Dynamic interplay between the co-opted Fis1 mitochondrial fission protein and membrane contact site proteins in supporting tombusvirus replication

Plus-stranded RNA viruses have limited coding capacity and have to co-opt numerous pro-viral host factors to support their replication. Many of the co-opted host factors support the biogenesis of the viral replication compartments and the formation of viral replicase complexes on subverted subcellular membrane surfaces. Tomato bushy stunt virus (TBSV) exploits peroxisomal membranes, whereas the closely-related carnation Italian ringspot virus (CIRV) hijacks the outer membranes of mitochondria. How these organellar membranes can be recruited into pro-viral roles is not completely understood. Here, we show that the highly conserved Fis1 mitochondrial fission protein is co-opted by both TBSV and CIRV via direct interactions with the p33/p36 replication proteins. Deletion of FIS1 in yeast or knockdown of the homologous Fis1 in plants inhibits tombusvirus replication. Instead of the canonical function in mitochondrial fission and peroxisome division, the tethering function of Fis1 is exploited by tombusviruses to facilitate the subversion of membrane contact site (MCS) proteins and peroxisomal/mitochondrial membranes for the biogenesis of the replication compartment. We propose that the dynamic interactions of Fis1 with MCS proteins, such as the ER resident VAP tethering proteins, Sac1 PI4P phosphatase and the cytosolic OSBP-like oxysterol-binding proteins, promote the formation and facilitate the stabilization of virus-induced vMCSs, which enrich sterols within the replication compartment. We show that this novel function of Fis1 is exploited by tombusviruses to build nuclease-insensitive viral replication compartment.     

Authentic In Vitro Replication of Two Tombusviruses in Isolated Mitochondrial and Endoplasmic Reticulum Membranes

Replication of plus-stranded RNA viruses takes place on membranous structures derived from various organelles in infected cells. Previous works with Tomato bushy stunt tombusvirus (TBSV) revealed the recruitment of either peroxisomal or endoplasmic reticulum (ER) membranes for replication. In case of Carnation Italian ringspot tombusvirus (CIRV), the mitochondrial membranes supported CIRV replication. In this study, we developed ER and mitochondrion-based in vitro tombusvirus replication assays. Using purified recombinant TBSV and CIRV replication proteins, we showed that TBSV could use the purified yeast ER and mitochondrial preparations for complete viral RNA replication, while CIRV preferentially replicated in the mitochondrial membranes. The viral RNA became partly RNase resistant after ∼40 to 60 min of incubation in the purified ER and mitochondrial preparations, suggesting that assembly of TBSV and CIRV replicases could take place in the purified ER and mitochondrial membranes in vitro. Using chimeric and heterologous combinations of replication proteins, we showed that multiple domains within the replication proteins are involved in determining the efficiency of tombusvirus replication in the two subcellular membranes. Altogether, we demonstrated that TBSV is less limited while CIRV is more restricted in utilizing various intracellular membranes for replication. Overall, the current work provides evidence that tombusvirus replication could occur in vitro in isolated subcellular membranes, suggesting that tombusviruses have the ability to utilize alternative organellar membranes during infection that could increase the chance of mixed virus replication and rapid evolution during coinfection.     

96 shRNAs designed for maximal coverage of HIV-1 variants

Background

Extensive studies of primary infection are crucial to our understanding of the course of HIV disease. In SIV-infected macaques, a model closely mimicking HIV pathogenesis, we used a combination of three markers -- viral RNA, 2LTR circles and viral DNA -- to evaluate viral replication and dissemination simultaneously in blood, secondary lymphoid tissues, and the gut during primary and chronic infections. Subsequent viral compartmentalization in the main target cells of the virus in peripheral blood during the chronic phase of infection was evaluated by cell sorting and viral quantification with the three markers studied.

Results

The evolutions of viral RNA, 2LTR circles and DNA levels were correlated in a given tissue during primary and early chronic infection. The decrease in plasma viral load principally reflects a large decrease in viral replication in gut-associated lymphoid tissue (GALT), with viral RNA and DNA levels remaining stable in the spleen and peripheral lymph nodes. Later, during chronic infection, a progressive depletion of central memory CD4+ T cells from the peripheral blood was observed, accompanied by high levels of viral replication in the cells of this subtype. The virus was also found to replicate at this point in the infection in naive CD4+ T cells. Viral RNA was frequently detected in monocytes, but no SIV replication appeared to occur in these cells, as no viral DNA or 2LTR circles were detected.

