Presence Of Microsomal Membranes Research Articles

BB0172, a Borrelia burgdorferi Outer Membrane Protein That Binds Integrin α 3 β 1

Lyme disease is a multisystemic disorder caused by Borrelia burgdorferi infection. Upon infection, some B. burgdorferi genes are upregulated, including members of the microbial surface components recognizing adhesive matrix molecule (MSCRAMM) protein family, which facilitate B. burgdorferi adherence to extracellular matrix components of the host. Comparative genome analysis has revealed a new family of B. burgdorferi proteins containing the von Willebrand factor A (vWFA) domain. In the present study, we characterized the expression and membrane association of the vWFA domain-containing protein BB0172 by using in vitro transcription/translation systems in the presence of microsomal membranes and with detergent phase separation assays. Our results showed evidence of BB0172 localization in the outer membrane, the orientation of the vWFA domain to the extracellular environment, and its function as a metal ion-dependent integrin-binding protein. This is the first report of a borrelial adhesin with a metal ion-dependent adhesion site (MIDAS) motif that is similar to those observed in eukaryotic integrins and has a similar function.

NS2B/3 proteolysis at the C-prM junction of the tick-borne encephalitis virus polyprotein is highly membrane dependent

The replication of tick-borne encephalitis virus (TBEV), like that of all flaviviruses, is absolutely dependent on proteolytic processing. Production of the mature proteins C and prM from their common precursor requires the activity of the viral NS2B/3 protease (NS2B/3pro) at the C-terminus of protein C and the host signal peptidase I (SPaseI) at the N-terminus of protein prM. Recently, we have shown in cell culture that the cleavage of protein C and the subsequent production of TBEV particles can be made dependent on the activity of the foot-and-mouth disease virus 3C protease, but not on the activity of the HIV-1 protease (HIV1pro) (Schrauf et al., 2012). To investigate this failure, we developed an in vitro cleavage assay to assess the two cleavage reactions performed on the C-prM precursor. Accordingly, a recombinant modular NS2B/3pro, consisting of the protease domain of NS3 linked to the core-domain of cofactor NS2B, was expressed in E. coli and purified to homogeneity. This enzyme could cleave a C-prM protein synthesised in rabbit reticulocyte lysates. However, cleavage was only specific when protein synthesis was performed in the presence of canine pancreatic microsomal membranes and required the prevention of signal peptidase I (SPaseI) activity by lengthening the h-region of the signal peptide. The presence of membranes allowed the concentration of NS2B/3pro used to be reduced by 10–20 fold. Substitution of the NS2B/3pro cleavage motif in C-prM by a HIV-1pro motif inhibited NS2B/3pro processing in the presence of microsomal membranes but allowed cleavage by HIV-1pro at the C-prM junction. This system shows that processing at the C-terminus of protein C by the TBEV NS2B/3pro is highly membrane dependent and will allow the examination of how the membrane topology of protein C affects both SPaseI and NS2B/3pro processing.

GLUT8, the enigmatic intracellular hexose transporter

GLUT8 is a class III sugar transporter predominantly expressed in testis and brain. In contrast to the class I and class II transporters, hydrophobicity plots predict a short extracellular loop between transmembrane domain (TM)1 and TM2 and a long extracellular loop between TM9 and TM10 that contains the only N-glycosylation site. In vitro translated GLUT8 migrates as a 35-kDa protein that is glycosylated in the presence of microsomal membranes. In heterologous expression systems, glucose transport activity (Km of 2 mM) was inhibited by fructose and galactose. The transporter carries an NH2-terminal endosomal/lysosomal targeting motif ([DE]XXXL[LI]). Accordingly, constitutive GLUT8 has been found to be associated with endosomes and lysosomes but also with membranes of the endoplasmic reticulum. A similar distribution was detected after overexpression of wild-type or tagged GLUT8 in different cell systems. In these cells, none of the conventional signals tested induced a translocation of GLUT8 to the plasma membrane. Therefore, GLUT8 appears to catalyze transport of sugars or sugar derivatives through intracellular membranes. Slc2a8 knockout mice were viable, developed normally, and showed mild alterations in brain (increased proliferation of neuronal cells in dentate gyrus of the hippocampus, hyperactivity), heart (impaired transmission of electrical wave through the atrium), and sperm cells (reduced number of motile sperm cells associated with reduced mitochondrial membrane potential and ATP levels in sperm). The links between molecular function, cellular localization and phenotype of the knockout mouse is unclear and remains to be determined.

