We have determined the nucleotide sequence at the extreme 5' and 3' termini of the hepatitis C virus (HCV) genome. Our analyses ofthese sequences show (t) the nucleotide sequence in the 5' untranslated region is highly conserved among HCV isolates of widely varying geographical orign, (i) within this region, there are blocks of nucleotide sequence homology with pestiviruses but not with other viruses, (ii) the relative position of short open reading frames present in the same region of the HCV genome is similar to that of the pestiviral genome, (iv) RNAs truncated at the 5' and 3' ends are found, but the origin and functions of these RNAs are unknown, and (v) poly(A) tails appear to be present on 3' subgenomic RNAs. (ORFs). The HCV genome, however, displays singular characteristics at each terminus. We detect a hairpin structure at the 5' end of the genome as well as 5' and 3' subgenomic RNAs, the latter of which are polyadenylylated. These are consistent with a polyadenylylated 3' terminus of the viral genome and perhaps of functional subgenomic RNAs. Our data provide insights into the organization of the HCV genome, which may have important ramifications regarding the replication strategy and evolution of the virus. MATERIALS AND METHODSRNA was extracted from a high-titer plasma of an experimentally infected chimpanzee (6) and plasma (or serum) from HCV-positive or negative blood donors by a low-temperature guanidinium thiocyanate method (7). Poly(A)+ RNA was isolated from the liver ofthe same infected chimpanzee by the guanidinium thiocyanate/urea method (8). cDNA was synthesized from RNA according to Han and Rutter (9) and amplified by polymerase chain reaction (PCR) according to Saiki et al. (10). Briefly, RNA isolated from about 500 Al of plasma or S Ag of poly(A)+ liver RNA was converted into single-stranded cDNA by reverse transcriptase (BRL) using 150 pmol of the appropriate cDNA primer. For 5' end characterization by primer extension (7), first-strand cDNA was precipitated by spermine (11) and tailed with dA (9). Tailed or untailed cDNA was converted into double-stranded cDNA using a second-strand cDNA primer (9). This doublestranded cDNA was amplified using the indicated HCVspecific PCR primers for 35 cycles (940C, 1.5 min; 60'C, 2 min; 720C, 3 min). PCR without cDNA template was routinely performed to check for possible contamination during PCR. The PCR product was analyzed by Southern blot hybridization using a 32P-labeled oligonucleotide probe. The sequences and locations in the HCV genome of various cDNA and PCR primers are shown in Figs. 2 and 5. Most primers were designed to contain a Not I site for subsequent cloning of the PCR products into pUC18S, which contains a polylinker derived from Agt22 (9). DNA sequence was obtained by the supercoil sequencing (12) or the direct PCR sequencing method (13). RESULTSBased on the sequence of the major part of the HCV genome determined from overlapping A clones (2), we devised a directed strategy for obtaining clones representing the remaini...

A cDNA clone of murine macrophage inflammatory protein 2 (MIP-2) has been isolated from a library prepared from lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and the nucleotide sequence determined. This cDNA was used to clone cDNAs for human homologues of MIP-2 from a library prepared from phorbol myristate acetate-treated and LPS-stimulated U937 cells. Two homologues were isolated and sequenced. Human MIP-2 alpha and MIP-2 beta are highly homologous to each other and to a previously isolated gene, human gro/melanoma growth-stimulating activity (MGSA). These three human genes, MIP-2 alpha, MIP-2 beta, and gro/MGSA, constitute a sub-family within the cytokine family represented by platelet factor 4 and interleukin 8.

Macrophages secrete a wide variety of proteins that mediate many aspects of acute and chronic inflammation (for review see reference 1). While some of these factors have been well characterized (e.g., IL-1 [2] and cachectin/TNF [3]), others remain poorly defined . Recently, we described the purification and characterization ofa new monokine found in the culture medium of an LPS-stimulated mouse macrophage tumor cell line (RAW264 .7) (4). This protein, termed macrophage inflammatory protein or MIP, has several properties indicative ofan endogenous mediator ofinflammation (e.g ., neutrophil attraction and activation) . Since MIP represents an important new addition to the family of activated macrophage products, it is important to investigate its structure and regulation on the molecular level. Here we describe the cloning and sequencing of the cDNA for murine MIP. Materials and MethodsConstruction ofthe cDNA Library. RAW264.7 cells were obtained from American Type Culture Collection (Rockville, MD) and grown in RPMI 1640 (Gibco Laboratories, Grand Island, NY) supplemented with 20 mM Hepes and 10% FCS (HyClone Laboratories, Logan, UT) until they reached confluency. The cells were then washed five times in HBSS (Gibco Laboratories) and the medium was replaced with serum-free RPMI supplemented with 1 Rg/ml of LPS W (Escherichia coli 0127:B8, Difco Laboratories, Detroit, MI). The cells were incubated at 37°C for 2 h and total RNA was extracted by the addition of6 M guanidinium thiocyanate (5) . Poly(A)' RNA was then isolated by two cycles of oligo-dT-cellulose chromatography, essentially as described by Maniatis et al. (6) . Double-stranded cDNA was prepared from the poly(A)' selected RNA as described by Gubler and Hoffman (7) . After methylation of the internal Eco RI sites and addition of Eco RI linkers, the cDNA was inserted into the Eco RI sites of the bacteriophage %gtl0 (8) .Construction of the Probe Pools . Oligonucleotide probe pools were synthesized as described by Warner et al. (9) against amino acids 22-30 of a partial NH2-terminal sequence . This portion ofthe polypeptide was selected because ofits lower degeneracy in the codon dictionary when compared with the remainder of the sequence . The resulting probe pools are two 512-fold degenerate pools of 26 nucleotides in length.

