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p53 is implicated in several cellular pathways such as induction of cell-cycle arrest, differentiation, senescence, and apoptosis. p53 is activated by a broad range of stress signals, including viral infections. While some viruses activate p53, others induce its inactivation, and occasionally p53 is differentially modulated during the replicative cycle. During calicivirus infections, apoptosis is required for virus exit and spread into the host; yet, the role of p53 during infection is unknown. By confocal microscopy, we found that p53 associates with FCV VP1, the protease-polymerase NS6/7, and the dsRNA. This interaction was further confirmed by proximity ligation assays, suggesting that p53 participates in the FCV replication. Knocked-down of p53 expression in CrFK cells before infection, resulted in a strong reduction of the non-structural protein levels and a decrease of the viral progeny production. These results indicate that p53 is associated with the viral replication complex and is required for an efficient FCV replication., Highlights • Host cell p53 protein levels and subcellular localization do not change during FCV infection. • Host cell p53 associates with FCV major viral capsid protein VP1, protease-polymerase NS6/7, and the dsRNA in FCV infected cells. • Host cell p53 is required for a FCV replication.

Alphavirus genome replication is carried out by the viral replication complex inside modified membrane structures called spherules. The viral nonstructural protein 1 (nsP1) is the only membrane-associated protein that anchors the replication complex to the cellular membranes. Although an internal amphipathic helix of nsP1 is critical for membrane association, the mechanism of nsP1 interaction with membranes and subsequent membrane reorganization is not well understood. We studied the membrane interaction of chikungunya virus (CHIKV) nsP1 and show that both the CHIKV nsP1 protein and the amphipathic peptide specifically bind to negatively charged phospholipid vesicles. Using cryo-electron microscopy, we further show that nsP1 forms a contiguous coat on lipid vesicles and induces structural reorganization, while the amphipathic peptide alone failed to deform the membrane bilayer. This suggests that although amphipathic helix of nsP1 is required for initial membrane binding, the remaining cytoplasmic domain of nsP1 is involved in the subsequent membrane reorganization.

Here we review the evolving story of the coronavirus endoribonuclease (EndoU). Coronavirus EndoU is encoded within the sequence of nonstructural protein (nsp) 15, which was initially identified as a component of the viral replication complex. Biochemical and structural studies revealed the enzymatic nature of nsp15/EndoU, which was postulated to be essential for the unique replication cycle of viruses in the order Nidovirales. However, the role of nsp15 in coronavirus replication was enigmatic as EndoU-deficient coronaviruses were viable and replicated to near wild-type virus levels in fibroblast cells. A breakthrough in our understanding of the role of EndoU was revealed in recent studies, which showed that EndoU mediates the evasion of viral double-stranded RNA recognition by host sensors in macrophages. This new discovery of nsp15/EndoU function leads to new opportunities for investigating how a viral EndoU contributes to pathogenesis and exploiting this enzyme for therapeutics and vaccine design against pathogenic coronaviruses.

Human papillomaviruses are causative agents in several human diseases ranging from genital warts to ano-genital and oropharyngeal cancers. Currently only symptoms of HPV induced disease are treated; there are no antivirals available that directly target the viral life cycle. Previously, we determined that the cellular protein TopBP1 interacts with the HPV16 replication/transcription factor E2. This E2-TopBP1 interaction is essential for optimal E1-E2 DNA replication and for the viral life cycle. The drug calcein disrupts the interaction of TopBP1 with itself and other host proteins to promote cell death. Here we demonstrate that calcein blocks HPV16 E1-E2 DNA replication via blocking the viral replication complex forming at the origin of replication. This occurs at non-toxic levels of calcein and demonstrates specificity as it does not block the ability of E2 to regulate transcription. We propose that calcein or derivatives could be developed as an anti-HPV therapeutic.

Double-stranded RNA (dsRNA) is an important type of RNA that plays essential roles in diverse cellular processes in eukaryotic organisms and a hallmark in infections by positive-sense RNA viruses. Currently, no in vivo technology has been developed for visualizing dsRNA in living cells. Here, we report a dsRNA binding-dependent fluorescence complementation (dRBFC) assay that can be used to efficiently monitor dsRNA distribution and dynamics in vivo. The system consists of two dsRNA-binding proteins, which are fused to the N- and C-terminal halves of the yellow fluorescent protein (YFP). Binding of the two fusion proteins to a common dsRNA brings the split YFP halves in close proximity, leading to the reconstitution of the fluorescence-competent structure and restoration of fluorescence. Using this technique, we were able to visualize the distribution and trafficking of the replicative RNA intermediates of positive-sense RNA viruses in living cells.