Conclusion

We demonstrated the persistence of viral replication and dissemination, mostly in secondary lymphoid tissues, during primary and early chronic infection. During chronic infection, the central memory CD4+ T cells were the major site of viral replication in peripheral blood, but viral replication also occurred in naive CD4+ T cells. The role of monocytes seemed to be limited to carrying the virus as a cargo because there was an observed lack of replication in these cells. These data may have important implications for the targeting of HIV treatment to these diverse compartments.     

Flock House Virus RNA Replicates on Outer Mitochondrial Membranes in Drosophila Cells

The identification and characterization of host cell membranes essential for positive-strand RNA virus replication should provide insight into the mechanisms of viral replication and potentially identify novel targets for broadly effective antiviral agents. The alphanodavirus flock house virus (FHV) is a positive-strand RNA virus with one of the smallest known genomes among animal RNA viruses, and it can replicate in insect, plant, mammalian, and yeast cells. To investigate the localization of FHV RNA replication, we generated polyclonal antisera against protein A, the FHV RNA-dependent RNA polymerase, which is the sole viral protein required for FHV RNA replication. We detected protein A within 4 h after infection of Drosophila DL-1 cells and, by differential and isopycnic gradient centrifugation, found that protein A was tightly membrane associated, similar to integral membrane replicase proteins from other positive-strand RNA viruses. Confocal immunofluorescence microscopy and virus-specific, actinomycin D-resistant bromo-UTP incorporation identified mitochondria as the intracellular site of protein A localization and viral RNA synthesis. Selective membrane permeabilization and immunoelectron microscopy further localized protein A to outer mitochondrial membranes. Electron microscopy revealed 40- to 60-nm membrane-bound spherical structures in the mitochondrial intermembrane space of FHV-infected cells, similar in ultrastructural appearance to tombusvirus- and togavirus-induced membrane structures. We concluded that FHV RNA replication occurs on outer mitochondrial membranes and shares fundamental biochemical and ultrastructural features with RNA replication of positive-strand RNA viruses from other families.     

The pro-apoptotic Bcl-2 family member tBid localizes to mitochondrial contact sites

Background

Following cleavage by caspase 8, the C-terminus of Bid translocates from the cytosol to the mitochondria that is dependent upon structures formed by the mitochondrial-specific lipid cardiolipin. Once associated with mitochondria, truncated Bid (tBid) causes the potent release of cytochrome c, endonuclease G, and smac.

Results

We investigated whether tBid localizes specifically to the contact sites of mitochondria purported to be rich in cardiolipin. A point mutation changing the glycine at position 94 to glutamic acid in the BH3 domain of tBid (tBid_G94E) was principally used because mitochondria treated with this mutant tBid displayed better preservation of the outer membrane than those treated with wild type tBid. Additionally, tBid_G94E lowers the cytochrome c releasing activity of tBid without affecting its targeting to mitochondria. Electron microscope tomography coupled with immunogold labeling was used as a new hybrid technique to investigate the three-dimensional distributions of tBid and tBid_G94E around the mitochondrial periphery. The statistics of spatial point patterns was used to analyze the association of these proteins with contact sites.

Conclusions

Immunoelectron tomography with statistical analysis confirmed the preferential association of tBid with mitochondrial contact sites. These findings link these sites with cardiolipin in tBid targeting and suggest a role for Bcl-2 family members in regulating the activity of contact sites in relation to apoptosis. We propose a mechanism whereby Bcl-2 proteins alter mitochondrial function by disrupting cardiolipin containing contact site membranes.     

Analysis of the sorting signals directing NADH-cytochrome b5 reductase to two locations within yeast mitochondria.

