Nelly Pante

The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

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Being non-pathogenic to humans, rodent parvoviruses (PVs) are naturally oncolytic viruses that have great potential as anti-cancer agents. Since these viruses replicate in the nucleus of the host cell, they have to overcome various cellular barriers, including the nuclear envelope (NE), before reaching the nucleus. The PV minute virus of mice (MVM) and several other PVs transiently disrupt the NE and enter the nucleus through the resulting breaks. However, the molecular basis of this unique nuclear entry pathway remains uncharacterized. Therefore, this thesis uses MVM as a model to identify the molecular mechanism by which PVs induce NE disruption during viral nuclear entry. By combining bioinformatics analyses, metabolic labeling assays and mutagenesis, we identified a functional internal N-myristoylation site starting with glycine (G) at position 78 in the unique portion of the viral structural protein VP1 (VP1u) of MVM. Performing proteolytic cleavage studies with a peptide containing this N-myristoylation site or with purified virions, we found that tryptophan at position 77 of MVM VP1u is susceptible to chymotrypsin cleavage, implying that this cleavage exposes G78 at the N-terminus of VP1u for N-myristoylation. Subsequent experiments were conducted in MVM-infected cells or using an NE permeabilization assay with inhibitors of N-myristoylation, chymotrypsin-like proteases, or the chymotrypsin-like activity of the proteasome. These results provide further evidence that protein N-myristoylation and a chymotrypsin-like activity, which may come from cellular proteasomes, are essential for MVM-induced NE disruption. In addition to MVM VP1, we investigated the role of proteolytic cleavage at the N-terminus of VP2 (the other structural protein of MVM) in viral nuclear entry. By generating and studying MVM with cleaved VP2 N-termini, we demonstrated that the VP2 N-terminus cleavage during early infection steps of MVM makes the virion more infectious than the wild-type one by enhancing its ability to disrupt the NE during viral nuclear entry. Similar to the wild-type MVM, the VP2 N-termini-cleaved virion also requires protein N-myristoylation and proteasomal processing to induce NE disruption. These findings on the mechanism of MVM nuclear entry would deepen our understanding of the basic biology of PVs and contribute to the development of PVs-based therapeutics.

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The nuclear pore complex (NPC) mediates the bidirectional transport of macromolecules across the nuclear envelope. Nup153 is a nucleoporin localized in the nuclear side of the NPC that was identified as a host factor required for influenza A virus (IAV) infection in two genome-wide RNA interference (RNAi) screens (Hao et al., 2008, Nature 454:890-893; König et al., 2010, Nature 463:813-817). Since IAV uses the NPC at different steps of its infective cycle, the hypothesis that Nup153 could be required for the nuclear transport of IAV mRNAs, viral proteins, and viral ribonucleoproteins (vRNPs) was tested. The main objective of this thesis was to investigate the role of Nup153 during IAV infection. This was studied by following the progression of IAV infection in HeLa cells where Nup153 was knocked down (KD) with siRNA. These cells produced less infectious particles than control cells; however, no significant changes were detected in the nuclear import of vRNPs and the influenza nucleoprotein. To explain why Nup153 KD cells produced less infectious viral particles, the sub-cellular localization of the viral proteins nucleoprotein, matrix 1, and hemagglutinin were analyzed. The results indicate deficiency in the cytoplasmic trafficking of these proteins. To understand the observed defects in IAV infected Nup153 KD cells, the cellular changes associated with transient RNAi depletion of Nup153 were studied. Strikingly, defects in the intracellular traffic of other host cell proteins and in the distribution of endocytic organelles and the cytoskeleton were found. In addition, pronounced blebbing of the plasma membrane and alterations of the nuclear and cellular architecture were observed. This is the first time that a systematic characterization of the plethora of cellular defects resulting from Nup153 RNAi has been conducted. Collectively, the results suggest that Nup153 depletion has an effect during the late stages of IAV infection and leads to defective vRNPs and/or the inadequate assembly or budding of progeny viral particles. This work adds to the cumulative evidence that nucleoporins have non-canonical roles, including roles during viral infection, and opens the possibility of considering nucleoporins as important factors for the development of future antiviral treatments.

