Coral Mucus Is a Hot Spot for Viral Infections

There is increasing suspicion that viral communities play a pivotal role in maintaining coral health, yet their main ecological traits still remain poorly characterized. In this study, we examined the seasonal distribution and reproduction pathways of viruses inhabiting the mucus of the scleractinians Fungia repanda and Acropora formosa collected in Nha Trang Bay (Vietnam) during an 11-month survey. The strong coupling between epibiotic viral and bacterial abundance suggested that phages are dominant among coral-associated viral communities. Mucosal viruses also exhibited significant differences in their main features between the two coral species and were also remarkably contrasted with their planktonic counterparts. For example, their abundance (inferred from epifluorescence counts), lytic production rates (KCN incubations), and the proportion of lysogenic cells (mitomycin C inductions) were, respectively, 2.6-, 9.5-, and 2.2-fold higher in mucus than in the surrounding water. Both lytic and lysogenic indicators were tightly coupled with temperature and salinity, suggesting that the life strategy of viral epibionts is strongly dependent upon environmental circumstances. Finally, our results suggest that coral mucus may represent a highly favorable habitat for viral proliferation, promoting the development of both temperate and virulent phages. Here, we discuss how such an optimized viral arsenal could be crucial for coral viability by presumably forging complex links with both symbiotic and adjacent nonsymbiotic microorganisms.     

Vaccinia Viral Protein A27 Is Anchored to the Viral Membrane via a Cooperative Interaction with Viral Membrane Protein A17

The vaccinia viral protein A27 in mature viruses specifically interacts with heparan sulfate for cell surface attachment. In addition, A27 associates with the viral membrane protein A17 to anchor to the viral membrane; however, the specific interaction between A27 and A17 remains largely unclear. To uncover the active binding sites and the underlying binding mechanism, we expressed and purified the N-terminal (18–50 residues) and C-terminal (162–203 residues) fragments of A17, which are denoted A17-N and A17-C. Through surface plasmon resonance, the binding affinity of A27/A17-N (K_A = 3.40 × 10⁸ m⁻¹) was determined to be approximately 3 orders of magnitude stronger than that of A27/A17-C (K_A = 3.40 × 10⁵ m⁻¹), indicating that A27 prefers to interact with A17-N rather than A17-C. Despite the disordered nature of A17-N, the A27-A17 interaction is mediated by a specific and cooperative binding mechanism that includes two active binding sites, namely ³²SFMPK³⁶ (denoted as F1 binding) and ²⁰LDKDLFTEEQ²⁹ (F2). Further analysis showed that F1 has stronger binding affinity and is more resistant to acidic conditions than is F2. Furthermore, A27 mutant proteins that retained partial activity to interact with the F1 and F2 sites of the A17 protein were packaged into mature virus particles at a reduced level, demonstrating that the F1/F2 interaction plays a critical role in vivo. Using these results in combination with site-directed mutagenesis data, we established a computer model to explain the specific A27-A17 binding mechanism.     

Viral Capsid Is a Pathogen-Associated Molecular Pattern in Adenovirus Keratitis

Human adenovirus (HAdV) infection of the human eye, in particular serotypes 8, 19 and 37, induces the formation of corneal subepithelial leukocytic infiltrates. Using a unique mouse model of adenovirus keratitis, we studied the role of various virus-associated molecular patterns in subsequent innate immune responses of resident corneal cells to HAdV-37 infection. We found that neither viral DNA, viral gene expression, or viral replication was necessary for the development of keratitis. In contrast, empty viral capsid induced keratitis and a chemokine profile similar to intact virus. Transfected viral DNA did not induce leukocyte infiltration despite CCL2 expression similar to levels in virus infected corneas. Mice without toll-like receptor 9 (Tlr9) signaling developed clinical keratitis upon HAdV-37 infection similar to wild type mice, although the absolute numbers of activated monocytes in the cornea were less in Tlr9^−/− mice. Virus induced leukocytic infiltrates and chemokine expression in mouse cornea could be blocked by treatment with a peptide containing arginine glycine aspartic acid (RGD). These results demonstrate that adenovirus infection of the cornea induces chemokine expression and subsequent infiltration by leukocytes principally through RGD contact between viral capsid and the host cell, possibly through direct interaction between the viral capsid penton base and host cell integrins.     