Mitochondrial NADH-cytochrome b5 reductase (Mcr1p) is encoded by a single nuclear gene and imported into two different submitochondrial compartments: the outer membrane and the intermembrane space. We now show that the amino-terminal 47 amino acids suffice to target the Mcr1 protein to both destinations. The first 12 residues of this sequence function as a weak matrix-targeting signal; the remaining residues are mostly hydrophobic and serve as an intramitochondrial sorting signal for the outer membrane and the intermembrane space. A double point mutation within the hydrophobic region of the targeting sequence virtually abolishes the ability of the precursor to be inserted into the outer membrane but increases the efficiency of transport into the intermembrane space. Import of Mcr1p into the intermembrane space requires an electrochemical potential across the inner membrane, as well as ATP in the matrix, and is strongly impaired in mitochondria lacking Tom7p or Tim11p, two components of the translocation machineries in the outer and inner mitochondrial membranes, respectively. These results indicate that intramitochondrial sorting of the Mcr1 protein is mediated by specific interactions between the bipartite targeting sequence and components of both mitochondrial translocation systems.     

Mitochondrial targeting and membrane anchoring of a viral replicase in plant and yeast cells

Replication of the Carnation Italian ringspot virus genomic RNA in plant cells occurs in multivesicular bodies which develop from the mitochondrial outer membrane during infection. ORF1 in the viral genome encodes a 36-kDa protein, while ORF2 codes for the 95-kDa replicase by readthrough of the ORF1 stop codon. We have shown previously that the N-terminal part of ORF1 contains the information leading to vesiculation of mitochondria and that the 36-kDa protein localizes to mitochondria. Using infection, in vivo expression of green fluorescent protein fusions in plant and yeast cells, and in vitro mitochondrial integration assays, we demonstrate here that both the 36-kDa protein and the complete replicase are targeted to mitochondria and anchor to the outer membrane with the N terminus and C terminus on the cytosolic side. Analysis of deletion mutants indicated that the anchor sequence is likely to correspond approximately to amino acids 84 to 196, containing two transmembrane domains. No evidence for a matrix-targeting presequence was found, and the data suggest that membrane insertion of the viral proteins is mediated by an import receptor-independent signal-anchor mechanism relying on the two transmembrane segments and multiple recognition signals present in the N-terminal part of ORF1.     

Involvement of p32 and microtubules in alteration of mitochondrial functions by rubella virus

The interaction of the rubella virus (RV) capsid (C) protein and the mitochondrial p32 protein is believed to participate in virus replication. In this study, the physiological significance of the association of RV with mitochondria was investigated by silencing p32 through RNA interference. It was demonstrated that downregulation of p32 interferes with microtubule-directed redistribution of mitochondria in RV-infected cells. However, the association of the viral C protein with mitochondria was not affected. When cell lines either pretreated with respiratory chain inhibitors or cultivated under (mild) hypoxic conditions were infected with RV, viral replication was reduced in a time-dependent fashion. Additionally, RV infection induces increased activity of mitochondrial electron transport chain complex III, which was associated with an increase in the mitochondrial membrane potential. These effects are outstanding among the examples of mitochondrial alterations caused by viruses. In contrast to the preferential localization of p32 to the mitochondrial matrix in most cell lines, RV-permissive cell lines were characterized by an almost exclusive membrane association of p32. Conceivably, this contributes to p32 function(s) during RV replication. The data presented suggest that p32 fulfills an essential function for RV replication in directing trafficking of mitochondria near sites of viral replication to meet the energy demands of the virus.     

Effects of turnip crinkle virus infection on the structure and function of mitochondria and expression of stress proteins in turnips

We have investigated the effect of turnip crinkle virus (TCV) infection on mitochondrial structure and function in turnips ( Brassica rapa cultivar Just Right ). TCV infection resulted in plants with small, mottled leaves with severely crinkled edges, and in a 46% reduction in storage root mass. TCV infection resulted in specific vesicularization of mitochondrial outer membranes where TCV replication is thought to occur, with no apparent affect on other cellular membrane systems. Immunoblot analysis of mitochondrial proteins from storage roots indicated that the TCV p28 protein, which is essential for viral replication, was associated with mitochondria and that mitochondrial heat shock protein 70 and cpn60 levels increased upon TCV infection. Isolation of mitochondrial outer membranes further showed TCV p28 protein enrichment in the outer membrane as compared with total mitochondrial proteins or total cellular proteins. Analysis of mitochondrial electron transport chain activities indicated that TCV infection resulted in a 54% decrease in exogenous NADH-dependent oxygen uptake and a 8% decrease in succinate-dependent oxygen uptake. Together these results indicate that TCV infection induces a stress response in mitochondria and a reduction in the ability of mitochondria to supply adenosine 5'-triphosphate to the cell.     

