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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2008 Oct 9;105(42):16326–16331. doi: 10.1073/pnas.0808720105

Effect of host cell lipid metabolism on alphavirus replication, virion morphogenesis, and infectivity

Ching G Ng *,†, Isabelle Coppens *, Dhanasekaran Govindarajan *, John Pisciotta *, Vladimir Shulaev ‡, Diane E Griffin *,§
PMCID: PMC2571027  PMID: 18845681

Abstract

The alphavirus Sindbis virus (SINV) causes encephalomyelitis in mice. Lipid-containing membranes, particularly cholesterol and sphingomyelin (SM), play important roles in virus entry, RNA replication, glycoprotein transport, and budding. Levels of SM are regulated by sphingomyelinases (SMases). Acid SMase (ASMase) deficiency results in the lipid storage disease type A Niemann-Pick disease (NPD-A), mimicked in mice by interruption of the ASMase gene. We previously demonstrated that ASMase-deficient mice are more susceptible to fatal SINV encephalomyelitis, with increased viral replication, spread, and neuronal death. To determine the mechanisms by which ASMase deficiency enhances SINV replication, we compared NPD-A fibroblasts (NPAF) to normal human fibroblasts (NHF). NPAF accumulated cholesterol- and sphingolipid-rich late endosomes/lysosomes in the perinuclear region. SINV replication was faster and reached higher titer in NPAF than in NHF, and NPAF died more quickly. SINV RNA and protein synthesis was greater in NHF than in NPAF, but virions budding from NPAF were 26 times more infectious and were regular dense particles whereas virions from NHF were larger particles containing substantial amounts of CD63. Cellular regulation of alphavirus morphogenesis is a previously unrecognized mechanism for control of virus replication and spread.

Keywords: acidic sphingomyelinase deficiency, CD63, Sindbis virus replication, cholesterol, sphingomyelin

Alphaviruses are enveloped, mosquito-borne plus-strand RNA viruses with Sindbis virus (SINV) as the prototype virus (1). SINV is widely distributed and cycles between mosquito and avian hosts. In humans, SINV infection causes fever, rash, and arthritis (2), and in mice it causes encephalomyelitis with neurons as the primary target cells (3). The severity of infection in mammals depends on both virus and host factors (4–6).

The 5′ two-thirds of genomic RNA encodes the nonstructural replicase proteins (nsP1–4), and the 3′ one-third encodes the structural virion proteins (capsid, pE2, 6K, and E1) (1). With accumulation, the nsP polyproteins are processed to become the replicase for plus-strand genomic RNA and a subgenomic RNA that encodes the structural proteins as a polyprotein (1). The N-terminal capsid protein is a serine protease that acts in cis to release itself. The rest of the polyprotein is cleaved by cellular signalase in the endoplasmic reticulum to form the transmembrane proteins pE2, 6K, and E1. pE2 is further cleaved by a furin-like protease in the trans-Golgi network (TGN) before arrival at the cell surface, virion assembly, and release.

Cellular membranes play important roles in alphavirus replication at the levels of entry, RNA synthesis, glycoprotein trafficking, and budding. Eukaryotic cell plasma membrane lipids consist primarily of sphingolipids, glycerophospholipids, and cholesterol. Sphingomyelin (SM) is enriched in the outer leaflet where it interacts with cholesterol to form microdomains (7). Sphingolipids are particularly abundant in the nervous systems (8), and the level of these lipids is highly regulated by sphingomyelinases (SMases) that cleave the phosphodiester bond between ceramide and phosphorylcholine (9, 10). The acid lysosomal SMase (ASMase) and the neutral SMases are well characterized and widely distributed (11). Defects in human ASMase activity result in the lipid storage disease type A Niemann-Pick disease (NPD-A) (12). NPD-A has been reproduced in mice by disruption of the ASMase gene (13). In both mice and humans, lack of SM degradation by ASMase results in increased intracellular levels of SM, cholesterol, lysobisphosphatidic acid, and glycosphingolipids (11, 13, 14).

