US3 Protein Research Articles

Identification of the US3 gene product of BHV-1 as a protein kinase and characterization of BHV-1 mutants of the US3 gene

We have identified the product of the US3 gene of bovine herpes virus type 1 (BHV-1), which is homologous to the herpes simplex virus type 1 (HSV-1) US3 protein kinase (PK) gene. The antibodies against the BHV-1 US3 gene product reacted with a 58 kDa polypeptide in BHV-1 infected cells and the 58 kDa polypeptide purified by immuno-precipitation demonstrated PK activity. Recent reports indicating that the US3 gene of HSV-1 is involved in the blockage of apoptosis in virus infected cells. As to the apoptosis in BHV-1 infected cells, we found following: (1) no apoptosis was observed in cells infected with wild type BHV-1 and the US3 mutants (2) the apoptosis induced by the osmotic shock of sorbitol treatment was blocked when cells were infected by the wild type BHV-1 (3) the US3 mutants of BHV-1 blocked the apoptosis of sorbitol treated cell, but the suppressive effect was delayed relative to that of wild type BHV-1 (4) the other BHV-1 mutants, with the intact US3 gene but with some other non-essential gene (genes) deleted behaved similar way to the US3 mutant. It is concluded that the US3 gene of BHV-1 is not directly involved in the blockage of apoptosis in infected cells.

US3 protein kinase of herpes simplex virus type 2 plays a role in protecting corneal epithelial cells from apoptosis in infected mice.

To clarify the biological role of US3 protein kinase of herpes simplex virus type 2 (HSV-2) in vivo, the expression of the viral antigen, the appearance of apoptotic bodies and DNA fragmentation were examined immunohistologically after corneal infection of mice with three different kinds of HSV-2 strain 186: the wild-type virus, a US3-deficient mutant (L1BR1) and its revertant (L1B-11). In both wild-type 186- and L1B-11-infected mice, viral antigen was diffusely found in the corneal epithelium; no apoptotic changes were detected in the epithelial cells. Whereas, in L1BR1-infected mice, HSV-immunoreactivity was localized around the virus-inoculated sites, and a large number of apoptotic bodies were observed in the corneal epithelium with dual-positive reactions for both HSV-immunostaining and TUNEL staining. These results suggest that the US3 protein kinase plays an important role in protecting HSV-2-infected cells from apoptotic death in vivo.

The herpes simplex virus US11 protein effectively compensates for the gamma1(34.5) gene if present before activation of protein kinase R by precluding its phosphorylation and that of the alpha subunit of eukaryotic translation initiation factor 2.

In herpes simplex virus-infected cells, viral gamma134.5 protein blocks the shutoff of protein synthesis by activated protein kinase R (PKR) by directing the protein phosphatase 1alpha to dephosphorylate the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2alpha). The amino acid sequence of the gamma134.5 protein which interacts with the phosphatase has high homology to a domain of the eukaryotic protein GADD34. A class of compensatory mutants characterized by a deletion which results in the juxtaposition of the alpha47 promoter next to US11, a gamma2 (late) gene in wild-type virus-infected cells, has been described. In cells infected with these mutants, protein synthesis continues even in the absence of the gamma134.5 gene. In these cells, PKR is activated but eIF-2alpha is not phosphorylated, and the phosphatase is not redirected to dephosphorylate eIF-2alpha. We report the following: (i) in cells infected with these mutants, US11 protein was made early in infection; (ii) US11 protein bound PKR and was phosphorylated; (iii) in in vitro assays, US11 blocked the phosphorylation of eIF-2alpha by PKR activated by poly(I-C); and (iv) US11 was more effective if present in the reaction mixture during the activation of PKR than if added after PKR had been activated by poly(I-C). We conclude the following: (i) in cells infected with the compensatory mutants, US11 made early in infection binds to PKR and precludes the phosphorylation of eIF-2alpha, whereas US11 driven by its natural promoter and expressed late in infection is ineffective; and (ii) activation of PKR by double-stranded RNA is a common impediment countered by most viruses by different mechanisms. The gamma134.5 gene is not highly conserved among herpesviruses. A likely scenario is that acquisition by a progenitor of herpes simplex virus of a portion of the cellular GADD34 gene resulted in a more potent and reliable means of curbing the effects of activated PKR. US11 was retained as a gamma2 gene because, like many viral proteins, it has multiple functions.