Like other positive-strand RNA viruses, plant potyviruses assemble viral replication complexes (VRCs) on modified cellular membranes. Potyviruses encode two membrane proteins, 6K2 and P3. The former is known to play pivotal roles in the formation of membrane-associated VRCs. However, P3 remains to be one of the least characterized potyviral proteins. The P3 cistron codes for P3 as well as P3N-PIPO which results from RNA polymerase slippage. In this study, we show that the P3N-PIPO of Turnip mosaic virus (TuMV) is required for viral cell-to-cell movement but not for viral replication. We demonstrate that the C-terminal region of P3 (P3C) is indispensable for P3 to form cytoplasmic punctate inclusions and target VRCs. We reveal that TuMV mutants that lack P3C are replication-defective. Taken together, these data suggest that the P3 cistron has two distinct functions: P3N-PIPO as a dedicated movement protein and P3 as an essential component of the VRC.

Replication of Tomato ringspot virus (ToRSV) occurs in association with endoplasmic reticulum (ER)-derived membranes. We have previously shown that the putative nucleotide triphosphate-binding protein (NTB) of ToRSV is an ER-targeted protein and that an intermediate polyprotein containing the domains for NTB and for the genome-linked viral protein (VPg) is associated with the replication complex. We now report the detection of a 95-kDa polyprotein that contains the domains for the RNA-dependent RNA polymerase (Pol), the proteinase (Pro) and the VPg. This polyprotein appears to be a truncated version of the full-length 111-kDa VPg-Pro-Pol polyprotein and was termed VPg-Pro-Pol′. A subpopulation of VPg-Pro-Pol′ was peripherally associated with ER-derived membranes active in viral replication. However, the VPg, Pro and Pol domains did not target to membranes in the absence of viral infection. We propose a model in which VPg-Pro-Pol′ is brought to the site of replication through interaction with a viral membrane protein.

The hepatitis C virus (HCV) nonstructural 5A (NS5A) phosphoprotein has been intensely studied due to its ability to subvert the host interferon-induced antiviral response. However, more recent studies suggest that it may also play an important regulatory role in HCV RNA replication as well as modulate host intracellular signaling pathways. Phosphorylation of NS5A appears to be a highly regulated process and several cellular protein kinases responsible for NS5A phosphorylation have been identified in vitro. Studies utilizing the HCV replicon cell culture system have suggested a provocative role for the differential phosphorylation of NS5A in the regulation of viral RNA replication through its association with the viral replication complex, including several host cell factors. Importantly, recent in vivo data linking loss of NS5A hyperphosphorylation to non-productive HCV replication in the chimpanzee model have provided high validation for targeting the cellular kinases involved, particularly the kinases responsible for NS5A phosphorylation, for antiviral therapeutic intervention. Understanding the process of NS5A phosphorylation and the definite identification of the culprit cellular protein kinase(s) will shed light on the mechanisms of HCV RNA replication and/or pathogenesis.

The Sindbis virus (SIN) nonstructural protein nsP4 possesses the RNA-dependent RNA polymerase activity required for the replication of the SIN genome and transcription of a subgenomic mRNA during infection. Isolation of this protein from other viral components of the RNA synthetic complex allowed the characterization of template requirements for nsP4-mediated genome replication. The major findings of this study are: (i) in the absence of other viral proteins nsP4 is capable of copying SIN plus- and minus-strand templates, but does not transcribe subgenomic RNA; (ii) mutations in the 3' conserved sequence element and poly(A) tail of the plus-strand template prevent nsP4-mediated de novo initiation of minus-strand RNA synthesis; (iii) nsP4-dependent terminal addition of nucleotides occurs on template RNA possessing certain mutations in the 3'CSE and polyadenylate tail ; (iv) nsP4 is capable of minus-strand synthesis independent of the sequence at the 5' end of the template; (v) an A-U rich sequence in the 3'CSE represents a binding site for a replicase component, probably nsP4; (vi) plus-strand genomic RNA synthesis is dependent on the 3' end of the minus-strand template. These studies begin to define the specific interactions with the viral RNA templates mediated by individual components of the viral replication complex and suggest a model for ternary complex formation during the initiation of minus-strand RNA synthesis.