The binding of SINV to poorly characterized receptors on the plasma membrane of the host cell (15) leads to endocytic uptake followed by low-pH-triggered cholesterol and sphingolipid-dependent fusion of the viral and host cell membranes (15–17). Fusion activates ASMase to cleave SM, leading to the release of ceramide that can initiate apoptosis, facilitate fusion, and expand lipid microdomains (18, 19). RNA synthesis takes place on the cytoplasmic surface of intracellular membranes derived from endosomes. The envelope proteins E1 and pE2 are synthesized at the endoplasmic reticulum (ER) and then transported through the Golgi, processed in the TGN, and delivered to the plasma membrane where cholesterol-dependent virus budding occurs (15, 16, 20, 21). Therefore, abnormalities of lipid metabolism may affect the rate and efficiency of the production of alphaviruses at multiple steps.

ASMase−/− mice are more susceptible to fatal SINV encephalitis than WT mice (22), and fibroblasts from patients with NPD-A (NPAF) are more susceptible to SINV-induced apoptosis than normal human fibroblasts (NHF) (19). In both the neurons of mice and fibroblasts of humans, susceptibility was associated with greater production of infectious virus. To determine how cellular ASMase deficiency influences the replication of SINV and the outcome of infection, we have compared infection and replication in NHF and NPAF. NHF have more efficient production of viral RNA and proteins than NPAF, but the virions produced by NPAF are more infectious and thus propagate viruses more efficiently than those produced by NHF.

Results

Intracellular Compartments of NPAFs Are Lipid-Loaded and Intersect the Endocytic Pathway.

To identify the differences between NPAF and NHF that might influence SINV replication, cells were examined by transmission electron microscopy (TEM) and fluorescence staining for intracellular lipids (Fig. 1). By TEM, NPAF had abundant membranous mulitilamellar inclusions that were not present in NHF (Fig. 1A). Filipin staining of β-hydroxysterols showed that NHF had small vesicular fluorescent structures within the cytoplasm, whereas NPAF had intense punctate fluorescence concentrated in large perinuclear compartments (Fig. 1B). The filipin staining pattern of NPAF was similar to that reported for NPD-A macrophages (23) and indicated an accumulation of cholesterol within vesicles in the perinuclear region of these cells. Boron difluoride dipyrromethane (BODIPY)-LacCer staining of sphingolipids in NPAF showed perinuclear staining that colocalized with filipin, indicating that the cholesterol-rich perinuclear NPAF compartments were also enriched in sphingolipids (Fig. 1B).

Fig. 1.

Fig. 1.

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Morphology and physiological features of cholesterol and SM-containing compartments. (A) TEM observations illustrating a representative field of the cytoplasm of NHF (i) and NPAF (ii). Note the abundance of membranous multilamellar inclusions in NPAFs (arrowheads and ii Inset). m, mitochondria. (B) Fluorescence microscopy for analysis of lipid distribution. Staining of NHF (i) and NPAF (ii) with filipin and phase microscopy (i′ and ii′) show cholesterol-laden organelles in NPAF. Cholesterol and sphingolipid colocalization is demonstrated by filipin (iii) and BODIPY-LacCer (iv) staining. The merged picture is presented in v (magnification: 100×). (C) Fluorescence microscopy (i–iii) and TEM (iv) showing the endocytic nature of the lipid-containing compartments. Incubation of NPAF with filipin (i) to identify cholesterol-containing organelles and with LysoTracker (ii) to track the endocytic structures resulted in a partial colocalization of the dyes (see merge in iii). In iv, NPAF have been incubated with BSA-gold particles (15 nm) for 16 h at 37°C. Arrows highlight BSA-containing structures that morphologically resemble cholesterol and SM-containing compartments. (Scale bars: A, 0.75 μm; C, 0.5 μm.)

To determine the nature of the cholesterol- and sphingolipid-rich vesicles that accumulate in NPAF, cells were costained with LysoTracker, which stains acidic organelles (Fig. 1C). LysoTracker staining colocalized with filipin staining, indicating lipid accumulation in late endosomes/lysosomes. NPAF were also incubated with albumin adsorbed to gold particles to label endocytic compartments for TEM observations. Gold particles were present in the membranous vesicles, further supporting the identification of the cholesterol- and sphingolipid-rich vesicles as late endosomes/lysosomes.

SINV Infection of NHF and NPAF.