Characterization of the herpes simplex virus type 2 (HSV-2) US2 gene product and a US2-deficient HSV-2 mutant.

The herpes simplex virus type 2 (HSV-2) US2 gene product was identified by using a rabbit polyclonal antiserum raised against a recombinant 6 x His-US2 fusion protein expressed in Escherichia coli. The antiserum reacted specifically with a 39 kDa protein in HSV-2 strain 186-infected cell lysates. The protein was not detectable in the presence of the virus DNA synthesis inhibitor phosphonoacetic acid. Indirect immunofluorescence studies localized the US2 protein in the cytoplasm and as discrete granules at late times post-infection within and at the periphery of the nucleus, and nuclear fractionation studies showed that the protein was partially associated with the nuclear matrix of infected cells. The protein was easily detected in purified virions. Also, a US2 insertion mutant was constructed which contained an ICP6-lacZ insertion in the US2 gene. This mutant was as virulent as wild-type virus in mice when inoculated by the footpad route. The importance of the US2 protein of HSV-2 in the virus life-cycle may be apparent only in the natural human host.

The genetic organization and transcriptional analysis of the short unique region in the genome of nononcogenic Marek's disease virus serotype 2

Studies on the Marek's disease virus (MDV) serotype 2 (MDV2) genome may be important for understanding the naturally nononcogenic nature of the virus. To determine the complete DNA sequence of MDV2 unique short (U s) region, genomic BamHI fragments F, M1 and R were sequenced. The MDV2 U s region is 12 109 bp long and contains 12 potential open reading frames (ORFs) likely to encode for proteins. Seven of them exhibit homologies to herpes simplex virus type 1 (HSV-1) US1 (ICP22), US2, US3 (protein kinase), US6 (gD), US7 (gI), US8 (gE) and US10 genes. These ORFs are conserved in a similar arrangement with those of HSV-1, except for US10 which is transposed in the U s regions of all three MDV serotypes. The predicted amino acid sequence of MDV2 ORF6 is homologous to SORF3 of the other serotypes of MDV serotype 1 (MDV1) and herpesvirus of turkeys (HVT) and to infectious laryngotracheitis virus SR1. In addition, four ORFs, which have been identified around the U s and inverted repeat junction regions, have no apparent relation to any other known herpesvirus genes. The identified ORFs in the MDV2 U s region were more colinear with their previously reported locations of MDV1 than with those of HVT and other alphaherpesviruses. Ten of the 12 ORFs in the MDV2 U s region were expressed and transcribed with 3′-coterminal transcripts and/or a unique transcript in the virus-infected cells. Compared to other MDV serotypes, the MDV2 U s-encoded proteins showed 46–70% and 33–59% identities with equivalent of MDV1 and HVT at the amino acid level, respectively. Our present data will be useful to understand the different pathogenicity among serotypes of MDV and to allow precise manipulation of the genes for a possible use in genetically engineered vaccines.

Translational control of viral and host protein synthesis during the course of herpes simplex virus type 1 infection: evidence that initiation of translation is the limiting step.

Herpes simplex virus type 1 (HSV-1) infection induces the selective shut-off of host protein synthesis, other than ribosomal proteins, and the successive synthesis of viral proteins. Because viral mRNAs persist in the cytoplasm after viral protein synthesis has been inhibited, we hypothesized that viral gene expression may be under translational control. Expression of genes encoding immediate early ICP27, early DBP and late US11 proteins, together with glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was monitored over the course of infection at the level of mRNA and protein synthesis. After an efficient synthesis beginning with the appearance of successive viral mRNAs in the cytoplasm, synthesis of viral proteins was shut off similarly to the synthesis of GAPDH. This shut-off was not achieved by mRNA degradation but by progressive shifts of viral mRNAs from large polyribosomes to smaller ones, then to 40S ribosomal subunits. Transient expression of the UL41 gene alone, directing synthesis of virion-associated host shut-off (VHS) protein, induced efficient mRNA degradation, but did not impair recruitment of the remaining GAPDH and beta-actin mRNAs into polyribosomes. These results indicate that HSV-1 induces a selective repression of initiation of mRNA translation which is probably the main cause of the shut-off of viral protein synthesis, and which contributes to the repression of host protein synthesis. VHS protein is not directly involved in this repression, at least in the absence of other viral proteins.

Functional analysis of the human cytomegalovirus US28 gene by insertion mutagenesis with the green fluorescent protein gene.