The Signal Peptide of the Mouse Mammary Tumor Virus Rem Protein Is Released from the Endoplasmic Reticulum Membrane and Accumulates in Nucleoli

N-terminal signal sequences mediate endoplasmic reticulum (ER) targeting and insertion of nascent secretory and membrane proteins and are, in most cases, cleaved off by signal peptidase. The mouse mammary tumor virus envelope protein and its alternative splice variant Rem have an unusually long signal sequence, which contains a nuclear localization signal. Although the envelope protein is targeted to the ER, inserted, and glycosylated, Rem has been described as a nuclear protein. Rem as well as a truncated version identical to the cleaved signal sequence have been shown to function as nuclear export factors for intron-containing transcripts. Using transiently transfected cells, we found that Rem is targeted to the ER, where the C-terminal portion is translocated and glycosylated. The signal sequence is cleaved off and accumulates in nucleoli. In a cell-free in vitro system, the generation of the Rem signal peptide depends on the presence of microsomal membranes. In vitro and in cells, the signal peptide initially accumulates in the membrane and is subsequently released into the cytosol. This release does not depend on processing by signal peptide peptidase, an intramembrane cleaving protease that can mediate the liberation of signal peptide fragments from the ER membrane. Our study suggests a novel pathway by which a signal peptide can be released from the ER membrane to fulfill a post-targeting function in a different compartment.

The Pestivirus Glycoprotein Erns Is Anchored in Plane in the Membrane via an Amphipathic Helix

E(rns) is a structural glycoprotein of pestiviruses found to be attached to the virion and to membranes within infected cells via its COOH terminus, although it lacks a hydrophobic anchor sequence. The COOH-terminal sequence was hypothesized to fold into an amphipathic alpha-helix. Alanine insertion scanning revealed that the ability of the E(rns) COOH terminus to bind membranes is considerably reduced by the insertion of a single amino acid at a wide variety of positions. Mutations decreasing the hydrophobicity of the apolar face of the putative helix led to reduction of membrane association. Proteinase K protection assays showed that E(rns) translated in vitro in the presence of microsomal membranes was protected, whereas a mutant with an artificial transmembrane region and a short cytosolic tag was shortened by the protease treatment. A tag fused to the COOH terminus of wild type E(rns) was not accessible for antibodies within digitonin-permeabilized cells, but the variant with the tag located downstream of the artificial transmembrane region was detected under the same conditions. These results are in accordance with the model that the COOH-terminal membrane anchor of E(rns) represents an amphipathic helix embedded in plane into the membrane. The integrity of the membrane anchor was found to be important for recovery of infectious virus.

Membrane insertion and topology of the p7B movement protein of Melon Necrotic Spot Virus (MNSV)

Cell-to-cell movement of the Melon Necrotic Spot Virus (MNSV) is controlled by two small proteins working in trans, an RNA-binding protein (p7A) and an integral membrane protein (p7B) separated by an amber stop codon. p7B contains a single hydrophobic region. Membrane integration of this region was observed when inserted into model proteins in the presence of microsomal membranes. Furthermore, we explored the topology and targeting mechanisms of full-length p7B. Here we present evidence that p7B integrates in vitro into the ER membrane cotranslationally and with an Nt-cytoplasmic/Ct-luminal orientation. The observed topology was monitored in vivo by fusing GFP to the Ct of p7B, enabling the overexpression in Escherichia coli cultures. Finally, the topology of a putative p14 movement protein was established by replacing the amber stop codon located between p7A and p7B.