Lymphotoxin Signaling Is Initiated by the Viral Polymerase in HCV-linked Tumorigenesis

Exposure to hepatitis C virus (HCV) typically results in chronic infection that leads to progressive liver disease ranging from mild inflammation to severe fibrosis and cirrhosis as well as primary liver cancer. HCV triggers innate immune signaling within the infected hepatocyte, a first step in mounting of the adaptive response against HCV infection. Persistent inflammation is strongly associated with liver tumorigenesis. The goal of our work was to investigate the initiation of the inflammatory processes triggered by HCV viral proteins in their host cell and their possible link with HCV-related liver cancer. We report a dramatic upregulation of the lymphotoxin signaling pathway and more specifically of lymphotoxin-β in tumors of the FL-N/35 HCV-transgenic mice. Lymphotoxin expression is accompanied by activation of NF-κB, neosynthesis of chemokines and intra-tumoral recruitment of mononuclear cells. Spectacularly, IKKβ inactivation in FL-N/35 mice drastically reduces tumor incidence. Activation of lymphotoxin-β pathway can be reproduced in several cellular models, including the full length replicon and HCV-infected primary human hepatocytes. We have identified NS5B, the HCV RNA dependent RNA polymerase, as the viral protein responsible for this phenotype and shown that pharmacological inhibition of its activity alleviates activation of the pro-inflammatory pathway. These results open new perspectives in understanding the inflammatory mechanisms linked to HCV infection and tumorigenesis.     

pelo Is Required for High Efficiency Viral Replication

Viruses hijack host factors for their high speed protein synthesis, but information about these factors is largely unknown. In searching for genes that are involved in viral replication, we carried out a forward genetic screen for Drosophila mutants that are more resistant or sensitive to Drosophila C virus (DCV) infection-caused death, and found a virus-resistant line in which the expression of pelo gene was deficient. Our mechanistic studies excluded the viral resistance of pelo deficient flies resulting from the known Drosophila anti-viral pathways, and revealed that pelo deficiency limits the high level synthesis of the DCV capsid proteins but has no or very little effect on the expression of some other viral proteins, bulk cellular proteins, and transfected exogenous genes. The restriction of replication of other types of viruses in pelo deficient flies was also observed, suggesting pelo is required for high level production of capsids of all kinds of viruses. We show that both pelo deficiency and high level DCV protein synthesis increase aberrant 80S ribosomes, and propose that the preferential requirement of pelo for high level synthesis of viral capsids is at least partly due to the role of pelo in dissociation of stalled 80S ribosomes and clearance of aberrant viral RNA and proteins. Our data demonstrated that pelo is a host factor that is required for high efficiency translation of viral capsids and targeting pelo could be a strategy for general inhibition of viral infection.     

Oligomerization of Baculovirus LEF-11 Is Involved in Viral DNA Replication

We have previously reported that baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV) late expression factor 11 (lef-11) is associated with viral DNA replication and have demonstrated that it potentially interacts with itself; however, whether LEF-11 forms oligomers and the impact of LEF-11 oligomerization on viral function have not been substantiated. In this study, we first demonstrated that LEF-11 is capable of forming oligomers. Additionally, a series of analyses using BmNPV LEF-11 truncation mutants indicated that two distinct domains control LEF-11 oligomerization (aa 42–61 and aa 72–101). LEF-11 truncation constructs were inserted into a lef-11-knockout BmNPV bacmid, which was used to demonstrate that truncated LEF-11 lacking either oligomerization domain abrogates viral DNA replication. Finally, site-directed mutagenesis was used to determine that the conserved hydrophobic residues Y58&I59 (representing Y58 and I59), I85 and L88&L89 (representing L88 and L89) are required for LEF-11 oligomerization and viral DNA replication. Collectively, these data indicate that BmNPV LEF-11 oligomerization influences viral DNA replication.     

Human Cytomegalovirus pUL93 Is Required for Viral Genome Cleavage and Packaging

Human cytomegalovirus (HCMV) pUL93 is essential for virus growth, but its precise function in the virus life cycle is unknown. Here, we characterize a UL93 stop mutant virus (UL93st-TB40/E-BAC) to demonstrate that the absence of this protein does not restrict viral gene expression; however, cleavage of viral DNA into unit-length genomes as well as genome packaging is abolished. Thus, pUL93 is required for viral genome cleavage and packaging.     

