/chemokines, stimulation was not confined to the Ly49H(+) population. At the single-cell level, the production of the five mediators showed strong positive correlation with each other. Interestingly, NK cells were a major source of these five cytokines/chemokines in vitro and in vivo, whereas infected macrophages produced only limited amounts of macrophage-inflammatory protein 1alpha, macrophage-inflammatory protein1beta, and RANTES. These findings suggest that both virus-specific and nonspecific NK cells play crucial roles in activating and directing other inflammatory cells during MCMV infection.     

Activation of natural killer (NK) T cells during murine cytomegalovirus infection enhances the antiviral response mediated by NK cells

NK1.1+ T (NKT) cells are efficient regulators of early host responses which have been shown to play a role in tumor surveillance. The relevance of NKT cells in immune surveillance of viral infections, however, is not well understood. In this study, we investigated the functional relevance of NKT cells in controlling herpesvirus infections by using challenge with murine cytomegalovirus (MCMV) as the study model. This model has proven to be one of the best systems for evaluating the role of NK cells during virus infection. Using gene-targeted mice and alpha-galactosylceramide (alpha-GalCer) as an exogenous stimulator of NKT cells, we have analyzed the role of these cells in the immune surveillance of MCMV infection. Our studies in NKT-cell-deficient, T-cell receptor Jalpha281 gene-targeted mice have established that classical NKT cells do not play a critical role in the early clearance of MCMV infection. Importantly, however, activation of NKT cells by alpha-GalCer resulted in reduced viral replication in visceral organs. Depletion studies, coupled with analysis of gene-targeted mice lacking perforin and gamma interferon (IFN-gamma), have revealed that the antiviral effects of alpha-GalCer involve NK cells and have clearly demonstrated that the antiviral activity of alpha-GalCer, unlike the antitumor one, is critically dependent on both perforin and IFN-gamma.     

Middle East respiratory syndrome coronavirus infection: virus-host cell interactions and implications on pathogenesis

Middle-East Respiratory Syndrome coronavirus (MERS-CoV) was identified to cause severe respiratory infection in humans since 2012. The continuing MERS epidemic with a case-fatality of more than 30 % poses a major threat to public health worldwide. Currently, the pathogenesis of human MERS-CoV infection remains poorly understood. We reviewed experimental findings from human primary cells and ex vivo human lung tissues, as well as those from animal studies, so as to understand the pathogenesis and high case-fatality of MERS. Human respiratory epithelial cells are highly susceptible to MERS-CoV and can support productive viral replication. However, the induction of antiviral cytokines and proinflammatory cytokines/chemokines are substantially dampened in the infected epithelial cells, due to the antagonistic mechanisms evolved by the virus. MERS-CoV can readily infect and robustly replicate in human macrophages and dendritic cells, triggering the aberrant production of proinflammatory cytokines/chemokines. MERS-CoV can also effectively infect human primary T cells and induce massive apoptosis in these cells. Although data from clinical, in vitro and ex vivo studies suggested the potential for virus dissemination, extrapulmonary involvement in MERS patients has not been ascertained due to the lack of autopsy study. In MERS-CoV permissive animal models, although viral RNA can be detected from multiple organs of the affected animals, the brain of human DPP4-transgenic mouse was the only extrapulmonary organ from which the infectious virus can be recovered. More research findings on the pathogenesis of MERS and the tissue tropisms of MERS-CoV may help to improve the treatment and infection control of MERS.     

Distinct roles for the NK cell-activating receptors in mediating interactions with dendritic cells and tumor cells

Hyperproduction of goblet cells and mucin in the airway epithelium is an important feature of airway inflammatory diseases. We investigated the involvement of Notch signaling in MUC5AC expression in NCI-H292 cells, a human lung carcinoma cell line. Epidermal growth factor (EGF) stimulated generation of the Notch intracellular domain (NICD) in a RBP-Jκ-dependent manner. Treatment with γ-secretase inhibitors L-685,458 or DAPT or introduction of small interfering RNA directed against Notch1 reduced EGF-induced MUC5AC expression. The inhibitory effect of L-685,458 on EGF-induced MUC5AC mRNA and protein expression was also observed in primary human bronchial epithelial cells. Blockage of Notch signaling with L-685,458 or Notch siRNA resulted in a decrease in EGF-induced phosphorylation of ERK. These results suggested that ERK activation is necessary for the regulation of EGF receptor (EGFR)-mediated MUC5AC expression by Notch signaling. Conversely, forced expression of NICD induced both EGFR and ERK phosphorylation with MUC5AC expression even in the absence of EGF. Treatment of the NICD-expressing cells with EGF further augmented ERK phosphorylation in an additive manner. The ERK phosphorylation induced by exogenous NICD was inhibited by treatment with an Ab that antagonizes EGFR activity as well as by inhibitors of EGFR and ERK, implying that Notch signaling induces MUC5AC expression by activating the EGFR pathway. Collectively, these results suggest that MUC5AC expression is regulated by a bidirectional circuit between Notch and EGFR signaling pathways.     