NHF and NPAF were infected with SINV, and viability was determined (Fig. 2A). Death of NHF occurred primarily between 24 and 48 h, whereas death of NPAF was more rapid. At 48 h, 27.7 ± 4.2% of NHF were intact whereas only 8.6 ± 1.5% of NPAF were alive (P < 0.001, ANOVA). Infectious virus was first detected in NHF supernatant fluid 8–12 h after infection, whereas for NPAF new virus was detected 6–8 h after infection (Fig. 2B). The amount of infectious virus released by infected NPAF at 24 h was ≈10-fold higher than that released by NHF (P < 0.001, ANOVA).

Fig. 2.

Fig. 2.

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Comparison of SINV infection in NHF and NPAF. (A) Viability of cells at various time points as determined by FACS analysis for failure to exclude propidium iodide. Cells were infected with SINV at an MOI of 50. Cells were analyzed in triplicate (two-way ANOVA). (B) Growth of SINV in NHF and NPAF. Cells were infected with SINV at an MOI of 50, and infectious virus released was assayed on BHK cells. Wells were analyzed in duplicate (two-way ANOVA).

Initiation of Infection.

To determine whether differences in lipid composition of the host cell membrane affect viral binding and entry, attachment was assessed by TEM and binding of 35S-labeled SINV to NHF and NPAF. No qualitative difference in viral attachment to the cell surface was observed and SINV entered through coated vesicles in both cell types (data not shown). Equivalence of attachment was confirmed by measurement of the binding of 35S-labeled SINV to NHF and NPAF. Kinetics were comparable, and after 4 h of incubation, virus bound to NHF was 29.9 ± 2.9% and that bound to NPAF was 26.1 ± 4.4% (P = 0.0668, two-way ANOVA). The rate of endocytosis during virus infection was assessed by using FM1–43, a styryl dye that reversibly labels endosomal membranes (24). NHF and NPAF had similar levels of endocytosis after 1-h exposure to the dye with log10 shifts in geometric median of fluorescence of 2.66 for NHF and 2.81 for NPAF.

nsP and RNA Synthesis.

After fusion and release of genomic RNA from the nucleocapsid, the synthesis of nsPs leads to formation of replication complexes and synthesis of viral RNAs. To measure nsP synthesis, cells were infected with a recombinant SINV nsP reporter, Toto1101/Luc (25) and cell lysates were collected at early times after infection (Fig. 3A). Luciferase activity, reflecting nsP synthesis from genomic RNA, was greater in NHF than NPAF with the level of luciferase activity in infected NPAF ≈60% of that in infected NHF 5 h after infection (P < 0.001, ANOVA).

Fig. 3.

Fig. 3.

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Analysis of viral nsP and RNA synthesis. (A) nsP synthesis. Lysates of NHF and NPAF infected with Toto1101/Luc (25) (MOI = 5) were collected at 2–5 h after infection, and the levels of luciferase activity were measured. Data are shown as the mean of duplicate measurements of two replicates (two-way ANOVA). (B) Viral RNA synthesis. NHF and NPAF infected with SINV (MOI = 50) were labeled with [3H]uridine for 3 h in the presence of actinomycin D at 2, 6, 10, 14, and 24 h postinfection. Newly synthesized viral RNA was examined by agarose gel electrophoresis and autoradiography. Ratios of genomic-to-subgenomic RNA were determined with Fuji image gauge software. M, mock-infected NHF; ND, not detected.

Viral RNA synthesis was investigated in infected cells in 3-h time blocks beginning 2 h after infection (Fig. 3B). The peak of RNA production for both cell types was at 10–13 h. At all times, there was more [H3]uridine-labeled viral RNA in the cell lysates of infected NHF than infected NPAF. Molar ratios of subgenomic to genomic RNA ranged from 1.9 to 2.5 in NHF and from 0.8 to 1.6 in NPAF.

Synthesis, Processing, and Release of Viral Envelope Proteins.

To monitor the synthesis and processing of viral envelope proteins, cells were pulsed with 35S 7 h after infection and chased for various times, and pE2/E2 was immunoprecipitated from cell lysates (Fig. 4A). More pE2/E2 was detected in infected NHF than NPAF. Maturation of pE2 to E2 was monitored by calculating the percentage of total pE2 + E2 present as E2. There were no differences in pE2 processing to E2. To monitor the release of viral particles, supernatant fluids were pelleted and analyzed by SDS/PAGE (Fig. 4B). E1 and E2 glycoproteins appeared in pellets at 90 min for both NHF and NPAF and accumulated through the rest of the chase period. Comparable amounts of labeled E1 and E2 were present in viral particles released from infected NHF and NPAF despite the higher levels of viral RNA and protein synthesis in NHF.