Specific Features of the Prion Protein Transmembrane Domain Regulate Nascent Chain Orientation

The sequence of a transmembrane (TM) domain and the adjacent regions are important for recognition, orientation, and integration at the translocon during membrane protein biosynthesis. However, the sequences of individual TM domains vary considerably. Although some general effects of electrostatic and hydrophobic interactions have been observed, it is still not clear what features of diverse sequences influence TM domain orientation. Here we utilized the ability of the prion protein (PrP) to be synthesized in multiple topological forms to assay the effects of substitutions and mutations on TM domain orientation. Several of the TM domains we tested appear to contain no inherent information regulating orientation. In contrast, we found that the middle region of the PrP TM domain significantly reduces the ability of the chain to invert its orientation in the translocon. We also observed that the C-terminal region of the PrP TM domain influences orientation, and we characterized the orientation differences between two forms of a physiologically relevant polymorphism in this region. Specifically, we found that the identity of a single amino acid, that at position 129, can significantly alter PrP TM domain orientation. Because position 129 is the location of the disease-associated Met/Val polymorphism, we discuss both how this small change may affect TMD orientation and the larger biological implications of these results.

Post-translational signal peptide cleavage controls differential epitope recognition in the QP-rich domain of recombinant Theileria parva PIM

The presence of the schizont stage of the obligate intracellular parasites Theileria parva or T. annulata in the cytoplasm of an infected leukocyte results in host cell transformation via a mechanism that has not yet been elucidated. Proteins, secreted by the schizont, or expressed on its surface, are of interest as they can interact with host cell molecules that regulate host cell proliferation and/or survival. The major schizont surface protein is the polymorphic immunodominant molecule, PIM, which contains a large glutamine- and proline-rich domain (QP-rd) that protrudes into the host cell cytoplasm. Analyzing QP-rd generated by in vitro transcription/translation, we found that the signal peptide was efficiently cleaved post-translationally upon addition of T cell lysate or canine pancreatic microsomes, whereas signal peptide cleavage of a control protein only occurred cotranslationally and in the presence of microsomal membranes. The QP-rd of PIM migrated anomalously in SDS–PAGE and removal of the 19 amino acids corresponding to the predicted signal peptide caused a decrease in apparent molecular mass of 24 kDa. The molecule was analyzed using monoclonal antibodies that recognize a set of previously defined PIM epitopes. Depending on the presence or the absence of the signal peptide, two conformational states could be demonstrated that are differentially recognized, with N-terminal epitopes becoming readily accessible upon signal peptide removal, and C-terminal epitopes becoming masked. Similar observations were made when the QP-rd of PIM was expressed in bacteria. Our observations could also be of relevance to other schizont proteins. A recent analysis of the proteomes of T. parva and T. annulata revealed the presence of a large family of potentially secreted proteins, characterized by the presence of large stretches of amino acids that are also particularly rich in QP-residues.

Double-spanning Plant Viral Movement Protein Integration into theEndoplasmic Reticulum Membrane Is Signal Recognition Particle-dependent,Translocon-mediated, andConcerted

The current model for cell-to-cell movement of plant viruses holds that transport requires virus-encoded movement proteins that intimately associate with endoplasmic reticulum membranes. We have examined the early stages of the integration into endoplasmic reticulum membranes of a double-spanning viral movement protein using photocross-linking. We have discovered that this process is cotranslational and proceeds in a signal recognition particle-dependent manner. In addition, nascent chain photocross-linking to Sec61alpha and translocating chain-associated membrane protein reveal that viral membrane protein insertion takes place via the translocon, as with most eukaryotic membrane proteins, but that the two transmembrane segments of the viral protein leave the translocon and enter the lipid bilayer together.