The protein encoded by the US28 gene of human cytomegalovirus (HCMV) has homology to G protein-coupled receptors (GCR). Previous studies demonstrated that recombinant US28 protein can bind the beta class of chemokines (K. Neote, D. DiGregorio, J. Y. Mak, R. Horuk, and T. J. Schall, Cell 72:415-425, 1993) and induce a rise in intracellular calcium after the binding of chemokines (J. L. Gao and P. M. Murphy, J. Biol. Chem. 269:28539-28542, 1994). In order to investigate the function of the US28 protein in virus-infected cells, a recombinant HCMV (HV5.8) was constructed, with the US28 open reading frame disrupted by the insertion of the Escherichia coli gpt gene and the gene for the green fluorescent protein. The US28 gene is not required for growth in human fibroblasts (HF). HF infected with wild-type HCMV bound RANTES at 24 h postinfection and demonstrated an intracellular calcium flux induced by RANTES. In cells infected with HV5.8, RANTES did not bind or induce a calcium flux, demonstrating that US28 is responsible for the beta-chemokine binding and induced calcium signaling in HCMV-infected cells. The ability of the US28 gene to bind chemokines was shown to cause a significant reduction in the concentration of RANTES in the medium of infected cells. Northern analysis of RNA from infected cells showed that US28 is an early gene, while US27 (another GCR) is a late gene.

Liquid chromatography/tandem mass spectrometry of synthesis products associated with the viral protein US11.

US11 is a small basic protein composed of 161 amino acid residues, and is among the most abundant viral proteins in cells infected with herpes simplex viruses HSV1 and HSV2. The amino acid sequence [91-121] is considered essential for the binding of this protein with RNA. Automated solid phase synthesis of this fragment resulted in a crude reaction mixture containing the desired sequence as well as a number of unknown side products. On-line liquid chromatography/electrospray mass spectrometry (LC/ES-MS) and LC/ES tandem mass spectrometry (MS/MS) allowed the identification of the separated components and furnished relevant sequencing information. The unusual sequences of the monitored components, which consist of a tandemly repeated three-amino-acid motif with the sequence Arg-X-Pro, where X is an acidic or uncharged polar amino acid residue, yielded product ion spectra lacking substantial sequence information and rich in fragment ions manifesting the neutral losses 17, 42 and 60 u.

Distinct domains in herpes simplex virus type 1 US11 protein mediate post-transcriptional transactivation of human T-lymphotropic virus type I envelope glycoprotein gene expression and specific binding to the Rex responsive element.

Herpes simplex virus type 1 (HSV- 1) US11 protein is an RNA-binding protein which is able to mediate post-transcriptional transactivation of human T-lymphotropic virus type I (HTLV-I) envelope glycoprotein gene expression by interacting with the Rex responsive element (XRE) located at the 3' end of the env mRNA. In view of this functional activity, and because US11 protein is capable of substituting for HTLV-I Rex protein, it was hypothesized that US11 protein should exhibit at least two functional domains, an RNA-binding domain for specific interaction with the target RNA, and an effector domain involved in transport and translation of this mRNA. Recombinant US11 wild-type and deleted proteins were tested for their ability (i) to bind to the XRE and to HSV-1 UL34 RNA, the natural target of US11 protein, and (ii) to transactivate HTLV-I env gene expression. The C-terminal half of US11 protein, consisting of 20-24 XPR repeats, was necessary and sufficient to mediate RNA-binding with a high affinity and specificity. Structure prediction analyses showed the likely conformation of this domain to be that of a polyproline type II helix. Localized within the first 40 amino acids of the N-terminal region of US11 protein was the effector domain, deletion of which created US11(delta1-40), a trans-dominant negative mutant. These results demonstrate structural differences between US11 protein and proteins like Rex and Rev, despite their functional similarities.

The Us9 gene product of pseudorabies virus, an alphaherpesvirus, is a phosphorylated, tail-anchored type II membrane protein.