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Influenza A virus exploits the cellular transport machinery during the early stages of infection. It enters cells by endocytosis and takes advantage of the endocytic trafficking to move towards the perinuclear region with the assistance of actin filaments and microtubules. A recent proteomic study identified vimentin as a putative interacting protein of influenza viral components. However, the role of vimentin during influenza A infection has not yet been determined. After endocytosis, the viral ribonucleopotein complexes (vRNPs), containing the RNA viral genome, the viral polymerases, and several copies of nucleoprotein, are released from late endosomes and enter the nucleus for replication. Two nuclear localization sequences (NLSs), NLS1 and NLS2, on nucleoprotein mediate the nuclear import of vRNPs. The function of NLS1 has been well studied, however, the role of NLS2 remains to be defined.This thesis has two major aims: to characterize the function of NLS2 and to determine the role of vimentin during influenza infection. For the first aim, I use a chimeric protein (5GFP) fused to NLS2, in combination with RNAi of several importin α isoforms and biochemical assays, and found that NLS2 is able to mediate the nuclear import of 5GFP by interacting with importin α1, α3, α5, and α7. NLS2 contains only a single amino acid difference at position 17 between different strains of influenza A virus, which could be either lysine (K) or arginine (R). I found that NLS2K induces more nuclear accumulation of 5GFP than NLS2R. Using site-directed mutagenesis I demonstrated that NLS2K contains two independent functional basic clusters, while NLS2R only has one. Moreover, my study also revealed that inhibiting the function of NLS1 and NLS2 impairs the nuclear import of vRNPs and further inhibits viral infection.For the second aim I followed influenza A virus infection in both vimentin null cells and vimentin RNAi knock-down cells, and found that vimentin plays a role in releasing vRNPs from endosomes. In summary, my work dissects the basic mechanisms involved in influenza A virus endocytic trafficking and nuclear import, which provide us with better insights into the viral-host interaction during influenza A virus infection.

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Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the archetype of the Baculoviridae family, is an enveloped, rod-shaped, double-stranded DNA virus that replicates in the nucleus of its host cells. Baculoviruses have been used extensively as pesticides and in biological systems. Despite their importance, the mechanism by which baculovirus deliver its genome into the nucleus has been the subject of considerable debate. Molecules
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There are three structurally and functionally distinct cytoskeleton components: actin filaments, microtubules, and intermediate filaments (IFs). Among the three cytoskeleton networks IFs are understudied; consequently, there is a lack of information about the role of IFs during early viral infection. IFs have long been known to serve structural functions within the cell, and recently, additional functions have been elucidated, including novel roles during infection by many viruses. During early infection with the parvovirus minute virus of mice (MVM), prior to viral replication, I have found that the virus induces dramatic morphological changes in mouse fibroblast cells. This observed change in the shape of infected cells may be a result of the virus using the host cytoskeleton to aid in the mechanism of intracellular trafficking. Thus, this thesis focuses on MVM and its effects on the cytoskeleton components, especially IFs, during infection. Using fluorescence microscopy techniques, I found that during early infection with MVM, after endosomal escape, the vimentin IF network was considerably altered, yielding collapsed immunofluorescence staining near the nuclear periphery. Furthermore, I found that vimentin plays an important role in the infection cycle of MVM. The number of cells successfully replicating MVM was reduced in infected cells in which the vimentin network was pharmacologically modified or in cells lacking a vimentin network; viral endocytosis, however, remained unaltered. Perinuclear accumulation of MVM-containing vesicles and progression of MVM through the endocytic pathway was reduced in cells lacking vimentin. Cells lacking vimentin, accumulated virions in early endosomes up to 2 h post-infection compared to wild type cells. Thus, my data supports a model where vimentin facilitates a productive MVM infection, presenting possibly a dual role: (1) during progression of MVM through the endocytic pathway and (2) following MVM escape from endosomes.