In vitro expression and analysis of secreted fowlpox virus CC chemokine-like proteins Fpv060, Fpv061, Fpv116 and Fpv121

The four CC chemokine-like proteins (Fpv060, Fpv061, Fpv116 and Fpv121) of fowlpox virus (FWPV) were over-expressed as His-tagged versions from a T7 promoter/EMCV IRES construct in vitro, by coupled transcription/translation, or in cell culture, by co-infection with two recombinant FWPVs (one expressing the chemokine-like protein and one expressing T7 RNA polymerase). All, except Fpv116, appeared to be glycosylated in the presence of microsomal membranes in vitro. In culture, all were secreted (even though secretion of Fpv061 was not predicted). Secreted forms of Fpv060 and Fpv121 were the most abundant forms of those two proteins. Glycosidase analysis of cellular and secreted forms confirmed that Fpv060, Fpv061 and Fpv121 were N-glycosylated and that the most abundant, cellular form of Fpv061 had been glycosylated but remained Endo H-sensitive (retained in the endoplasmic reticulum or Golgi). N-terminal sequence analysis of His-tagged Fpv060 and Fpv121 showed that they were processed at the predicted signal cleavage sites. Fpv060- and Fpv061-specific antipeptide sera allowed confirmation that the expression, processing and secretion of the native proteins were as determined for the His-tagged proteins. Isolation of knock-out mutants showed that all four proteins were non-essential for replication in tissue culture.

Transport of the intracisternal A-type particle Gag polyprotein to the endoplasmic reticulum is mediated by the signal recognition particle.

Intracisternal A-type particles (IAP) are defective endogenous retroviruses that accumulate in the endoplasmic reticulum (ER) of rodent cells. The enveloped particles are produced by assembly and budding of IAP Gag polyproteins at the ER membrane. In this study, we analyzed the specific ER transport of the Gag polyprotein of the IAP element MIA14. To this end, we performed in vitro translation of Gag in the presence of microsomal membranes or synthetic proteoliposomes followed by membrane sedimentation or flotation. ER binding of IAP Gag occurred mostly cotranslationally, and Gag polyproteins interacted specifically with proteoliposomes containing only signal recognition particle (SRP) receptor and the Sec61p complex, which form the minimal ER translocation apparatus. The direct participation of SRP in ER targeting of IAP Gag was demonstrated in cross-linking and immunoprecipitation experiments. The IAP polyprotein was not translocated into the ER; it was found to be tightly associated with the cytoplasmic side of the ER membrane but did not behave as an integral membrane protein. Substituting the functional signal peptide of preprolactin for the hydrophobic sequence at the N terminus of IAP Gag also did not result in translocation of the chimeric protein into the ER lumen, and grafting the IAP hydrophobic sequence onto preprolactin failed to yield luminal transport as well. These results suggest that the N-terminal hydrophobic region of the IAP Gag polyprotein functions as a transport signal which mediates SRP-dependent ER targeting, but polyprotein translocation or integration into the membrane is prevented by the signal sequence itself and by additional regions of Gag.

'Piggy-back' transport of Xenopus hyaluronan synthase (XHAS1) via the secretory pathway to the plasma membrane.

Hyaluronan is the sole glycosaminoglycan whose biosynthesis takes place directly at the plasma membrane. The mechanism by which hyaluronan synthase (HAS) becomes inserted there, as well as the question of how the enzyme discriminates between particular membrane species in polarized cells, are largely unknown. In vitro translation of HAS suggested that the nascent protein becomes stabilized in the presence of microsomal membranes, but would not insert spontaneously into membranes after being translated in the absence of those. We therefore monitored the membrane attachment of enzymatically active fusion proteins consisting of Xenopus HAS1 and green fluorescent protein shortly after de novo synthesis in Vero cells. Our data strongly suggest that HAS proteins are directly translated on the ER membrane without exhibiting an N-terminal signal sequence. From there the inactive protein is transferred to the plasma membrane via the secretory pathway. For unknown reasons, HAS inserted into membranes other than the plasma membrane remains inactive.