The Us9 gene is highly conserved among the alphaherpesviruses sequenced to date, yet its function remains unknown. In this report, we demonstrate that the pseudorabies virus (PRV) Us9 protein is present in infected cell lysates as several phosphorylated polypeptides ranging from 17 to 20 kDa. Synthesis is first detected at 6 h postinfection and is sensitive to the DNA synthesis inhibitor phosphonoacetic acid. Unlike the herpes simplex virus type 1 Us9 homolog, which was reported to be associated with nucleocapsids in the nuclei of infected cells (M. C. Frame, D. J. McGeoch, F. J. Rixon, A. C. Orr, and H. S. Marsden, Virology 150:321-332, 1986), PRV Us9 localizes to the secretory pathway (predominately to the Golgi apparatus) and not to the nucleus. By fusing the enhanced green fluorescent protein (EGFP) reporter molecule to the carboxy terminus of Us9, we demonstrated that Us9 not only is capable of targeting a Us9-EGFP fusion protein to the Golgi compartment but also is able to direct efficient incorporation of such chimeric molecules into infectious viral particles. Moreover, through protease digestion experiments with Us9-EGFP-containing viral particles, we demonstrated that the Us9 protein is inserted into the viral envelope as a type II, tail-anchored membrane protein.

Subcellular localization of the US3 protein kinase of herpes simplex virus type 2.

One supposes that herpes simplex virus US3 gene product possessing serine/threonine protein kinase activity is a cytoplasmic enzyme. To determine its subcellular localization during viral replication we prepared an antiserum to a synthetic oligopeptide corresponding to the N-terminal region of the US3 protein of HSV type 2 strain 186. The US3 protein first appeared in the cytoplasm of infected cell at 4 h postinfection but strong fluorescence was detected in the nuclei at 8 h postinfection. At 12 h postinfection fluorescence was mainly detected in the cytoplasm, again. Further, the US3 protein expressed alone was widely distributed throughout the cell, indicating that the US3 protein by itself can be localized in the nuclei even in the absence of any other viral proteins. These observations suggest that the HSV-2 US3 protein kinase may function not only in the cytoplasm but also in the nuclei.

The antiviral xanthate compound D609 inhibits herpes simplex virus type 1 replication and protein phosphorylation

The mechanism of antiviral action of tricyclodecan-9-yl-xanthogenate (D609) was investigated in vitro. D609 inhibited herpes simplex virus type 1 (HSV-1) replication without apparent cytotoxicity. It reduced phosphorylation of virus-infected cell polypeptides and inhibited the HSV-1 encoded protein kinase (US3 PK) and, to a lesser extent, cellular protein kinase C in vitro. Virus production was reduced by D609 at concentrations greater than 3.8 μM, with complete inhibition at 75.2 μM at an MOI of 1 PFU/cell or less. Addition of D609 could be delayed until 7 h post-infection and still inhibit virus replication. Phosphorylation of infected cell viral polypeptides of 34 (similar molecular weight to the substrate of the viral US3 protein kinase) and 69 kDa was inhibited at 18.4 μM. Treatment of infected or uninfected cells with 37.6 μM D609 reduced protein phosphorylation to background levels. A concentration of 1.9 μM D609 in vitro inhibited the viral US3-encoded PK, which had been purified from infected cell lysates by affinity chromatography and identified by specific antibody. Purified cellular protein kinase C was inhibited at 75.2 μM D609 whereas other cellular kinases including casein kinase 1 and cAMP dependent kinase were not inhibited at concentrations as high as 188 μM D609. Collectively these data indicate that the mechanism of antiviral action of D609 is by inhibition of protein kinases and protein phosphorylation affecting a late step in HSV replication.

Herpes simplex virus Us11 protein enhances recovery of protein synthesis and survival in heat shock treated HeLa cells.

One of the herpes simplex virus type 1 (HSV-1) true late gene products, Us11 protein, is brought into the cell by the infecting virion and may play a role in the virally-induced post-transcriptional control of gene expression. Us11 protein forms large oligomers, exhibits RNA binding features, concentrates into the nucleolus and is able to replace Rex protein in post-transcriptional control of human T-cell leukemia/lymphoma virus type I (HTLV-I) expression. As heat shock drastically alters protein synthesis, and because HSV-1 infection stimulates heat shock protein (Hsp) expression, we analyzed the consequence of heat shock in HeLa cells expressing Us11 alone, either transiently or constitutively. No detectable modification of the overall pattern of protein synthesis was observed in cells growing at normal temperatures, including no induction of Hsp expression or accumulation. However, Us11 protein expression induced an enhanced recovery of protein synthesis after heat shock. Moreover, the level of Us11 protein-mediated protection of protein synthesis was similar to that observed for cells made thermotolerant, but only when submitted to a mild heat shock. Finally, Us11 protein expression induced in cells an enhanced survival to heat shock.