Flock house virus RNA polymerase is a transmembrane protein with amino-terminal sequences sufficient for mitochondrial localization and membrane insertion

Localization of RNA replication to intracellular membranes is a universal feature of positive-strand RNA viruses. Replication complexes of flock house virus (FHV), the best-studied alphanodavirus, are located on outer mitochondrial membranes in infected Drosophila melanogaster cells and are associated with the formation of membrane-bound spherules, similar to structures found for many other positive-strand RNA viruses. To further study FHV replication complex formation, we investigated the subcellular localization, membrane association, and membrane topology of protein A, the FHV RNA-dependent RNA polymerase, in the yeast Saccharomyces cerevisiae, a host able to support full FHV RNA replication and virion formation. Confocal immunofluorescence revealed that protein A localized to mitochondria in yeast, as in Drosophila cells, and that this mitochondrial localization was independent of viral RNA synthesis. Nycodenz gradient flotation and dissociation assays showed that protein A behaved as an integral membrane protein, a finding consistent with a predicted N-proximal transmembrane domain. Protease digestion and selective permeabilization after differential epitope tagging demonstrated that protein A was inserted into the outer mitochondrial membrane with the N terminus in the inner membrane space or matrix and that the C terminus was exposed to the cytoplasm. Flotation and immunofluorescence studies with deletion mutants indicated that the N-proximal region of protein A was important for both membrane association and mitochondrial localization. Gain-of-function studies with green fluorescent protein fusions demonstrated that the N-terminal 46 amino acids of protein A were sufficient for mitochondrial localization and membrane insertion. We conclude that protein A targets and anchors FHV RNA replication complexes to outer mitochondrial membranes, in part through an N-proximal mitochondrial localization signal and transmembrane domain.     

Negative regulation of mitochondrial VDAC channels by C-Raf kinase

Background

Growth of cancer cells results from the disturbance of positive and negative growth control mechanisms and the prolonged survival of these genetically altered cells due to the failure of cellular suicide programs. Genetic and biochemical approaches have identified Raf family serine/threonine kinases B-Raf and C-Raf as major mediators of cell survival. C-Raf cooperates with Bcl-2/Bcl-X_L in suppression of apoptosis by a mechanism that involves targeting of C-Raf to the outer mitochondrial membrane and inactivation of the pro-apoptotic protein Bad. However, apoptosis suppression by C-Raf also occurs in cells lacking expression of Bad or Bcl-2.

Results

Here we show that even in the absence of Bcl-2/Bcl-X_L, mitochondria-targeted C-Raf inhibits cytochrome c release and caspase activation induced by growth factor withdrawal. To clarify the mechanism of Bcl-2 independent survival control by C-Raf at the mitochondria a search for novel mitochondrial targets was undertaken that identified voltage-dependent anion channel (VDAC), a mitochondrial protein (porin) involved in exchange of metabolites for oxidative phosphorylation. C-Raf forms a complex with VDAC in vivo and blocks reconstitution of VDAC channels in planar bilayer membranes in vitro.

Conclusion

We propose that this interaction may be responsible for the Raf-induced inhibition of cytochrome c release from mitochondria in growth factor starved cells. Moreover, C-Raf kinase-induced VDAC inhibition may regulate the metabolic function of mitochondria and mediate the switch to aerobic glycolysis that is common to cancer cells.     

Localization of the tomato bushy stunt virus replication protein p33 reveals a peroxisome-to-endoplasmic reticulum sorting pathway

Tomato bushy stunt virus (TBSV), a positive-strand RNA virus, causes extensive inward vesiculations of the peroxisomal boundary membrane and formation of peroxisomal multivesicular bodies (pMVBs). Although pMVBs are known to contain protein components of the viral membrane-bound RNA replication complex, the mechanisms of protein targeting to peroxisomal membranes and participation in pMVB biogenesis are not well understood. We show that the TBSV 33-kD replication protein (p33), expressed on its own, targets initially from the cytosol to peroxisomes, causing their progressive aggregation and eventually the formation of peroxisomal ghosts. These altered peroxisomes are distinct from pMVBs; they lack internal vesicles and are surrounded by novel cytosolic vesicles that contain p33 and appear to be derived from evaginations of the peroxisomal boundary membrane. Concomitant with these changes in peroxisomes, p33 and resident peroxisomal membrane proteins are relocalized to the peroxisomal endoplasmic reticulum (pER) subdomain. This sorting of p33 is disrupted by the coexpression of a dominant-negative mutant of ADP-ribosylation factor1, implicating coatomer in vesicle formation at peroxisomes. Mutational analysis of p33 revealed that its intracellular sorting is also mediated by several targeting signals, including three peroxisomal targeting elements that function cooperatively, plus a pER targeting signal resembling an Arg-based motif responsible for vesicle-mediated retrieval of escaped ER membrane proteins from the Golgi. These results provide insight into virus-induced intracellular rearrangements and reveal a peroxisome-to-pER sorting pathway, raising new mechanistic questions regarding the biogenesis of peroxisomes in plants.     