The vaccinia virus B9R protein is a 6 kDa intracellular protein that is non-essential for virus replication and virulence.

Vaccinia virus (VV) strain Western Reserve gene B9R is shown to encode an intracellular 6 kDa protein that is expressed late during the infectious cycle. In vitro transcription and translation produced two polypeptides in the presence of microsomal membranes, but only the larger protein in the absence of membranes. The smaller protein sedimented with microsomes during centrifugation, suggesting it was inserted into the lipid membrane or into the microsomal lumen via the N-terminal hydrophobic signal sequence that was subsequently cleaved proteolytically. A VV mutant lacking B9R was constructed and found to replicate normally in cell culture and two in vivo models.

Dopamine β-Monooxygenase Signal/Anchor Sequence Alters Trafficking of Peptidylglycine α-Hydroxylating Monooxygenase

Dopamine beta-monooxygenase (DBM) and peptidylglycine alpha-hydroxylating monooxygenase (PHM) are essential for the biosynthesis of catecholamines and amidated peptides, respectively. The enzymes share a conserved catalytic core. We studied the role of the DBM signal sequence by appending it to soluble PHM (PHMs) and expressing the DBMsignal/PHMs chimera in AtT-20 and Chinese hamster ovary cells. PHMs produced as part of DBMsignal/PHMs was active. In vitro translated and cellular DBMsignal/PHMs had similar masses, indicating that the DBM signal was not removed. DBMsignal/PHMs was membrane-associated and had the properties of an intrinsic membrane protein. After in vitro translation in the presence of microsomal membranes, trypsin treatment removed 2 kDa from DBMsignal/PHMs while PHMs was entirely protected. In addition, a Cys residue in DBMsignal/PHMs was accessible to Cys-directed biotinylation. Thus the chimera adopts the topology of a type II membrane protein. Pulse-chase experiments indicate that DBMsignal/PHMs turns over rapidly after exiting the trans-Golgi network. Although PHMs is efficiently localized to secretory granules, DBMsignal/PHMs is largely localized to the endoplasmic reticulum in AtT-20 cells. On the basis of stimulated secretion, the small amount of PHMs generated is stored in secretory granules. In contrast, the expression of DBMsignal/PHMs in PC12 cells yields protein that is localized to secretory granules.

GLUTX1, a Novel Mammalian Glucose Transporter Expressed in the Central Nervous System and Insulin-sensitive Tissues

Based on homology with GLUT1-5, we have isolated a cDNA for a novel glucose transporter, GLUTX1. This cDNA encodes a protein of 478 amino acids that shows between 29 and 32% identity with rat GLUT1-5 and 32-36% identity with plant and bacterial hexose transporters. Unlike GLUT1-5, GLUTX1 has a short extracellular loop between transmembrane domain (TM) 1 and TM2 and a long extracellular loop between TM9 and TM10 that contains the only N-glycosylation site. When expressed in Xenopus oocytes, GLUTX1 showed strong transport activity only after suppression of a dileucine internalization motif present in the amino-terminal region. Transport activity was inhibited by cytochalasin B and partly competed by D-fructose and D-galactose. The Michaelis-Menten constant for glucose was approximately 2 mM. When translated in reticulocytes lysates, GLUTX1 migrates as a 35-kDa protein that becomes glycosylated in the presence of microsomal membranes. Western blot analysis of GLUTX1 transiently expressed in HEK293T cells revealed a diffuse band with a molecular mass of 37-50 kDa that could be converted to a approximately 35-kDa polypeptide following enzymatic deglycosylation. Immunofluorescence microscopy detection of GLUTX1 transfected into HEK293T cells showed an intracellular staining. Mutation of the dileucine internalization motif induced expression of GLUTX1 at the cell surface. GLUTX1 mRNA was detected in testis, hypothalamus, cerebellum, brainstem, hippocampus, and adrenal gland. We hypothesize that, in a similar fashion to GLUT4, in vivo cell surface expression of GLUTX1 may be inducible by a hormonal or other stimulus.