Partial Substitution of the Functions of the Herpes Simplex Virus 1 UL13 Gene by the Human Cytomegalovirus UL97 Gene

The predicted amino acid sequence of the human cytomegalovirus UL97 protein bears partial homology to the herpes simplex virus 1 UL13 protein, especially in regions that are homologous to conserved domains characteristic of protein kinases. Earlier studies showed that UL13 mediated the posttranslational processing of several herpes simplex virus 1 proteins including ICP22. Whereas no kinase activity has been specifically attributed to UL13, it has been shown that UL97 can phosphorylate ganciclovir. To examine whether UL97 can substitute for UL13, we constructed a herpes simplex virus 1 recombinant virus, R4970, in which UL13 was replaced by UL97 and in addition, the thymidine kinase gene was deleted. Characterization of this recombinant virus showed the following: (1) The recombinant virus grew as well as the wild-type virus in BHKTK+ cells, which restricted the growth of the UL13−virus. (2) UL97 could partially mediate the posttranslational modification of HSV-1 ICP22. This modification correlated with the restoration of the amounts of ICP0 and US11 proteins, which were down regulated in the UL13−virus-infected cells. (3) The recombinant virus was sensitive to ganciclovir in Vero- and KHOS-infected cells but not in the 143 thymidine kinase minus cells derived from KHOS cells. Vero cells infected with this recombinant virus phosphorylated ganciclovir. We conclude that UL97 partially compensates for UL13 functions.

Two distinct upstream regulatory domains containing multicopy cellular transcription factor binding sites provide basal repression and inducible enhancer characteristics to the immediate-early IES (US3) promoter from human cytomegalovirus.

The US3 gene of human cytomegalovirus (HCMV) is expressed at immediate-early (IE) times in permissive HF cells, but not in nonpermissive rodent cells, and encodes several proteins that have been reported to have regulatory characteristics, although they are dispensable for growth in cell culture. Both spliced and unspliced forms of US3 IE transcripts are associated with the second of only two known large and complex upstream enhancer domains within the 229-kb HCMV genome, which we refer to as the IES cis-acting control region. Only the 260-bp proximal segment (from -313 to -55) of the 600-bp IES control domain, which contains multicopy NF-kappaB binding sites, proved to be necessary to transfer both high basal expression plus phorbol ester- and okadaic acid-inducible characteristics to heterologous promoters in transient assays in U-937 and K-562 cells. However, the IES control region contains a distinctive 280-bp distal domain, characterized by the presence of seven interspersed repeats of a 10-bp TGTCGCGACA palindromic consensus motif that encompasses a NruI site. This far upstream Nru repeat region (from -596 to -314) imparted up to 20-fold down-regulation effects onto strong basal heterologous promoters as well as onto the IES enhancer plus minimal promoter region in both U-937 and K-562 cells. Functional Nru repressor elements (NREs) could not be generated by multimerizing either the palindromic (P) Nru motifs alone or adjacent degenerate interrupted (I Nru motifs alone. However, multimerized forms of the combined P plus I elements reconstituted the full 20-fold cis-acting down-regulation phenotype of the intact NRE domain. The P and I forms of the Nru elements each bound independently and specifically to related cellular DNA-binding factors to form differently migrating A or B complexes, respectively, whereas the combined P plus I elements bound cooperatively to both the A and B complexes with high affinity. Interestingly, nuclear extracts from U-937, K-562, HeLa, and Vero cells all formed both the A and B NRE binding factor complexes, whereas those from HF cells produced only A complexes, and Raji, HL60, and BALB/c 3T3 cells lacked both types of binding factor complexes. The core pentameric CGACA and CGATA half sites present in both the P and I Nru motifs are related to recently described Drosophila chromosomal insulator binding sites. Therefore, in addition to its cis-repression or silencer characteristics, the NRE domain appears likely to act to shield adjacent segments of the viral genome from the chromatin-reorganizing effects of the IES-inducible enhancer. We speculate that differential expression and regulation of the IES enhancer-controlled US3 protein, either in concert with or separately from the major IE (MIE) enhancer-controlled IE1 and IE2 transactivator proteins, may play a critical role in determining HCMV permissiveness in some cell types and perhaps also in the establishment of or reactivation from latency.