Fig. 4.

Fig. 4.

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Analysis of the synthesis, processing, and release of viral envelope proteins. (A) Production of viral envelope proteins. NHF and NPAF were labeled with [35S] for 1 h beginning 7 h after infection and then chased. 35S-labeled viral proteins in cell lysates were immunoprecipitated with α-E2 mAb. (B) Release of viral particles from infected NHF and NPAF. Supernatant fluids from the pulse–chase experiment were collected, pelleted, and analyzed by SDS/PAGE and autoradiography.

SINV Particles Produced by NPAF Were More Infectious and Distinct from Those Produced by NHF.

To determine whether the virions produced differed in infectivity, the particle-to-pfu ratios of SINV isolated from infected NHF and NPAF were calculated. The particle-to-pfu ratio for NHF SINV was 4,725 ± 590 and for NPAF SINV it was 181 ± 48. To determine why the NHF virions were less infectious, cells were examined by TEM and fluorescence microscopy (Fig. 5). TEM showed larger numbers of nucleocapsids attached to the cytoplasmic surface of intracellular vesicles and tubular structures (type II cytopathic vacuoles) (26) in NPAF than in NHF (Fig. 5A). Fluorescence microscopy demonstrated that E2 partially colocalized with the tetraspanin protein CD63 in NHF, whereas E2 was more abundant at the NPAF periphery and showed a marginal codistribution with CD63 (Fig. 5B). ImmunoEM demonstrated that SINV development intersected with CD63-containing compartments to a lesser extent in NPAF than in NHF (Fig. 5C).

Fig. 5.

Fig. 5.

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Differences in SINV development in NPAFs and NHFs. (A) TEM observations showed large numbers of nucleocapsids (arrows) attached to intracellular membranes in NPAF. Viral development is on the cytoplasmic surface of vesicles (i and ii) or tubular structures (ii and iii). Infected NPAF were incubated with BSA-gold particles to label endocytic compartments (arrowheads). n, nucleus. (B) Fluorescence microscopy showing partial colocalization of E2 (i and iv) and CD63 (ii and v) in NHF and NPAF (yellow circles). Merged pictures are shown in iii and vi (magnification: 100×). (C) Immunoelectron microscopy of NHF (i–iii) and NPAF (iv–vi) showing the viral development in CD63-containing compartments. i, ii, iv, and v represent uninfected cells immunolabeled with anti-CD63 antibodies to identify endosomal compartments. iii and vi are infected cells with SINV identified by anti-E2 antibodies (small gold particles). Viral maturation occurs in organelles positively immunolabeled for CD63 (large gold particles). (Scale bars: A, Cii, Ciii, and Cvi, 100 nm; Ci, Civ, and Cv, 500 nm.)

Ultrastructural analysis showed that the virions budding from the surface of NPAF were small and regular, whereas virions budding from NHF were larger and more heterogeneous (Fig. 6A). NPAF and NHF virions had a mean diameter of 16 + 4 and 21 + 2 nm, respectively (Fig. 6B). To determine the protein composition of the virus particles released, purified virions were examined for viral and cellular proteins (Fig. 6C). Virions contained similar levels of E1/E2 and capsid, but the particles from NHFs also contained a large amount of CD63. By mass spectrometry, no differences were found in the lipid composition of the virions (data not shown).

Fig. 6.

Fig. 6.

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Differences in SINV morphology and protein composition. (A) TEM observations of virus particles budding from the plasma membrane of NPAF and NHF showing differences in size of virions originating from the two host cell types. SINV egressing from NPAF (single arrowhead) have a higher electron-density and are homogenous in size (arrowhead in i and ii). By contrast, SINV budding from NHF display two different sizes, either a size similar to NPAF-derived SINV (arrowhead) or a larger size (double arrowhead in iii and iv). (B) Negative staining of purified virions observed by TEM. (C) Western blotting on the same preparation of purified virions as in B showing similar amounts of viral proteins E1/E2 and C, but larger amounts of CD63 in NHF virions. (Scale bars: A, 100 nm; B Upper, 20 nm; B Lower, 10 nm.)