Autophagy is a cellular process used to eliminate intracellular pathogens. Many viruses however are able to manipulate this cellular process for their own advantage. Here we demonstrate that Mouse Norovirus (MNV) infection induces autophagy but does not appear to utilise the autophagosomal membrane for establishment and formation of the viral replication complex. We have observed that MNV infection results in lipidation and recruitment of LC3 to the autophagosome membrane but prevents subsequent fusion of the autophagosomes with lysosomes, as SQSTM1 (an autophagy receptor) accumulates and Lysosome-Associated Membrane Protein1 is sequestered to the MNV replication complex (RC) rather than to autophagosomes. We have additionally observed that chemical modulation of autophagy differentially affects MNV replication. From this study we can conclude that MNV infection induces autophagy, however suppresses the final maturation step of this response, indicating that autophagy induction contributes to MNV replication independently of RC biogenesis.

The small hydrophobic protein 3AB of the picornaviruses, encompassing the replication primer 3B, has been suggested to anchor the viral replication complex to membranes. For hepatitis A virus (HAV) 3AB, we have previously demonstrated its ability to form stable homodimers, to bind to membranes, and to interact specifically with RNA, implicating its multiple involvement in viral replication. In the present report, we show that HAV 3AB additionally interacts with HAV protein 3CD, a feature also described for the corresponding polypeptide of poliovirus. By assessing the interactions of three deletion mutants, distinct domains of HAV 3AB were mapped. The hydrophobic domain and the 3B moiety were found to be essential for the 3AB interaction with 3CD. Both electrostatic and hydrophobic forces are involved in this interaction. The cluster of charged amino acid residues at the C terminus of 3A seems to determine the specificity of 3AB interaction with RNA structures formed at either terminus of the HAV genome. Furthermore, our data implicate that 3A can interact with HAV RNA. Compared with poliovirus 3AB, which by itself is a nonspecific RNA-binding protein, HAV 3AB specifically recognizes HAV RNA structures that might be of relevance for initiation of viral RNA replication.

The structural proteins of poliovirus can assemble into a series of different configurations (capsid-related particles, CRP). Only some seem to be true capsid precursors and the role of most CRP in morphogenesis is unclear. We used electron microscopic immunocytochemistry with monoclonal antibodies recognizing different CRP [protomers, pentamers, 65S empty capsids (EC), 74S-EC, and virions] to locate CRP in subcellular fractions containing virus-induced vesicles associated with the viral replication complex. We found pentamer antigenic CRP to be associated with the replication complex. The same pentamer antigenicity was exhibited by novel, “capsid-like” structures attached to the surface of the virus-induced vesicles. Upon solubilization of the vesicular fraction, mainly 65S-EC and only negligible amounts of pentamers were found by sucrose gradient analysis and by immunoprecipitation. We show that the pentamer antigenic particles are converted into 65S-EC when their membranous support is dissolved. We propose that the vesicular membrane prevents the assembly of 65S-EC and keeps the pentamer antigenic CRP in the appropriate concentration and configuration for association with the nascent progeny RNA.

The adenovirus DNA binding protein (DBP) functions in a number of diverse processes that include viral DNA replication and host range determination. A subpopulation of DBP has been found to exist in a high-molecular-weight (HMWDBP) complex of greater than 650 kDa. This complex was stable to high salt and low levels of detergent in the presence of DTT at 4°. While the complex prepared in high salt was devoid of nucleic acid, it retained its ability to bind to ssDNA. Electron microscopic (EM) analysis in the presence of M13 ssDNA verified its interaction with ssDNA. The complex exists as particles approximately 25-30 nm based on EM analysis of negatively stained samples in the absence of DNA. No viral replication components were detected in the HMWDBP complex, suggesting that it is not a viral replication complex. The HMWDBP complex prepared under a variety of infection conditions contained a consistent set of proteins. HMWDBP complex was also detected in the absence of viral infection in gmDBP6 cells (a HeLa cell derivative that expresses DBP from the dexamethasone-inducible MMTV promoter). These results suggest that the components of the complex, other than DBP, are host encoded.