IL-10 restricts activation-induced death of NK cells during acute murine cytomegalovirus infection

IL-10 is an immunomodulatory cytokine that acts to antagonize T cell responses elicited during acute and chronic infections. Thus, the IL-10R signaling pathway provides a potential therapeutic target in strategies aimed at combating infectious diseases. In this study, we set out to investigate whether IL-10 expression had an effect on NK cells. Murine CMV infection provides the best characterized in vivo system to evaluate the NK cell response, with NK cells being critical in the early control of acute infection. Blockade of IL-10R during acute murine CMV infection markedly reduced the accumulation of cytotoxic NK cells in the spleen and lung, a phenotype associated with a transient elevation of virus DNA load. Impaired NK cell responsiveness after IL-10R blockade was attributed to elevated levels of apoptosis observed in NK cells exhibiting an activated phenotype. Therefore, we conclude that IL-10 contributes to antiviral innate immunity during acute infection by restricting activation-induced death in NK cells.     

Comparing the kinetics of NK cells, CD4, and CD8 T cells in murine cytomegalovirus infection

NK cells recognize virus-infected cells with germline-encoded activating and inhibitory receptors that do not undergo genetic recombination or mutation. Accordingly, NK cells are often considered part of the innate immune response. The innate response comprises rapid early defenders that do not form immune memory. However, there is increasing evidence that experienced NK cells provide increased protection to secondary infection, a hallmark of the adaptive response. In this study, we compare the dynamics of the innate and adaptive immune responses by examining the kinetic profiles of the NK and T cell response to murine CMV infection. We find that, unexpectedly, the kinetics of NK cell proliferation is neither earlier nor faster than the CD4 or CD8 T cell response. Furthermore, early NK cell contraction after the peak of the response is slower than that of T cells. Finally, unlike T cells, experienced NK cells do not experience biphasic decay after the response peak, a trait associated with memory formation. Rather, NK cell contraction is continuous, constant, and returns to below endogenous preinfection levels. This indicates that the reason why Ag-experienced NK cells remain detectable for a prolonged period after adoptive transfer and infection is in part due to the high precursor frequency, slow decay rate, and low background levels of Ly49H(+) NK cells in recipient DAP12-deficient mice. Thus, the quantitative contribution of Ag-experienced NK cells in an endogenous secondary response, with higher background levels of Ly49H(+) NK cells, may be not be as robust as the secondary response observed in T cells.     

Proteomic analysis of virus-host interactions in an infectious context using recombinant viruses

RNA viruses exhibit small-sized genomes encoding few proteins, but still establish complex networks of interactions with host cell components to achieve replication and spreading. Ideally, these virus-host protein interactions should be mapped directly in infected cell culture, but such a high standard is often difficult to reach when using conventional approaches. We thus developed a new strategy based on recombinant viruses expressing tagged viral proteins to capture both direct and indirect physical binding partners during infection. As a proof of concept, we engineered a recombinant measles virus (MV) expressing one of its virulence factors, the MV-V protein, with a One-STrEP amino-terminal tag. This allowed virus-host protein complex analysis directly from infected cells by combining modified tandem affinity chromatography and mass spectrometry analysis. Using this approach, we established a prosperous list of 245 cellular proteins interacting either directly or indirectly with MV-V, and including four of the nine already known partners of this viral factor. These interactions were highly specific of MV-V because they were not recovered when the nucleoprotein MV-N, instead of MV-V, was tagged. Besides key components of the antiviral response, cellular proteins from mitochondria, ribosomes, endoplasmic reticulum, protein phosphatase 2A, and histone deacetylase complex were identified for the first time as prominent targets of MV-V and the critical role of the later protein family in MV replication was addressed. Most interestingly, MV-V showed some preferential attachment to essential proteins in the human interactome network, as assessed by centrality and interconnectivity measures. Furthermore, the list of MV-V interactors also showed a massive enrichment for well-known targets of other viruses. Altogether, this clearly supports our approach based on reverse genetics of viruses combined with high-throughput proteomics to probe the interaction network that viruses establish in infected cells.     

