Feline Calicivirus Infection Disrupts Assembly of Cytoplasmic Stress Granules and Induces G3BP1 Cleavage.

UNLABELLED: In response to stress such as virus infection, cells can stall translation by storing mRNAs away in cellular compartments called stress granules (SGs). This defense mechanism favors cell survival by limiting the use of energy and nutrients until the stress is resolved. In some cases it may also block viral propagation as viruses are dependent on the host cell resources to produce viral proteins. Human norovirus is a member of the Caliciviridae family responsible for gastroenteritis outbreaks worldwide. Previous studies on caliciviruses have identified mechanisms by which they can usurp the host translational machinery, using the viral protein genome-linked VPg, or regulate host protein synthesis through the mitogen-activated protein kinase (MAPK) pathway. Here, we examined the effect of feline calicivirus (FCV) infection on SG accumulation. We show that FCV infection impairs the assembly of SGs despite an increased phosphorylation of eukaryotic initiation factor eIF2α, a hallmark of stress pathway activation. Furthermore, SGs did not accumulate in FCV-infected cells that were stressed with arsenite or hydrogen peroxide. FCV infection resulted in the cleavage of the SG-nucleating protein Ras-GTPase activating SH3 domain-binding protein (G3BP1), which is mediated by the viral 3C-like proteinase NS6(Pro) Using mutational analysis, we identified the FCV-induced cleavage site within G3BP1, which differs from the poliovirus 3C proteinase cleavage site previously identified. Finally, we showed that NS6(Pro)-mediated G3BP1 cleavage impairs SG assembly. In contrast, murine norovirus (MNV) infection did not impact arsenite-induced SG assembly or G3BP1 integrity, suggesting that related caliciviruses have distinct effects on the stress response pathway. IMPORTANCE: Human noroviruses are a major cause of viral gastroenteritis, and it is important to understand how they interact with the infected host cell. Feline calicivirus (FCV) and murine norovirus (MNV) are used as models to understand norovirus biology. Recent studies have suggested that the assembly of stress granules is central in orchestrating stress and antiviral responses to restrict viral replication. Overall, our study provides the first insight on how caliciviruses impair stress granule assembly by targeting the nucleating factor G3BP1 via the viral proteinase NS6(Pro) This work provides new insights into host-pathogen interactions that regulate stress pathways during FCV infection.

Functional analysis of Kaposi's sarcoma-associated herpesvirus vFLIP expression reveals a new mode of IRES-mediated translation.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus, the etiological agent of Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). One of the key viral proteins that contributes to tumorigenesis is vFLIP, a viral homolog of the FLICE inhibitory protein. This KSHV protein interacts with the NFκB pathway to trigger the expression of antiapoptotic and proinflammatory genes and ultimately leads to tumor formation. The expression of vFLIP is regulated at the translational level by an internal ribosomal entry site (IRES) element. However, the precise mechanism by which ribosomes are recruited internally and the exact location of the IRES has remained elusive. Here we show that a 252-nt fragment directly upstream of vFLIP, within a coding region, directs translation. We have established its RNA structure and demonstrate that IRES activity requires the presence of eIF4A and an intact eIF4G. Furthermore, and unusually for an IRES, eIF4E is part of the complex assembled onto the vFLIP IRES to direct translation. These molecular interactions define a new paradigm for IRES-mediated translation.

Polypyrimidine tract binding protein functions as a negative regulator of feline calicivirus translation.

BACKGROUND: Positive strand RNA viruses rely heavily on host cell RNA binding proteins for various aspects of their life cycle. Such proteins interact with sequences usually present at the 5' or 3' extremities of the viral RNA genome, to regulate viral translation and/or replication. We have previously reported that the well characterized host RNA binding protein polypyrimidine tract binding protein (PTB) interacts with the 5'end of the feline calicivirus (FCV) genomic and subgenomic RNAs, playing a role in the FCV life cycle. PRINCIPAL FINDINGS: We have demonstrated that PTB interacts with at least two binding sites within the 5'end of the FCV genome. In vitro translation indicated that PTB may function as a negative regulator of FCV translation and this was subsequently confirmed as the translation of the viral subgenomic RNA in PTB siRNA treated cells was stimulated under conditions in which RNA replication could not occur. We also observed that PTB redistributes from the nucleus to the cytoplasm during FCV infection, partially localizing to viral replication complexes, suggesting that PTB binding may be involved in the switch from translation to replication. Reverse genetics studies demonstrated that synonymous mutations in the PTB binding sites result in a cell-type specific defect in FCV replication. CONCLUSIONS: Our data indicates that PTB may function to negatively regulate FCV translation initiation. To reconcile this with efficient virus replication in cells, we propose a putative model for the function of PTB in the FCV life cycle. It is possible that during the early stages of infection, viral RNA is translated in the absence of PTB, however, as the levels of viral proteins increase, the nuclear-cytoplasmic shuttling of PTB is altered, increasing the cytoplasmic levels of PTB, inhibiting viral translation. Whether PTB acts directly to repress translation initiation or via the recruitment of other factors remains to be determined but this may contribute to the stimulation of viral RNA replication via clearance of ribosomes from viral RNA.

Murine norovirus-1 cell entry is mediated through a non-clathrin-, non-caveolae-, dynamin- and cholesterol-dependent pathway.