Structure and function in the herpes simplex virus 1 RNA-binding protein U(s)11: mapping of the domain required for ribosomal and nucleolar association and RNA binding in vitro

The herpes simplex virus 1 US11 protein is an RNA-binding regulatory protein that specifically and stably associates with 60S ribosomal subunits and nucleoli and is incorporated into virions. We report that US11/ beta-galactosidase fusion protein expressed in bacteria bound to rRNA from the 60S subunit and not the 40S subunit. This binding reflects the specificity of ribosomal subunit association. Analyses of deletion mutants of the US11 gene showed that specific RNA binding activity, nucleolar localization, and association with 60S ribosomal subunits were found to map to the amino acid sequences of the carboxyl terminus of US11 protein, suggesting that these activities all reflect specific binding of US11 to large subunit rRNA. The carboxyl-terminal half of the protein consists of a regular tripeptide repeat of the sequence RXP and constitutes a completely novel RNA-binding domain. All of the mutant US11 proteins could be incorporated into virus particles, suggesting that the signal for virion incorporation either is at the amino-terminal four amino acids or is redundant in the protein.

In situ hybridization and immuno-electron microscope analyses of the Us11 gene of herpes simplex virus type 1 during transient expression.

The distribution of Us11 RNA and of its encoded protein have been investigated at the ultrastructural level in HeLa cells transiently expressing the Us11 gene of herpes simplex virus type 1. In these transfected cells, Us11 protein accumulates at sites identical to those of lytically infected cells, i.e., in nucleoli and in regions of the cytoplasm that contain ribosomes. Us11 RNA and polyadenylated RNA are scattered over the ribosome-rich areas of the cytoplasm. They also accumulate in the nucleoplasm on clustered ribonucleoprotein (RNP) fibrils but also in clusters of interchromatin granules, some of them contiguous to nucleoli. However they are never found in nucleoli. These data reveal the involvement of interchromatin granules in some steps of Us11 mRNA maturation and/or transport.

Post-transcriptional transactivation of human retroviral envelope glycoprotein expression by herpes simplex virus Us11 protein.

Herpes simplex virus type 1 (HSV-1) Us11 protein, a true late gene product packaged within the virion, is delivered into cells after infection, exhibits a nucleocytoplasmic localization at early times, and later accumulates in the nucleoli. This RNA-binding basic phosphoprotein, capable of oligomerization, is supposed to be involved in post-transcriptional regulation of gene expression after HSV-1 infection. Expression of human T-cell leukaemia/lymphoma virus type-I (HTLV-I) and of human immunodeficiency virus type 1 (HIV-1) is post-transcriptionally regulated by Rex and Rev, respectively. These proteins are required for the cytoplasmic expression of unspliced gag-pol and singly spliced env transcripts. Here we show that HSV-1 Us11 protein is able to bind Rex- and Rev-responsive elements and to transactivate envelope retroviral glycoprotein expression.

The US3 protein kinase of herpes simplex virus type 2 is associated with phosphorylation of the UL12 alkaline nuclease in vitro.

Herpes simplex virus type 2 (HSV-2) gene US3 encodes a serine-threonine protein kinase. We previously described the isolation of a US3-inactivated mutant which is able to replicate in Vero cells but not in murine macrophages. To learn more about the biological role of the US3 protein kinase, we have sought to identify the target proteins of the enzyme. Studies of in vitro phosphorylation with extracts of infected cells demonstrate that the US3 protein kinase is involved in phosphorylation of the UL12 alkaline nuclease in vitro, suggesting that the nuclease is a possible target of the protein kinase.

The Cytomegalovirus US28 Protein Binds Multiple CC Chemokines with High Affinity

Human cytomegalovirus encodes several proteins with high similarity to seven transmembrane domain receptors. We investigated the ability of one of these proteins, the product of the US28 open reading frame, to bind various chemoattractant ligands. When transfected into COS-7 cells, the US28 product conferred high affinity binding to the labeled chemokines monocyte chemoattractant protein-1 (MCP-1) (Kd = 6.0 × 10−10 M) and RANTES (Kd = 2.7 × 10−10 M). Binding of these labeled ligands could be competed by the unlabeled macrophage inflammatory proteins MIP-1α and MIP-1β, with Kd values in the range 1.2 × 10−9 to 7.5 × 10−9 M. Comparisons of the sequences of US28 and other receptors that bind chemokines should help to define regions responsible for receptor-ligand interactions.