A number of macrophage-derived mediators have been implicated in the vascular changes of inflammation. We recently reported the isolation of a novel monokine, macrophage inflammatory protein 1 (MIP-1), which causes local inflammatory responses in vivo, and induces superoxide production by neutrophils in vitro. Purified native MIP-1 comprises two peptides with very similar physical characteristics. We report here the resolution of MIP-1 into component peptides by SDS-hydroxylapatite chromatography, and compare the NH2-terminal sequences of the two peptides, now referred to as MIP-1 alpha and MIP-1 beta. A synthetic oligonucleotide probe pool corresponding to the NH2-terminal amino acid sequence of MIP-1 beta was used to isolate a cDNA clone containing its coding sequence. The sequence codes for a 109 amino acid-long polypeptide, of which 69 amino acids correspond to the mature product. Comparison of this MIP-1 beta cDNA with our previously cloned MIP-1 alpha sequence reveals that the MIP-1 peptides, members of a growing family of potential inflammatory mediators, are distinct but highly homologous (58.9% sequence identity) products of different genes.

To determine the regions of interleukin-8 (IL-8) that allow high affinity and interleukin-8 receptor type 1 (IL8R1)-specific binding of chemokines, we produced chimeric proteins containing structural domains from IL-8, which binds to both IL8R1 and interleukin-8 receptor type 2 (IL8R2) with high affinity, and from GRO␥, which does not bind to IL8R1 and binds to IL8R2 with reduced affinity.

We have previously shown that treatment of endothelial cells with minimally modified LDL (MM-LDL) induces the binding of monocytes to unknown endothelial receptor molecules. We now report that a member of the GRO family of chemokines plays a role in MM-LDL-induced monocyte binding. A cDNA library made from rabbit aortic endothelial cells (RAEC) treated with MM-LDL was expression screened for molecules inducing binding of a human monocyte cell line (THP-1). A cDNA was isolated with 75% homology to GRO. GRO mRNA levels were significantly elevated after exposure of RAEC or human aortic endothelial cells (HAEC) to MM-LDL. HAEC treated with MM-LDL displayed an increase in a surface-associated protein that bound to antibody against GRO despite low levels of GRO in the medium. Antibody to GRO significantly inhibited the binding of monocytes to MM-LDL-treated RAEC and HAEC. The increase in GRO expression and monocyte binding were reduced by incubating MM-LDL-treated endothelial cells with heparin (in a method that releases heparan sulfate bound molecules from the cell surface). These results suggest that GRO related chemokines are bound to the surface of MM-LDL-treated endothelial cells and may contribute to the monocyte adhesion induced by MM-LDL. (J. Clin.

Purified recombinant (r) macrophage inflammatory proteins (MIPs) 1 alpha, 1 beta, and 2 were assessed for effects on murine (mu) and human (hu) marrow colony-forming unit-granulocyte-macrophage (CFU-GM) and burst-forming unit-erythroid (BFU-E) colonies. Recombinant MIP-1 alpha, -1 beta, and -2 enhanced muCFU-GM colonies above that stimulated with 10 to 100 U natural mu macrophage-colony-stimulating factor (M-CSF) or rmuGM-CSF, with enhancement seen on huCFU-GM colony formation stimulated with suboptimal rhuM-CSF or rhuGM-CSF; effects were neutralized by respective MIP-specific antibodies. Macrophage inflammatory proteins had no effects on mu or huBFU-E colonies stimulated with erythropoietin (Epo). However, natural MIP-1 and rMIP-1 alpha, but not rMIP-1 beta or -2, suppressed muCFU-GM stimulated with pokeweed mitogen spleen-conditioned medium (PWMSCM), huCFU-GM stimulated with optimal rhuGM-CSF plus rhu interleukin-3 (IL-3), muBFU- E and multipotential progenitors (CFU-GEMM) stimulated with Epo plus PWMSCM, and huBFU-E and CFU-GEMM stimulated with Epo plus rhuIL-3 or rhuGM-CSF. The suppressive effects of natural MIP-1 and rMIP-1 alpha were also apparent on a population of BFU-E, CFU-GEMM, and CFU-GM present in cell-sorted fractions of human bone marrow (CD34 HLA-DR+) highly enriched for progenitors with cloning efficiencies of 42% to 75%. These results, along with our previous studies, suggest that MIP-1 alpha, -1 beta, and -2 may have direct myelopoietic enhancing activity for mature progenitors, while MIP-1 alpha may have direct suppressing activity for more immature progenitors.

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