The African Swine Fever Virus Prenyltransferase Is an Integral Membrane trans-Geranylgeranyl-diphosphate Synthase

Abstract

In a previous study, it was shown that the protein encoded by the gene B318L of African swine fever virus (ASFV) is a trans-prenyltransferase that catalyzes in vitro the condensation of farnesyl diphosphate and isopentenyl diphosphate to synthesize geranylgeranyl diphosphate and longer chain prenyl diphosphates (Alejo, A., Yá ñ ez, R. J., Rodríguez, J. M., Viñ uela, E., and Salas, M. L. (1997) J. Biol. Chem. 272, 9417-9423). To investigate the in vivo function of the viral enzyme, we have determined, in this work, its subcellular localization and activity in cell extracts. Two systems were used in these studies: cells infected with ASFV and cells infected with a recombinant pseudo-Sindbis virus carrying the complete B318L gene. In this latter system, the trans-prenyltransferase was found to colocalize with the endoplasmic reticulum marker protein-disulfide isomerase, whereas in cells infected with ASFV, the viral enzyme was present in cytoplasmic viral assembly sites, associated with precursor viral membranes derived from the endoplasmic reticulum. In addition, after subcellular fractionation, the viral enzyme partitioned into the membrane fraction. Extraction of membrane proteins with alkaline carbonate and Triton X-114 indicated that the ASFV enzyme behaved as an integral membrane protein. The membrane enzyme synthesized predominantly all-trans-geranylgeranyl diphosphate from farnesyl diphosphate and isopentenyl diphosphate. These results indicate that the viral B318L protein is a transgeranylgeranyl-diphosphate synthase, being the only enzyme of this type that is known to have a membrane localization.In the biosynthetic pathway of isoprenoid compounds, different enzymes belonging to the family of prenyltransferases catalyze the condensation of isopentenyl diphosphate (IPP) 1 with allylic diphosphates to give rise to prenyl diphosphates of different chain lengths and double-bond stereochemistry (1-3). Thus, farnesyl-diphosphate synthase and geranylgeranyldiphosphate synthase produce the 15-carbon all-trans-FPP and the 20-carbon all-trans-GGPP isoprenoids, respectively, which can serve as substrates for protein farnesylation or geranylgeranylation (4, 5). FPP is also the direct precursor for the synthesis of cholesterol and serves as allylic substrate for cisand trans-polyprenyl-diphosphate synthases involved in the synthesis of the long chain isoprenoids with cis-or trans-stereochemistry, which are the precursors of dolichols and the lateral chain of ubiquinones, respectively (6, 7).Prenyltransferases have been described in a wide variety of organisms, both prokaryotic and eukaryotic, where their isoprenoid products play essential roles in cellular processes. More recently, it has been shown that prenylation of cellular or viral proteins is required for the multiplication of certain viruses (8 -11). In this connection, we have previously reported (12) the characterization of the first viral trans-prenyltransferase encoded by African swine fever virus (ASFV), a large enveloped DNA virus with an icosahedral morp...

African Swine Fever Virus Protein p17 Is Essential for the Progression of Viral Membrane Precursors toward Icosahedral Intermediates

“…We have shown that p17 has an intrinsic affinity for the ER that it shares with the proteins that localize at this structural domain (59) or that transiently interact with viral membrane precursors (1). A similar behavior has been described for the VV membrane proteins that localize at the membrane of the IV (20), and this is probably related to the necessity of these proteins to integrate early in assembly into ER-derived membranes that have not been completely modified by the viral machinery.…”

“…ASFV structural proteins that reside at the inner viral envelope, like p54 (59), or nonstructural proteins that localize at the precursor membranes, like the viral prenyltransferase (1), have an intrinsic affinity for the ER (1,59). To test whether protein p17 can associate with ER membranes when expressed outside the context of ASFV infection, we analyzed its subcellular localization in Vero cells transfected with a plasmid containing the D117L ORF under the control of a T7 RNA polymerase promoter and infected with a recombinant VV that expresses the T7 RNA polymerase.…”

Disruption of hepatitis C virus RNA replication through inhibition of host protein geranylgeranylation

“…Inasmuch as 25-hydroxycholesterol lowers transcription of the HMG CoA reductase gene (25), 25-hydroxycholesterol may inhibit HCV replication by depleting cells of geranylgeranyl pyrophosphate, the mevalonate-derived donor of protein geranylgeranylation (7,10). A more general role for host cell geranylgeranylation in viral replication may be inferred from the finding that African swine fever virus, an icosahedral DNA virus, encodes a membrane-bound prenyltransferase that synthesizes geranylgeranyl pyrophosphate (26).…”

Small Rho GTPases and Cholesterol Biosynthetic Pathway Intermediates in African Swine Fever Virus Infection

“…The reduction of virus progeny when geranylation was inhibited after virus entry suggests that intact pools of GGPP or geranylated proteins during a later stage of ASFV infection are also crucial for correct viral replication. In this regard, ASFV encodes a transprenyltransferase (open reading frame B318L) that catalyzes the condensation of farnesyl diphosphate and isopentenyl diphosphate to GGPP and longer chain prenyl diphosphates (1,2). B318L is an essential gene whose expression occurs late during virus infection, and its protein remains associated with precursor viral membranes derived from the endoplasmic reticulum at the viral assembly sites.…”