Membrane Topology of Alzheimer's Disease-related Presenilin 1: EVIDENCE FOR THE EXISTENCE OF A MOLECULAR SPECIES WITH A SEVEN MEMBRANE-SPANNING AND ONE MEMBRANE-EMBEDDED STRUCTURE

A significant member of early-onset familial type of Alzheimer's disease cases has been shown to be caused by dominant mutations in either of the two genes encoding presenilin 1 (PS1) and presenilin 2 (PS2). These two proteins are highly homologous to each other and have been reported to be mainly localized to the membranes of intracellular compartments such as the endoplasmic reticulum. Information about the membrane topological structures of these proteins is indispensable for understanding their physiological and pathological roles. Although several models have been proposed previously, their precise membrane topologies remain unknown. In this study, we examined this issue in detail by expressing a series of C-terminally deleted PS1 mutants fused to the hydrophilic portion of Escherichia coli leader peptidase in vitro using a reticulocyte lysate in the presence of microsomal membranes. Our results predict that PS1 exists mainly in a seven membrane-spanning structure with its C-terminal end exposed to the luminal space. This was also confirmed by expressing these fusion proteins in cultured cells. We further showed that a ninth hydrophobic segment is tightly bound to the membrane without spanning it. Based on the above observations, we propose a novel "seven membrane-spanning and one membrane-embedded" topological model for presenilins.

Multi-ubiquitination of a nascent membrane protein produced in a rabbit reticulocyte lysate.

During a large-scale in vitro translation analysis of a human full-length cDNA bank, we found many clones producing in vitro translation products showing ladder bands on a fluorogram with the equidistance of about 9 kDa at the position larger than the molecular mass expected from the open reading frame. We have analyzed a clone showing a typical pattern of the ladder bands. This clone encoded a 188-amino acid polypeptide containing a putative transmembrane domain. A green fluorescent protein-tagged polypeptide expressed in COS7 cells was localized in the endoplasmic reticulum and the Golgi apparatus. The ladder bands were observed in a rabbit reticulocyte lysate system, but not in a wheat germ extract system. Addition of the glutathione S-transferase-fused ubiquitin into the lysate caused upward shifts of the ladder bands. Addition of microsomal membranes prevented the formation of the ladder bands. Time course experiments demonstrated that the in vitro translation products increased in the presence of microsomal membranes, but were gradually degraded in their absence. These results suggest that the ladder formation resulted from the ubiquitination of misfolded polypeptide that failed to translocate to its proper position, and that an exclusion mechanism of misfolded membrane protein works in the rabbit reticulocyte lysate system.

Effects of Missense Mutations and Deletions on Membrane Anchoring and Enzyme Function of Human Steroid 21-Hydroxylase (P450c21)

We studied membrane binding and enzyme function of six variant forms of human steroid 21-hydroxylase (P450c21), a mutant (P30Q) from a patient with congenital adrenal hyperplasia, four artificial deletions in the amino terminal region (delS1 and del S2; the first and second hydrophobic segment, delS3; the region in between, delS4; the combination of these), and one naturally ocurring polymorphism in a region implicated to be critical for membrane integration (delL10). Enzyme function was assayed after transient expression in COS-1 cells, and membrane binding was studied by coupledin vitrotranscription-translation in the presence of microsomal membranes. P450c21(delS1) retained some enzyme activity but showed severely reduced membrane binding. P450c21(P30Q), P450c21 (delS2), P450c21(delS3), and P450c21(delS4) had abolished enzyme function. P450c21(P30Q) and P450c21 (delS2) did not affect membrane binding, P450c21 (delS3) had slightly reduced binding with a qualitative difference suggested by the absence of a glycosylated form of the protein, and P450c21(delS4) had abolished membrane integration. No significant differences could be identified for the delL10 variant. These data support that P450c21 spans the membrane through its first hydrophobic domain only, and that the protein lacking this segment retains sufficiently normal structure to enable catalysis. They also confirm that P30Q is responsible for the severe phenotype of the patient in which it was found, and indicate that the common delL10 polymorphism does not have a major effect on enzyme function.

