Functional analysis of the vaccinia virus entry protein L1

Vaccinia virus (VACV) L1 is a myristoylated envelope protein which is required for ceL1 entry and low-pH-triggered fusion of virus infected cells. L1 also associates with members of the poxvirus entry-fusion complex (EFC), but is not required for the assembly or stability of the core complex. Despite some recent progress, the specific function(s) of L1 during entry remain poorly defined. It is currently unclear if L1 functions during or prior to membrane fusion, such as attaching to cell surface molecules. In addition, the host cell receptor(s) responsible for triggering internalization and/or membrane fusion of VACV have not been identified. In this dissertation, I present my efforts to identify the specific role that L1 plays in entry and to characterize the regions on this protein which are important for its function. In the first chapter of this dissertation, I show that a recombinant form of the L1 ectodomain binds to cell surfaces and inhibits entry of VACV independently of glycosaminoglycans. These observations suggest that L1 is a viable candidate for the VACV receptor-binding protein. In the second chapter of this dissertation, I engineered six L1 mutants which are defective in complementation and/or N-myristoylation. I demonstrate that two acylated L1 mutants which are impaired in complementation are properly incorporated into mature virions. These results indicate that the conserved amino terminus of L1 is important for its myristoylation and for VACV entry. In the third chapter of this dissertation, I performed truncations and a deletion in the conserved C-terminal endodomain of L1. I discovered that several components of the poxvirus EFC do not interact with the truncated mutants, providing the first evidence that the endodomain of L1 is required for EFC association. I also found that a L1 mutant with a two residue deletion in its endodomain associates normally with EFC proteins and is packaged into mature virions, yet it does not function in entry. These observations indicate that the endodomain of L1 is a critical functional region required for EFC association and for virus entry

Functional analysis of the vaccinia virus entry protein L1

Vaccinia virus (VACV) L1 is a myristoylated envelope protein which is required for ceL1 entry and low-pH-triggered fusion of virus infected cells. L1 also associates with members of the poxvirus entry-fusion complex (EFC), but is not required for the assembly or stability of the core complex. Despite some recent progress, the specific function(s) of L1 during entry remain poorly defined. It is currently unclear if L1 functions during or prior to membrane fusion, such as attaching to cell surface molecules. In addition, the host cell receptor(s) responsible for triggering internalization and/or membrane fusion of VACV have not been identified. In this dissertation, I present my efforts to identify the specific role that L1 plays in entry and to characterize the regions on this protein which are important for its function. In the first chapter of this dissertation, I show that a recombinant form of the L1 ectodomain binds to cell surfaces and inhibits entry of VACV independently of glycosaminoglycans. These observations suggest that L1 is a viable candidate for the VACV receptor-binding protein. In the second chapter of this dissertation, I engineered six L1 mutants which are defective in complementation and/or N-myristoylation. I demonstrate that two acylated L1 mutants which are impaired in complementation are properly incorporated into mature virions. These results indicate that the conserved amino terminus of L1 is important for its myristoylation and for VACV entry. In the third chapter of this dissertation, I performed truncations and a deletion in the conserved C-terminal endodomain of L1. I discovered that several components of the poxvirus EFC do not interact with the truncated mutants, providing the first evidence that the endodomain of L1 is required for EFC association. I also found that a L1 mutant with a two residue deletion in its endodomain associates normally with EFC proteins and is packaged into mature virions, yet it does not function in entry. These observations indicate that the endodomain of L1 is a critical functional region required for EFC association and for virus entry

Topoisomerase 1 inhibition promotes cyclic GMP-AMP synthase-dependent antiviral responses

ABSTRACT Inflammatory responses, while essential for pathogen clearance, can also be deleterious to the host. Chemical inhibition of topoisomerase 1 (Top1) by low-dose camptothecin (CPT) can suppress transcriptional induction of antiviral and inflammatory genes and protect animals from excessive and damaging inflammatory responses. We describe the unexpected finding that minor DNA damage from topoisomerase 1 inhibition with low-dose CPT can trigger a strong antiviral immune response through cyclic GMP-AMP synthase (cGAS) detection of cytoplasmic DNA. This argues against CPT having only anti-inflammatory activity. Furthermore, expression of the simian virus 40 (SV40) large T antigen was paramount to the proinflammatory antiviral activity of CPT, as it potentiated cytoplasmic DNA leakage and subsequent cGAS recruitment in human and mouse cell lines. This work suggests that the capacity of Top1 inhibitors to blunt inflammatory responses can be counteracted by viral oncogenes and that this should be taken into account for their therapeutic development. IMPORTANCE Recent studies suggest that low-dose DNA-damaging compounds traditionally used in cancer therapy can have opposite effects on antiviral responses, either suppressing (with the example of CPT) or potentiating (with the example of doxorubicin) them. Our work demonstrates that the minor DNA damage promoted by low-dose CPT can also trigger strong antiviral responses, dependent on the presence of viral oncogenes. Taken together, these results call for caution in the therapeutic use of low-dose chemotherapy agents to modulate antiviral responses in humans

Genome-wide siRNA Screening at Biosafety Level 4 Reveals a Crucial Role for Fibrillarin in Henipavirus Infection.