Discussion

Fibroblasts from humans with NPD-A provide an in vitro model for investigation of the effect of altered lipid metabolism on SINV replication. NPAF accumulated large amounts of cholesterol and SM in perinuclear acidic organelles that were not seen in NHF. SINV replicated faster and to higher titers in NPAF than NHF, and NPAF were more susceptible to SINV-induced cell death. There were no differences in the ability of SINV to bind or be endocytosed, but viral RNA and protein synthesis was initiated more rapidly and was greater in NHF. There was no difference in the rate at which SINV glycoproteins were produced, processed, or released from infected cells. However, virions budding from NPAF were smaller, more compact, and 26 times more infectious than virions budding from NHF. NHF-produced virions assembled predominantly in a CD63-positive compartment and incorporated this cellular tetraspanin protein. These studies show that absence of ASMase activity with accumulation of SM and cholesterol in endosomes/lysosomes resulted in decreased SINV RNA and protein synthesis, but improved virion infectivity by excluding CD63 from budding virions. Cellular regulation of alphavirus morphogenesis is a previously unrecognized mechanism for control of virus replication and spread and explains the increased susceptibility of ASMase−/− mice to SINV encephalomyelitis.

The failure to degrade SM in ASMase-deficient cells affects the lipid composition of the membranes in several cellular compartments (27). Virus binding and endocytosis occurred normally, but subsequent steps in virus replication were affected. Synthesis of nsPs, reflecting the establishment of replication complexes, which occurs on modified endosomal membranes (26), was much less in NPAF. Less viral RNA was produced at all times, and the ratio of viral subgenomic-to-genomic RNA was lower in NPAF. These findings indicate that replication complexes are not efficiently established. Modification of endosomal membranes is required for tethering of the replication complexes and establishment of type 1 cytopathic vacuoles (CPVs) where RNA synthesis occurs in close association with the ER. The endosomal membranes of NPAF are altered by accumulation of large amounts of cholesterol and SM, and it is likely that the necessary nsP modifications of these membranes are less efficient than in NHF.

In agreement with the limited production of subgenomic RNA, viral structural proteins were produced in smaller amounts in NPAF than NHF. E1 and pE2 heterodimerize in the ER and are transported through the Golgi to the plasma membrane, and an important step in SINV maturation is the cleavage of pE2 to E2 and E3 catalyzed by a furin-like protease after the protein reaches the TGN (28, 29). However, there was no difference in the rate at which pE2 was processed to E2. This finding indicated that the abnormal accumulation of cholesterol and sphingolipid in NPAF did not alter glycoprotein trafficking to a furin-containing compartment.

SINV was released from NHF and NPAF at similar rates, suggesting that particles were being efficiently packaged and released from NPAF. Unlike most enveloped viruses, alphaviruses are composed of two highly organized icosahedral protein shells with an associated membrane (30, 31). The outer shell contains trimers of the glycoprotein E1 and E2 heterodimers. The inner shell contains the capsid protein assembled around the genomic RNA (32). The structure is stabilized through E1–E1 interactions and anchoring of the outer shell to the inner shell through a 1:1 interaction of the E2 endodomain with the hydrophobic cleft of the nucleocapsid across the host cell membrane (31, 33–38). Icosahedral symmetry is maintained by lateral contacts between the glycoproteins (39). This rigid assembly process produces an icosahedral, enveloped virus that includes host cell lipids, but usually excludes host cell proteins. Icosahedral symmetry is imposed either by the formation of ribonucleocapsids in the cytoplasm and subsequent interaction with the membrane glycoproteins or by lateral interactions of the viral glycoproteins at the plasma membrane and subsequent assembly of the capsid with RNA (38, 40, 41). Numerous assembled nucleocapsids were seen in association with membranous vesicles (type 2 CPV) in the cytoplasm, suggesting that nucleocapsids form first in these fibroblasts.

In NHF, a cell that produces SINV inefficiently, E1/E2 trafficked to a CD63-positive compartment, and virion assembly was modified by the inclusion of CD63 into viral particles budding from the surface as larger spherules. Virions from both cells incorporated similar proportions of E1/E2 and capsid, but NHF virions included larger amounts of CD63. CD63 is a widely expressed tetraspanin protein that is normally resident in the late endosome/lysosome compartment, but also cycles to secretory vesicles, multivesicular bodies, and the plasma membrane (42, 43). Virions produced by NPAF incorporated little CD63 probably because CD63 was retained in the expanded cholesterol and SM-rich endosomes/lysosomes and was less available in other compartments.