Localization of mRNAs coding for mitochondrial proteins in the yeast Saccharomyces cerevisiae

Targeted mRNA localization is a likely determinant of localized protein synthesis. To investigate whether mRNAs encoding mitochondrial proteins (mMPs) localize to mitochondria and, thus, might confer localized protein synthesis and import, we visualized endogenously expressed mMPs in vivo for the first time. We determined the localization of 24 yeast mMPs encoding proteins of the mitochondrial matrix, outer and inner membrane, and intermembrane space and found that many mMPs colocalize with mitochondria in vivo. This supports earlier cell fractionation and microarray-based studies that proposed mMP association with the mitochondrial fraction. Interestingly, a number of mMPs showed a dependency on the mitochondrial Puf3 RNA-binding protein, as well as nonessential proteins of the translocase of the outer membrane (TOM) complex import machinery, for normal colocalization with mitochondria. We examined the specific determinants of ATP2 and OXA1 mRNA localization and found a mutual dependency on the 3' UTR, Puf3, Tom7, and Tom70, but not Tom20, for localization. Tom6 may facilitate the localization of specific mRNAs as OXA1, but not ATP2, mRNA was mislocalized in tom6Δ cells. Interestingly, a substantial fraction of OXA1 and ATP2 RNA granules colocalized with the endoplasmic reticulum (ER) and a deletion in MDM10, which mediates mitochondria-ER tethering, resulted in a significant loss of OXA1 mRNA localization with ER. Finally, neither ATP2 nor OXA1 mRNA targeting was affected by a block in translation initiation, indicating that translation may not be essential for mRNA anchoring. Thus, endogenously expressed mRNAs are targeted to the mitochondria in vivo, and multiple factors contribute to mMP localization.     

Three-dimensional analysis of the association of viral particles with mitochondria during the replication of Rice gall dwarf virus

Examination of cultured insect vector cells that had been infected with Rice gall dwarf virus (RGDV), using transmission electron microscopy and confocal microscopy, revealed the presence of clusters of virus-coated mitochondria around viroplasms in which replication and assembly of RGDV occurred, suggesting a role for mitochondria in supplying the energy required for viral morphogenetic processes. Electron tomography revealed that RGDV particles on the surface of mitochondria are arrayed in an orderly but loose manner, unlike tightly packaged particles in vesicular compartments, suggesting the presence of counterpart molecules on the surface of mitochondria. The viral particles in close proximity to mitochondria were aligned along intermediate filaments, which might serve as scaffolds for the anchorage of these particles. RGDV has a putative mitochondrion-targeting sequence on the outer surface of the outer-capsid protein P8. The arrangement of RGDV particles around mitochondria suggests that the region of the P8 protein containing the mitochondrion-targeting sequence might attach to a molecule like a receptor on the outer mitochondrial membrane. Our analysis demonstrates the three-dimensional arrangement and molecular basis for the mitochondrial proximity of RGDV particles during viral replication.     

The cellular chaperone heat shock protein 90 facilitates Flock House virus RNA replication in Drosophila cells