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The minute virus of mice prototype (MVMp) is a non-enveloped single stranded DNA virus of the family Parvoviridae. MVMp is one of the smallest viruses and shows intriguing abilities to preferentially infect and kill cancer cells (oncotropism/oncolytism), suggesting a potential for MVMp as an anti-cancer agent. Unfortunately, there is a lack of knowledge of the early events of MVMp infection cycle, such as binding to the cell surface and subsequent endocytosis. In an attempt to identify cellular partners of MVMp infection, our lab performed a mass spectrometry analysis of MVMp potential binding partners. Following this analysis, the galactose-binding lectin (galectin) 3 (Gal-3) was identified as binding partner for MVMp. Given the involvement of this extra-cellular matrix protein in the clustering and endocytosis of cell surface receptors, and its up-regulation in various aggressive tumor cells, I hypothesized that Gal-3 could play a role in MVMp cell entry, and potentially in its oncotropism. Using siRNA knockdown of Gal-3 in different cells followed by immunofluorescence microscopy analysis, I found that Gal-3 is necessary for an efficient MVMp cell entry and infection in different cells. Moreover, I discovered that the Golgi enzyme β1,6-acetylglucosaminyltransferase 5 (Mgat5), whose role is the addition of complex N-glycosylation to various cell surface receptors for Gal-3 binding, is required for MVMp infection. I also found that cancer cells with higher Gal-3 expression are more susceptible to MVMp infection than cells with lower Gal-3 levels.Next I used a combination of flow cytometry, immuno-fluorescence and transmission electron microscopy to characterize the early events of MVMp infection in various tissue-culture cell lines. My results show that many crucial parameters of the mesenchymal cell migration process regulate MVMp cellular entry and infection. I found that MVMp relies on cell protrusions to cluster at the leading edge of migrating cells rapidly after binding to the plasma membrane, from where it is subsequently endocytosed. Moreover, transmission electron microscopy analysis revealed that MVMp uses various endocytic pathways, which was confirmed using drug inhibitors of endocytosis. Finally, I found that epithelial-mesenchymal transition, an inducer of cancer cell migration, triggers MVMp infection in highly dividing non-permissive cancer cells.

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In order to promote infection, viruses must target their genomes to specific compartments within the host cell. I have used the parvovirus minute virus of mice (MVM) as a model to study the trafficking of non-enveloped viruses. Parvoviruses are single-stranded DNA viruses which replicate in the nucleus of the cell. Most viruses that replicate in the nucleus transport their genomes through nuclear pore complexes, large protein assemblies that mediate nucleocytoplasmic transport. However, previous studies have shown that MVM can induce disruption of the nuclear membranes, called the nuclear envelope (NE). This led to the hypothesis that MVM enters the nucleus by an unusual mechanism: disruption of the NE and entry through the resulting breaks.The objectives of this thesis were to: (1) characterize the effect of MVM on the NE, (2) define the molecular mechanism used by MVM to induce NE disruption, (3) determine the role of NE disruption in the MVM replication cycle, and (4) identify host proteins involved in MVM infection. I found that MVM causes small, transient disruptions of the NE early during infection. I tested the hypothesis that viral enzymatic activity is necessary for MVM-induced NE disruption and found that this was not the case. Next I tested the hypothesis that MVM hijacks a cellular program for NE breakdown, and found that MVM utilizes apoptotic proteases called caspases to facilitate these disruptions. Caspase inhibition prevents NE disruption in MVM-infected cells, reduces viral gene expression, and prevents entry of MVM capsids into the nucleus. I propose that NE disruption involving caspases facilitates parvovirus genome delivery into the nucleus. NE disruption also alters the compartmentalization of host proteins, which may be favorable for the virus. I have shown that MVM uses a novel nuclear entry strategy, unlike those previously described for any virus or cellular protein. It will be of great interest to determine whether this strategy is shared by other viruses. Parvoviruses are not considered a serious threat as human pathogens. However, they may prove useful as vectors for gene therapy. An understanding of the basic biology of parvoviruses could help in the development of parvovirus-based therapeutics.

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