It is not clear why CD63-containing virions were less infectious, but it is possible that binding or the highly coordinated acid-induced conformational changes in the trimers of E1/E2 heterodimers that lead to fusion with the endosomal membrane (40) are less efficient with this protein present. The differences in SINV titer are approximately the same as the 26-fold difference in virion infectivity. This difference in infectivity likely accounts for the more efficient replication and more rapid spread of virus in the nervous system of ASMase-deficient mice (22). These less infectious SINV virions were produced by a primary cell with 100- to 1,000-fold lower production of infectious virus than more traditionally studied vertebrate cell lines (e.g., BHK). However, replication in NHF may be more representative of alphavirus replication in vivo. For instance, in mature neurons budding of typical alphavirus virions is seen infrequently, whereas the production of “spherules” has been frequently described (44, 45). The current studies suggest that cellular production of less infectious virus may be a previously unappreciated mechanism for host restriction of virus replication that influences disease outcome.

Materials and Methods

Cells.

BHK-21 cells were grown in DMEM supplemented with 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin (DMEM/10% FBS). NHF and NPAF were obtained from the Mental Retardation Research Center (The Kennedy Krieger Institute, Baltimore) and grown in MEM supplemented with 10% FBS (MEM/10% FBS).

Viruses.

The TE strain of SINV was grown and assayed by plaque formation in BHK cells (5). Toto1101/Luc, which expresses firefly luciferase as an in-frame fusion within nsP3, was a gift from Margaret MacDonald (The Rockefeller University, New York) (25). To obtain virus from plasmids, RNA was transcribed in vitro from linearized pToto1101/Luc by using the mMessage mMachine SP6 kit (Ambion) and transfected into BHK cells by using Lipofectin (Invitrogen). The supernatant fluid was collected, and virus was concentrated by PEG precipitation (46).

Fibroblasts were infected at a multiplicity of infection (MOI) of 50 for 1 h at 37°C and washed before adding fresh medium. Virus was assayed by plaque formation on BHK cells. Cells were stained with 1 μg/ml propidium iodide to determine viability by FACS analysis.

To prepare 35S-labeled SINV, BHK cells were infected at an MOI of 1. At 5 h after infection, the medium was replaced with Met- and Cys-free DMEM (ICN)/2% FBS and 33 μCi/ml TRANS35S-LABEL (≈1,000 Ci/mmol; ICN). Virus was concentrated by PEG precipitation, banded on a 15–45% (wt/vol) potassium tartrate gradient, and pelleted through a 15% sucrose cushion at 260,000 × g (46). The final pellet was suspended in DMEM/1% FBS (47).

Cell Staining.

Cells grown in chamber slides (Nalge–Nunc) were stained with the polyene antibiotic filipin (Sigma) to visualize the distribution of β-hydroxysterols (48, 49), with LysoTracker (Molecular Probes) to identify acidic cellular compartments, and with BODIPY-labeled lactosyl ceramide (B-LacCer; Molecular Probes) to visualize the distribution of sphingolipids (50).

Binding and Endocytosis Assays.

Binding assays were carried out as described (47) with binding buffer of PBS supplemented with 1 mM MgCl2, 1 mM CaCl2, 0.5% BSA, and 10% SuperBlock (Pierce). 35S-labeled SINV (7,000 cpm) in binding buffer was added to wells of confluent cells. Plates were rocked at 4°C for 30–240 min, washed twice with binding buffer, and lysed in 1% SDS. Cell lysates and pooled washes were counted to determine the percentage of virus bound. Assays were in triplicate with three separate aliquots of labeled virus.

Endocytosis assays used FM1–43 (Molecular Probes) at a concentration of 2.5 μg/ml (24). The cells were incubated in medium with or without FM1–43 for 15 min at 37°C before adding SINV (MOI = 1), with or without FM1–43, for 1 h. Cells were washed, trypsinized, and washed twice. The amount of FM1–43 uptake was quantified by FACS analysis.

Analysis of the Synthesis of Viral RNA and Protein.