Distinct renin isoforms generated by tissue-specific transcription initiation and alternative splicing.

The aspartyl protease renin catalyzes the initial and rate-limiting step in the formation of the biologically active peptide angiotensin II. It is mainly synthesized in the kidney as a preprohormone and secreted via constitutive and regulated pathways. We identified a novel transcript of the rat renin gene, renin b, characterized by the presence of an alternative first exon (exon 1b) that is spliced to exon 2 of the known transcript, termed renin a. We demonstrated that renin b is exclusively expressed in the brain. In contrast, renin a was not expressed in the brain. Using primer extension assays, we mapped the transcriptional start site of this novel mRNA within intron 1 of the rat genomic sequence, suggesting the presence of a brain-specific promoter within intron 1. The presence of a brain-specific renin isoform is evolutionally conserved, as demonstrated by the finding of renin b isoforms in mice and humans. The predicted protein renin b lacks the prefragment as well as a significant portion of the profragment and is therefore predicted not to be a secreted protein, unlike the classically described isoform renin a. As shown by in vitro translation of full-length renin b mRNA in the presence of microsomal membranes, renin b was not targeted into the endoplasmatic reticulum and remained intracellularly in transiently transfected AtT-20 cells. These findings provide evidence for a novel pathway of intracellular angiotensin generation that occurs exclusively in the brain.

Human Herpesvirus-8 ORF K8.1 Gene Encodes Immunogenic Glycoproteins Generated by Spliced Transcripts

A cDNA library from phorbol ester-induced human herpesvirus-8 (HHV-8) carrying BCBL-1 cells was screened with an HIV+KS+ serum, and several cDNA clones encoding HHV-8 proteins were identified. Sequence analysis of two full-length cDNA clones show open reading frames (ORFs) encoded by spliced messages originating from the HHV-8 K8.1 gene. One cDNA encodes an ORF of 228 amino acids, designated K8.1.A, with a cleavable signal sequence, a transmembrane domain, and fourN-glycosylation sites. The splicing event generated the transmembrane domain in the ORF not seen in the genomic K8.1 ORF. Another cDNA encodes an ORF of 167 amino acids, designated K8.1.B, that shares similar amino and carboxyl termini with ORF K8.1.A but with an in-frame deletion. The primary translation products of ORF K8.1A (34 kDa) and K8.1B (20 kDa) in thein vitro–transcription–translation experiments shifted into glycosylated species of 43 and 32 kDa, respectively, in the presence of microsomal membranes. This suggested that the ORF K8.1A and K8.1B encode for glycoproteins. Riboprobes from the K8.1A cDNA insert hybridized with an HHV-8-specific 0.9-kb abundant transcript from BCBL-1 cells. Synthesis of this RNA was eliminated in the presence of a DNA synthesis inhibitor, suggesting that this RNA was a late gene transcript. Because ORFs K8.1A and K8.1B are unique for HHV-8, human sera were tested in Western blot reactions for antibodies against glutathione-S-transferase–ORF K8.1A fusion protein. All sera that were positive for HHV-8 antibodies in immunofluorescence assays with phorbol ester-induced BCBL-1 cells were also positive for anti-ORF K8.1A antibodies. This suggests that measurement of anti-ORF K8.1A antibodies would provide an HHV-8-specific serological assay. Further work is needed to define the biological role of the HHV-8 ORF K8.1A and K8.1B glycoproteins.