Hendra and Nipah viruses (genus Henipavirus, family Paramyxoviridae) are highly pathogenic bat-borne viruses. The need for high biocontainment when studying henipaviruses has hindered the development of therapeutics and knowledge of the viral infection cycle. We have performed a genome-wide siRNA screen at biosafety level 4 that identified 585 human proteins required for henipavirus infection. The host protein with the largest impact was fibrillarin, a nucleolar methyltransferase that was also required by measles, mumps and respiratory syncytial viruses for infection. While not required for cell entry, henipavirus RNA and protein syntheses were greatly impaired in cells lacking fibrillarin, indicating a crucial role in the RNA replication phase of infection. During infection, the Hendra virus matrix protein co-localized with fibrillarin in cell nucleoli, and co-associated as a complex in pulldown studies, while its nuclear import was unaffected in fibrillarin-depleted cells. Mutagenesis studies showed that the methyltransferase activity of fibrillarin was required for henipavirus infection, suggesting that this enzyme could be targeted therapeutically to combat henipavirus infections

Dual microRNA Screens Reveal That the Immune-Responsive miR-181 Promotes Henipavirus Entry and Cell-Cell Fusion

<div><p>Hendra and Nipah viruses (family <i>Paramyxoviridae</i>, genus <i>Henipavirus</i>) are bat-borne viruses that cause fatal disease in humans and a range of other mammalian species. Gaining a deeper understanding of host pathways exploited by henipaviruses for infection may identify targets for new anti-viral therapies. Here we have performed genome-wide high-throughput agonist and antagonist screens at biosafety level 4 to identify host-encoded microRNAs (miRNAs) impacting henipavirus infection in human cells. Members of the miR-181 and miR-17~93 families strongly promoted Hendra virus infection. miR-181 also promoted Nipah virus infection, but did not affect infection by paramyxoviruses from other genera, indicating specificity in the virus-host interaction. Infection promotion was primarily mediated via the ability of miR-181 to significantly enhance henipavirus-induced membrane fusion. Cell signalling receptors of ephrins, namely EphA5 and EphA7, were identified as novel negative regulators of henipavirus fusion. The expression of these receptors, as well as EphB4, were suppressed by miR-181 overexpression, suggesting that simultaneous inhibition of several Ephs by the miRNA contributes to enhanced infection and fusion. Immune-responsive miR-181 levels was also up-regulated in the biofluids of ferrets and horses infected with Hendra virus, suggesting that the host innate immune response may promote henipavirus spread and exacerbate disease severity. This study is the first genome-wide screen of miRNAs influencing infection by a clinically significant mononegavirus and nominates select miRNAs as targets for future anti-viral therapy development.</p></div

Select Eph receptors inhibit HeV infection and cell-cell-fusion and are miR-181 target genes.

<p>(A) Relative mRNA levels of indicated target genes in HeLa cells 72 h post-transfection with siRNAs (40 nM). ***p<0.001 compared to siNEG (B) Relative percentage of cells infected with HeV (24 h, MOI 0.5), after 72 h transfection with siRNAs targeting indicated molecules. *p<0.05, ***p<0.001 compared to siNEG. (C) Relative mRNA levels of Eph receptors A4, A5, A7 and B4 in HeLa cells, 72 h post transfection with miRNA agonists (25 nM). N.s. not significant; **p<0.01, ***p≤0.001, compared to control agonist. (D) Cell-cell fusion of HeV-F and–G expressing HEK-293T cells to HeLa cells treated with indicated siRNAs. Values are normalised as a percentage to siNEG or control agonist.</p

miR-181d promotes henipavirus infection specifically.

<p>Infectivity assays were applied to assess changes in virus production or virus infection of HeLa cells infected with NiV, MeV, MuV, RSV or influenza A/WSN/33 virus for 24 h. Cells were previously transfected with miR-181d or negative control agonists (miNEG) for 72 h. **p≤0.01; n.s. not significant.</p

miR-181 significantly enhances HeV RNA synthesis and F- and G-mediated cell-cell fusion.

<p>(A) qRT-PCR measurements of intracellular viral RNA copy number in HeLa cells infected with HeV (MOI 5). ***p≤0.001; *p≤0.05. HeV RNA values were normalised to cellular 18S levels. (B) Cell-to-cell fusion of HeV-F and HeV-G-expressing HEK-293T effector cells to HeLa cells treated with indicated siRNA or miRNA agonists. Syncytia were imaged using automated fluorescence microscopy. Nuclei are shown in blue (DAPI), effector cells in green (HeV-G staining) and target cells red (DiO lipid dye). (f1) and (f2) are images of cells transfected with miR-181d from two different microscopy fields. (C) Quantification of fusion events by counting all nuclei present in all syncytia. Values are normalised as a percentage to siNEG or miNEG. *p ≤ 0.05, ***p ≤0.001 compared to respective negative controls.</p

Expression levels of miR-181 in biofluids of animals infected with HeV are increased.

<p>Sixteen ferrets were infected with HeV at BSL-4. At predetermined time-points, ten different tissues were harvested and analysed for viral RNA loads by qRT-PCR (A). (B) qRT-PCR analysis of miR-181d levels in the serum samples of the ferrets. Values were normalized to the U6 RNA. *p≤0.05 compared to day 0. (C) qRT-PCR analysis of miR-181d in blood of horses from a published HeV infection study [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005974#ppat.1005974.ref052" target="_blank">52</a>].</p