Cells infected with Toto1101/Luc were lysed between 2 and 5 h after infection with cell culture lysis buffer [25 mM Tris-phosphate (pH 7.8), 2 mM DTT, 2 mM 1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid, 10% glycerol, 1% Triton X-100] and assayed for luciferase expression according to the manufacturer's recommendation (Promega).

RNA synthesis was assessed as described (51). Cells were labeled for 3 h with DMEM containing 20 μCi [5,6-3H] uridine/ml and 1 μg actinomycin D/ml beginning 2, 6, 10, 14, and 24 h after infection.

To assess protein synthesis, at 7 h after infection cells were incubated with Met- and Cys-free DMEM for 1 h followed by addition of 50 μCi/ml TRANS[35S]LABEL for 20 min and then chase medium (DMEM/10% FBS plus 15 mg/liter l-methionine). At various times after chase, cells were washed with cold PBS and lysed with RIP buffer (150 mM NaCl, 10 mM EDTA, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris·Cl (pH 8.0)]. Samples were stored at −80°C and analyzed simultaneously. pE2/E2 was immunoprecipitated with G5 E2-specific mAb (52) and protein G-conjugated beads. For analysis of SINV released from cells, supernatant fluids were concentrated over a 15% sucrose cushion (47), and the pellet was resuspended in PBS/1% BSA and analyzed by 10% SDS/PAGE.

Viral Particle Analysis.

Viruses grown in NHF and NPAF were purified and concentrated as described above (47). PFU were measured on BHK cells, and viral RNA was purified with a QIAamp Viral RNA kit (Qiagen). The number of viral particles present was determined by quantitative RT-PCR (qRT-PCR). The primers and probe were from the E2 region (forward: 8732–5′-TGG GAC GAA GCG GAC GAT AA-3′-8752; reverse: 8805–5′-CTG CTC CGC TTT GGT CGT AT-3′-8786; TaqMan probe: 8760–5′-[FAM]-CGC ATA CAG ACT TCC GCC CAG T-[TAMRA]-3′-8781) (Applied Biosystems). Five microliters of purified viral RNA was used as template to synthesize cDNA by using SuperScript III RT (Invitrogen) and the reverse primer. qRT-PCR to determine the copies of cDNA, and in turn the number of viral particles, was carried out with TaqMan Universal PCR Master Mix, 1 μM of each primer, 250 nM of TaqMan probe, and 5 μl of cDNA template in a 25-μl reaction mixture. All reactions were performed in triplicate. A standard curve for threshold cycle (CT) values was run in parallel by using serial 10-fold dilutions of 108 copies of a pGEM-3Z vector (Promega) containing nucleotides 8638–8912 of the E2 coding region (6). Data were analyzed with Sequence Detector software version 1.7 (Applied Biosystems).

For analysis of the proteins in virions, equal amounts of purified virions were separated by SDS/PAGE (10%) and transferred to nitrocellulose membranes (Hybond-C Extra; Amersham Biosciences). These membranes were separately probed with a rabbit polyclonal antibody to SINV (3) or mAb to CD63 (Millipore) followed by horseradish peroxidase-conjugated secondary antibodies. Blots were developed with a chemiluminescent system (Cell Signaling) and visualized with ChemiDoc. The intensities of the bands were quantified with Quantity One software (Bio-Rad).

For analysis of lipids, virions were extracted with chloroform/methanol and processed as lithium adducts for electrospray ionization (ESI)-MS as described (53). Samples were infused at a flow rate of 5 μl/min into a Thermo-Finnigan TSQ quantum triple-stage quadrapole ESI-MS.

TEM.

Protocols for thin-section TEM and immunoEM procedures of cultured cells were as described (54). For negative staining, viral preparations were absorbed onto carbon-coated copper EM grids as described (55).

Statistical Analysis.

Student's t test and two-way ANOVA were done by using R (GNU Project, www.gnu.org).

Acknowledgments.

We thank Drs. Carolyn Machamer and David Sullivan for helpful discussions regarding these studies. This work was supported in part by National Institutes of Health Grants NS18596 (to D.E.G.) and AI060767 (to I.C.). C.G.N. was supported by the Agency for Science, Technology, and Research of Singapore. NPAF and NHF were supplied by the Kennedy/Hopkins National Institute of Child Health and Human Development Mental Retardation Research Center Core (HD24061).

Footnotes

The authors declare no conflict of interest.

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