Immunogold labelling of tumour cells during NK/target cell interactions

Using a system of immunocolloidal gold labelling, we have monitored the expression and distribution of transferrin receptors (TfRs) within the K562 cell line, during NK/target cell interactions. An indirect method of immunolabelling was used to effectively immunolabel tumour cells without disrupting the natural effector:target interactions. Successful localization of TfRs demonstrates the potential of the described technique for discerning antigenic distribution of other cell:cell interactions. Immunolabelling has also provided a useful method for demonstrating receptor down-regulation within NK target cells, as a proposed cause of reduced receptor expression by TPA-treated cells. Following 30 and 60 min incubation periods with TPA, approximately 15 and 30%, respectively, of the gold/antibody complexes were relocated from the surface membrane to an intracellular location within endocytotic vesicles. The demonstration of receptor down-regulation is important as a proposed cause of TPA-induced tumour cell resistance to NK-mediated cytolysis.     

Genome-resolved viral and cellular metagenomes revealed potential key virus-host interactions in a deep freshwater lake

Metagenomics has dramatically expanded the known virosphere, but freshwater viral diversity and their ecological interaction with hosts remain poorly understood. Here, we conducted a metagenomic exploration of planktonic dsDNA prokaryotic viruses by sequencing both virion (<0.22 μm) and cellular (0.22–5.0 μm) fractions collected spatiotemporally from a deep freshwater lake (Lake Biwa, Japan). This simultaneously reconstructed 183 complete (i.e., circular) viral genomes and 57 bacterioplankton metagenome-assembled genomes. Analysis of metagenomic read coverage revealed vertical partitioning of the viral community analogous to the vertically stratified bacterioplankton community. The hypolimnetic community was generally stable during stratification, but occasionally shifted abruptly, presumably due to lysogenic induction. Genes involved in assimilatory sulfate reduction were encoded in 20 (10.9%) viral genomes, including those of dominant viruses, and may aid viral propagation in sulfur-limited freshwater systems. Hosts were predicted for 40 (21.9%) viral genomes, encompassing 10 phyla (or classes of Proteobacteria) including ubiquitous freshwater bacterioplankton lineages (e.g., Ca. Fonsibacter and Ca. Nitrosoarchaeum). Comparison with viral genomes derived from published metagenomes revealed viral phylogeographic connectivity in geographically isolated habitats. Notably, analogous to their hosts, actinobacterial viruses were among the most diverse, ubiquitous and abundant viral groups in freshwater systems, with potential high lytic activity in surface waters.     

A highly efficient system to produce infectious human papillomavirus: Elucidation of natural virus-host interactions

A simple, efficient system has been developed to produce high titers of infectious human papillomavirus type 18 (HPV-18) in organotypic raft cultures of primary human keratinocytes (PHKs). Molecular characterization elucidated key early and late events in the reproductive program. The system obviates the need for immortalized cells and allows the analyses of mutant HPV genomes not previously possible. An E6 deletion mutant incapable of causing p53 degradation is defective in viral DNA amplification and capsid protein production. The high levels of p53 protein which accumulated in numerous cells did not lead to apoptosis over a prolonged duration. Time course and metabolic labeling experiments revealed novel interactions with the host. Notably, post-mitotic, differentiated cells are induced by HPV E7 expression to reenter S phase, whereupon host chromosomes replicate, but HPV DNA does not amplify until the cells have progressed to and are arrested in G2 phase. Here, we present data that strongly suggest that the abundant cytoplasmic viral E1^E4 protein is not responsible for this G2 arrest, as described in the literature upon ectopic expression in cell lines. We provide additional insights into the viral life cycle and contrast them to conclusions derived from experiments in cell lines.     

Antibody-dependent cell lysis by NK cells is preserved after sarcoma-induced inhibition of NK cell cytotoxicity

Osteosarcoma and Ewing’s sarcoma tumor cells are susceptible to IL15-induced or antibody-mediated cytolytic activity of NK cells in short-term cytotoxicity assays. When encountering the tumor environment in vivo, NK cells may be in contact with tumor cells for a prolonged time period. We explored whether a prolonged interaction with sarcoma cells can modulate the activation and cytotoxic activity of NK cells. The 40 h coculture of NK cells with sarcoma cells reversibly interfered with the IL15-induced expression of NKG2D, DNAM-1 and NKp30 and inhibited the cytolytic activity of NK cells. The inhibitory effects on receptor expression required physical contact between NK cells and sarcoma cells and were independent of TGF-β. Five days pre-incubation of NK cells with IL15 prevented the down-regulation of NKG2D and cytolytic activity in subsequent cocultures with sarcoma cells. NK cell FcγRIIIa/CD16 receptor expression and antibody-mediated cytotoxicity were not affected after the coculture. Inhibition of NK cell cytotoxicity was directly linked to the down-regulation of the respective NK cell-activating receptors. Our data demonstrate that the inhibitory effects of sarcoma cells on the cytolytic activity of NK cells do not affect the antibody-dependent cytotoxicity and can be prevented by pre-activation of NK cells with IL15. Thus, the combination of cytokine-activated NK cells and monoclonal antibody therapy may be required to improve tumor targeting and NK cell functionality in the tumor environment.