The assembly of viral RNA replication complexes on intracellular membranes represents a critical step in the life cycle of positive-strand RNA viruses. We investigated the role of the cellular chaperone heat shock protein 90 (Hsp90) in viral RNA replication complex assembly and function using Flock House virus (FHV), an alphanodavirus whose RNA-dependent RNA polymerase, protein A, is essential for viral RNA replication complex assembly on mitochondrial outer membranes. The Hsp90 chaperone complex transports cellular mitochondrial proteins to the outer mitochondrial membrane import receptors, and thus we hypothesized that Hsp90 may also facilitate FHV RNA replication complex assembly or function. Treatment of FHV-infected Drosophila S2 cells with the Hsp90-specific inhibitor geldanamycin or radicicol potently suppressed the production of infectious virions and the accumulation of protein A and genomic, subgenomic, and template viral RNA. In contrast, geldanamycin did not inhibit the activity of preformed FHV RNA replication complexes. Hsp90 inhibitors also suppressed viral RNA and protein A accumulation in S2 cells expressing an FHV RNA replicon. Furthermore, Hsp90 inhibition with either geldanamycin or RNAi-mediated chaperone downregulation suppressed protein A accumulation in the absence of viral RNA replication. These results identify Hsp90 as a host factor involved in FHV RNA replication and suggest that FHV uses established cellular chaperone pathways to assemble its RNA replication complexes on intracellular membranes.     

Tomato bushy stunt virus co-opts the RNA-binding function of a host metabolic enzyme for viral genomic RNA synthesis

Tomato bushy stunt virus (TBSV), a plus-stranded [(+)] RNA plant virus, incorporates the host metabolic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) into the viral replicase complex. Here, we show that, during TBSV replication in yeast, the yeast GAPDH Tdh2p moves from the cytosol to the peroxisomal membrane surface, the site of viral RNA synthesis. In yeast cells lacking Tdh2p, decreasing the levels of its functionally redundant homolog Tdh3p inhibited TBSV replication and resulted in equivalent levels of (+) and minus-stranded [(-)] viral RNA, in contrast to the hallmark excess of (+)RNA. Tdh2p specifically bound an AU pentamer sequence in the (-)RNA, suggesting that GAPDH promotes asymmetric RNA synthesis by selectively retaining the (-)RNA template in the replicase complex. Downregulation of GAPDH in a natural plant host decreased TBSV genomic RNA accumulation. Thus, TBSV co-opts the RNA-binding function of a metabolic protein, helping convert the host cell into a viral factory.     

Biogenesis of mitochondrial outer membrane proteins

Mitochondria are surrounded by two distinct membranes: the outer and the inner membrane. The mitochondrial outer membrane mediates numerous interactions between the mitochondrial metabolic and genetic systems and the rest of the eukaryotic cell. Proteins of this membrane are nuclear-encoded and synthesized as precursor proteins in the cytosol. They are targeted to the mitochondria and inserted into their target membrane via various pathways. This review summarizes our current knowledge of the sorting signals for this specific targeting and describes the mechanisms by which the mitochondrial import machineries recognize precursor proteins, mediate their membrane integration and facilitate assembly into functional complexes.     

Engineered retargeting of viral RNA replication complexes to an alternative intracellular membrane

Positive-strand RNA virus replication complexes are universally associated with intracellular membranes, although different viruses use membranes derived from diverse and sometimes multiple organelles. We investigated whether unique intracellular membranes are required for viral RNA replication complex formation and function in yeast by retargeting protein A, the Flock House virus (FHV) RNA-dependent RNA polymerase. Protein A, the only viral protein required for FHV RNA replication, targets and anchors replication complexes to outer mitochondrial membranes in part via an N-proximal sequence that contains a transmembrane domain. We replaced the FHV protein A mitochondrial outer membrane-targeting sequence with the N-terminal endoplasmic reticulum (ER)-targeting sequence from the yeast NADP cytochrome P450 oxidoreductase or inverted C-terminal ER-targeting sequences from the hepatitis C virus NS5B polymerase or the yeast t-SNARE Ufe1p. Confocal immunofluorescence microscopy confirmed that protein A chimeras retargeted to the ER. FHV subgenomic and genomic RNA accumulation in yeast expressing ER-targeted protein A increased 2- to 13-fold over that in yeast expressing wild-type protein A, despite similar protein A levels. Density gradient flotation assays demonstrated that ER-targeted protein A remained membrane associated, and in vitro RNA-dependent RNA polymerase assays demonstrated an eightfold increase in the in vitro RNA synthesis activity of the ER-targeted FHV RNA replication complexes. Electron microscopy showed a change in the intracellular membrane alterations from a clustered mitochondrial distribution with wild-type protein A to the formation of perinuclear layers with ER-targeted protein A. We conclude that specific intracellular membranes are not required for FHV RNA replication complex formation and function.