For many viruses, endocytosis and exposure to the low pH within acidic endosomes is essential for infection. It has previously been reported that feline calicivirus uses clathrin-mediated endocytosis for entry into mammalian cells. Here, we report that infection of RAW264.7 macrophages by the closely related murine norovirus-1 (MNV-1) does not require the clathrin pathway, as infection was not inhibited by expression of dominant-negative Eps15 or by knockdown of the adaptin-2 complex. Further, infection was not inhibited by reagents that raise endosomal pH. RAW264.7 macrophages were shown not to express caveolin, and flotillin depletion did not inhibit infection, suggesting that caveolae and the flotillin pathway are not required for cell entry. However, MNV-1 infection was inhibited by methyl-beta-cyclodextrin and the dynamin inhibitor, dynasore. Addition of these drugs to the cells after a period of virus internalization did not inhibit infection, suggesting the involvement of cholesterol-sensitive lipid rafts and dynamin in the entry mechanism. Macropinocytosis (MPC) was shown to be active in RAW264.7 macrophages (as indicated by uptake of dextran) and could be blocked by 5-(N-ethyl-N-isopropyl) amiloride (EIPA), which is reported to inhibit this pathway. However, infection was enhanced in the presence of EIPA. Similarly, actin disruption, which also inhibits MPC, resulted in enhanced infection. These results suggest that MPC could contribute to virus degradation or that inhibition of MPC could lead to the upregulation of other endocytic pathways of virus uptake.

Structural insights into the transcriptional and translational roles of Ebp1.

The ErbB3-binding protein 1 (Ebp1) is an important regulator of transcription, affecting eukaryotic cell growth, proliferation, differentiation and survival. Ebp1 can also affect translation and cooperates with the polypyrimidine tract-binding protein (PTB) to stimulate the activity of the internal ribosome entry site (IRES) of foot-and-mouth disease virus (FMDV). We report here the crystal structure of murine Ebp1 (p48 isoform), providing the first glimpse of the architecture of this versatile regulator. The structure reveals a core domain that is homologous to methionine aminopeptidases, coupled to a C-terminal extension that contains important motifs for binding proteins and RNA. It sheds new light on the conformational differences between the p42 and p48 isoforms of Ebp1, the disposition of the key protein-interacting motif ((354)LKALL(358)) and the RNA-binding activity of Ebp1. We show that the primary RNA-binding site is formed by a Lys-rich motif in the C terminus and mediates the interaction with the FMDV IRES. We also demonstrate a specific functional requirement for Ebp1 in FMDV IRES-directed translation that is independent of a direct interaction with PTB.

Feline calicivirus replication: requirement for polypyrimidine tract-binding protein is temperature-dependent.

The interaction of host-cell nucleic acid-binding proteins with the genomes of positive-stranded RNA viruses is known to play a role in the translation and replication of many viruses. To date, however, the characterization of similar interactions with the genomes of members of the family Caliciviridae has been limited to in vitro binding analysis. In this study, Feline calicivirus (FCV) has been used as a model system to identify and characterize the role of host-cell factors that interact with the viral RNA. It was demonstrated that polypyrimidine tract-binding protein (PTB) interacts specifically with the 5' sequences of the FCV genomic and subgenomic RNAs. Using RNA interference it was shown that PTB is required for efficient FCV replication in a temperature-dependent manner. siRNA-mediated knockdown of PTB resulted in a 15- to 100-fold reduction in virus titre, as well as a concomitant reduction in viral RNA and protein synthesis at 32 degrees C. In addition, virus-induced cytopathic effect was significantly delayed as a result of an siRNA-mediated reduction in PTB levels. A role for PTB in the calicivirus life cycle was more apparent at temperatures above and below 37 degrees C, fitting with the hypothesis that PTB functions as an RNA chaperone, potentially aiding the folding of RNA into functional structures. This is the first functional demonstration of a host-cell protein interacting with a calicivirus RNA.

Stabilized baculovirus vector expressing a heterologous gene and GP64 from a single bicistronic transcript.

The efficient scale-up of recombinant protein production in insect-cell bioreactors using baculovirus expression vectors is hampered by reductions in yield with increasing viral passage, the so-called passage effect. This phenomenon is characterized by the generation and subsequent accumulation of defective interfering baculoviruses (DIs), which interfere with the replication of genomically intact virus. A novel baculovirus expression vector is presented equipped with a bicistronic expression cassette that allows the simultaneous expression of the recombinant gene (GFP, first cistron) and an essential baculovirus gene (GP64, second cistron) from a single messenger RNA (mRNA). The translation of GP64 is mediated by an internal ribosome entry site (IRES) element from Rhopalosiphum padi virus (RhPV) while the native GP64 gene is deleted. In this way, a dominant selection pressure is placed on the entire bicistronic mRNA and hence on the maintenance of the foreign gene. The bicistronic expression vector was superior to the control baculovirus vector in that GFP expression remained at much higher levels upon continued virus passage. The versatility of this stabilized vector was demonstrated by its ability to propagate in a number of cell lines including Sf21, Sf9 and High Five cells. This novel baculovirus vector is especially valuable for large-scale recombinant protein production in insect-cell bioreactors where the number of viral passages is high.