Viral Membrane Protein Topology Is Dictated by Multiple Determinants in Its Sequence

The targeting, insertion, and topology of membrane proteins have been extensively studied in both prokaryotes and eukaryotes. However, the mechanisms used by viral membrane proteins to generate the correct topology within cellular membranes are less well understood. Here, the effect of flanking charges and the hydrophobicity of the N-terminal hydrophobic segment on viral membrane protein topogenesis are examined systematically. Experimental data reveal that the classical topological determinants have only a minor effect on the overall topology of p9, a plant viral movement protein. Since only a few individual sequence alterations cause an inversion of p9 topology, its topological stability is robust. This result further indicates that the protein has multiple, and perhaps redundant, structural features that ensure that it always adopts the same topology. These critical topogenic sequences appear to be recognized and acted upon from the initial stages of protein biosynthesis, even before the ribosome ends protein translation.     

Polarized Budding of Measles Virus Is Not Determined by Viral Surface Glycoproteins

For viruses that mature by a budding process, the envelope glycoproteins are considered the major determinants for the site of virus release from polarized epithelial cells. Viruses are usually released from that membrane domain where the viral surface glycoproteins are transported to. We here report that measles virus has developed a different maturation strategy. Measles virus was found to be released from the apical membrane domain of polarized epithelial cells, though the surface glycoproteins H and F were transported in a nonpolarized fashion and to the basolateral membrane domain, respectively.     

MORC3 Is a Target of the Influenza A Viral Protein NS1

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Human Cytomegalovirus UL36 Protein Is Dispensable for Viral Replication in Cultured Cells

Consistent with earlier analyses of human cytomegalovirus UL36 mRNA, we find that the UL36 protein is present throughout infection. In fact, it is delivered to the infected cell as a constituent of the virion. Curiously, much less UL36 protein accumulated in cells infected with the AD169 strain of human cytomegalovirus than in cells infected with the Towne or Toledo strain, and localization of the protein in cells infected with AD169 is strikingly different from that in cell infected with the Towne or Toledo strain. The variation in steady-state level of the proteins results from different stabilities of the proteins. The UL36 proteins from the three viral strains differ by several amino acid substitutions. However, this variability is not responsible for the different half-lives because the AD169 and Towne proteins, which exhibit very different half-lives within infected cells, exhibit the same half-life when introduced into uninfected cells by transfection with expression plasmids. We demonstrate that the UL36 protein is nonessential for growth in cultured cells, and we propose that the ability of the virus to replicate in the absence of UL36 function likely explains the striking strain-specific variation in the half-life and intracellular localization of the protein.     

HIV-1 Nef Dimerization Is Required for Nef-Mediated Receptor Downregulation and Viral Replication

Nef, a human immunodeficiency virus type 1 (HIV-1) accessory factor capable of interaction with a diverse array of host cell signaling molecules, is essential for high-titer HIV replication and AIDS progression. Previous biochemical and structural studies have suggested that Nef may form homodimers and higher-order oligomers in HIV-infected cells, which may be required for both immune and viral receptor downregulation as well as viral replication. Using bimolecular fluorescence complementation, we provide the first direct evidence for Nef dimers within HIV host cells and identify the structural requirements for dimerization in vivo. Bimolecular fluorescence complementation analysis shows that the multiple hydrophobic and electrostatic interactions found within the dimerization interface of the Nef X-ray crystal structure are essential for dimerization in cells. Nef dimers localized to the plasma membrane as well as the trans-Golgi network, two subcellular localizations essential for Nef function. Mutations in the Nef dimerization interface dramatically reduced both Nef-induced CD4 downregulation and HIV replication. Viruses expressing dimerization-defective Nef mutants were disabled to the same extent as HIV that fails to express Nef in terms of replication. These results identify the Nef dimerization region as a potential molecular target for antiretroviral drug discovery.     

The Lytic Enzyme of Bacteriophage PRD1 Is Associated with the Viral Membrane

Bacteriophage PRD1 encodes two proteins (P7 and P15) that are associated with a muralytic activity. Protein P15 is a soluble beta-1,4-N-acetylmuramidase that causes phage-induced host cell lysis. We demonstrate here that P15 is also a structural component of the PRD1 virion and that it is connected to the phage membrane. Small viral membrane proteins P20 and P22 modulate incorporation of P15 into the virion and may connect it to the phage membrane. The principal muralytic protein involved in PRD1 DNA entry seems to be the putative lytic transglycosylase protein P7, as the absence of protein P15 did not delay initiation of phage DNA replication in the virus-host system used. The incorporation of two different lytic enzymes into virions may reflect the broad host range of bacteriophage PRD1.