Using high resolution electron microscopic autoradiography and immunocytochemistry with monoclonal antibodies against poliovirus proteins of the P2 genomic region, the location of these proteins in respect to the virus-induced vesicle formation and the viral RNA synthesis was followed during the viral replication cycle. It was found that P2 proteins become rER associated soon after their synthesis. At the site of protein and rER interaction, electron-dense patches appear. Simultaneously, membrane protrusions grow and form vesicles which finally budd off, carrying the patches on their outer surface. As shown by autoradiography, these patches are the site of viral RNA replication and, therefore, they represent the poliovirus replication complex. The vesicles with the replication complex, including replicating and replicated viral RNA, move away from the rER to form a continuously growing vesiculated area in the center of the infected cell, where virus maturation takes place. A likely function of the 2C protein is to attach the replication complex, or some of its components, to the vesicular membranes.

Purification of the putative cowpea mosaic virus (CPMV) RNA replicase previously started with an RNA-dependent RNA polymerase activity which had been solubilized from a crude membrane fraction of CPMV-infected cowpea leaves by extraction with a Mg2+-deficient buffer. This led to the identification of a host-encoded, 130,000-dalton monomeric enzyme, the activity of which was highly enhanced upon infection. As the role of this enzyme in viral replication was questionable, we reverted to the template-associated RNA-dependent RNA polymerase in the crude membrane fraction in order to characterize in detail its in vitro products. We now demonstrate that the crude membrane fraction of CPMV-infected cowpea leaves harbors two functionally different, RNA-dependent RNA polymerase activities that are both associated with endogenous template RNA and can be separated from each other without affecting their distinct properties. One of the RNA polymerase activities was specific for CPMV-infected leaves and constituted a CPMV RNA replication complex; enzyme activity in vitro allowed for the completion of nascent chains initiated in vivo. Full-length viral RNAS (B- and M-RNA) were produced which were recovered mainly in double-stranded form. Solution- and Northern blot hybridization demonstrated that the in vitro-labeled RNA chains were viral RNAs of positive polarity. The other template-associated RNA-dependent RNA polymerase activity occurred in both uninfected and infected leaves and transcribed in vitro endogenous plant and viral RNAs only into small RNAs (4-5 S) of negative polarity. Northern blot analysis revealed the RNA products of plant origin to be transcribed from two major RNA templates of approximately 0.26 and 0.14 X 106 daltons, respectively. Washing of the crude membrane fraction in a Mg2+-deficient buffer did accomplish the complete release of the low-molecular-weight RNA-synthesizing activity but did not solubilize the CPMV RNA replication complex. We tentatively conclude that the RNA-dependent RNA polymerase which has previously been purified from the buffer-soluble fraction and has been identified as a host-encoded enzyme is not involved in viral RNA replication. Solubilization of the viral replication complex was achieved with Triton X-100. By taking advantage of the characteristic conformation of the replication complex, we applied Sepharose 2B chromatography as a highly efficient and simple means for purifying the detergent-solubilized complex. We anticipate that this purification step should also be applicable to replication complexes of other RNA viruses.

Infection of interferon-treated L cells by mengovirus increased a ribonuclease activity in a sedimentable fraction known to contain the viral replication complex. The enhanced in vitro phosphorylation of a 67,000 M r protein was also observed in the same fraction.

Two DNA-binding proteins, designated FP11 and FP14, have been purified from the viral replication complex which forms in the cytoplasm of HeLa cells during productive infection with vaccina virus. Polypeptide FP11 has an apparent molecular weight of 34,000 (by SDS-PAGE) and binds strongly to denatured DNA-cellulose columns, eluting as a narrow peak centered at 0.25 M NaCl. This polypeptide represents 30–40% of the [3H]lysine but only 17% of the [3H]methionine which can be incorporated into the complex. Polypeptide FP14 has an apparent molecular weight of 28,000 (by SDS-PAGE) and elutes from denatured DNA-cellulose as a relatively broad peak over the range 0.45–0.80 M NaCl. FP14 accounts for approximately 10% of incorporated radioactivity, regardless of whether lysine or methionine is employed as the radioactive precursor.