Abstract: Murine gammaherpesvirus 68 (gammaHV68) infects mice, thus providing a tractable small-animal model for analysis of the acute and chronic pathogenesis of gammaherpesviruses. To facilitate molecular analysis of gammaHV68 pathogenesis, we have sequenced the gammaHV68 genome. The genome contains 118,237 bp of unique sequence flanked by multiple copies of a 1,213-bp terminal repeat. The GC content of the unique portion of the genome is 46%, while the GC content of the terminal repeat is 78%. The unique portion of the genome is estimated to encode at least 80 genes and is largely colinear with the genomes of Kaposi's sarcoma herpesvirus (KSHV; also known as human herpesvirus 8), herpesvirus saimiri (HVS), and Epstein-Barr virus (EBV). We detected 63 open reading frames (ORFs) homologous to HVS and KSHV ORFs and used the HVS/KSHV numbering system to designate these ORFs. gammaHV68 shares with HVS and KSHV ORFs homologous to a complement regulatory protein (ORF 4), a D-type cyclin (ORF 72), and a G-protein-coupled receptor with close homology to the interleukin-8 receptor (ORF 74). One ORF (K3) was identified in gammaHV68 as homologous to both ORFs K3 and K5 of KSHV and contains a domain found in a bovine herpesvirus 4 major immediate-early protein. We also detected 16 methionine-initiated ORFs predicted to encode proteins at least 100 amino acids in length that are unique to gammaHV68 (ORFs M1 to 14). ORF M1 has striking homology to poxvirus serpins, while ORF M11 encodes a potential homolog of Bcl-2-like molecules encoded by other gammaherpesviruses (gene 16 of HVS and KSHV and the BHRF1 gene of EBV). In addition, clustered at the left end of the unique region are eight sequences with significant homology to bacterial tRNAs. The unique region of the genome contains two internal repeats: a 40-bp repeat located between bp 26778 and 28191 in the genome and a 100-bp repeat located between bp 98981 and 101170. Analysis of the gammaHV68, HVS, EBV, and KSHV genomes demonstrated that each of these viruses have large colinear gene blocks interspersed by regions containing virus-specific ORFs. Interestingly, genes associated with EBV cell tropism, latency, and transformation are all contained within these regions encoding virus-specific genes. This finding suggests that pathogenesis-associated genes of gammaherpesviruses, including gammaHV68, may be contained in similarly positioned genome regions. The availability of the gammaHV68 genomic sequence will facilitate analysis of critical issues in gammaherpesvirus biology via integration of molecular and pathogenetic studies in a small-animal model.

Abstract: We have investigated the organization and function of human immunodeficiency virus type 1 (HIV-1) preintegration complexes (PICs), the large nucleoprotein particles that carry out cDNA integration in vivo. PICs can be isolated from HIV-1-infected cells, and such particles are capable of carrying out integration reactions in vitro. We find that although the PICs are large, the cDNA must be condensed to fit into the measured volume. The ends of the cDNA are probably linked by a protein bridge, since coordinated joining of the two ends is not disrupted by cleaving the cDNA internally with a restriction enzyme. cDNA ends in PICs were protected from digestion by added exonucleases, probably due to binding of proteins. The intervening cDNA, in contrast, was susceptible to attack by endonucleases. Previous work has established that the virus-encoded integrase protein is present in PICs, and we have reported recently that the host protein HMG I(Y) is also present. Here we report that the viral matrix and reverse transcriptase (RT) proteins also cofractionated with PICs through several steps whereas capsid and nucleocapsid proteins dissociated. These data support a model of PIC organization in which the cDNA is condensed in a partially disassembled remnant of the viral core, with proteins tightly associated at the apposed cDNA ends but loosely associated with the intervening cDNA. In characterizing the structure of the cDNA ends, we found that the U5 DNA ends created by RT were ragged, probably due to the terminal transferase activity of RT. Only molecules correctly cleaved by integrase protein at the 3' ends were competent to integrate, suggesting that one role for terminal cleavage by integrase may be to create a defined end at otherwise heterogeneous cDNA termini.

Abstract: The recent discovery of DNA sequences of a new human herpesvirus in Kaposi's sarcoma (KS) has fueled speculation that this virus might cause KS. The mere presence, however, of a virus in a complex multicellular tumor like KS could just as well be construed as evidence of a passenger agent. We sought stronger evidence linking the KS-associated herpesvirus (KSHV) to tumor formation by using in situ hybridization to investigate the specificity, constancy, and timing of KSHV gene expression in KS tumor cells. Here we document expression of a 700-nucleotide viral RNA in every KS tumor examined, from the earliest histologically recognizable stage to advanced tumors in which the vast majority of identifiable spindle tumor cells contain this transcript. Two other KSHV RNAs were also detected in a smaller fraction of the tumor cells in all but the earliest lesion. These viral RNAs were expressed to relatively low levels in this subset; because one of these RNAs encodes a major viral capsid protein, these cells may be producing KSHV. We did not find these KSHV genes expressed in a variety of other tumors and proliferative processes, but we did detect viral gene expression in prostatic tissue, supporting a possible mechanism for sexual transmission of KSHV. The close relationship between KS and KSHV gene expression is consistent with the hypothesis that KSHV is directly involved in the etiology and pathogenesis of KS.

Abstract: The biological phenotype of primary human immunodeficiency virus type 1 (HIV-1) isolates varies according to the severity of the HIV infection. Here we show that the two previously described groups of rapid/high, syncytium-inducing (SI) and slow/low, non-syncytium-inducing (NSI) isolates are distinguished by their ability to utilize different chemokine receptors for entry into target cells. Recent studies have identified the C-X-C chemokine receptor CXCR4 (also named fusin or Lestr) and the C-C chemokine receptor CCR5 as the principal entry cofactors for T-cell-line-tropic and non-T-cell-line-tropic HIV-1, respectively. Using U87.CD4 glioma cell lines, stably expressing the chemokine receptor CCR1, CCR2b, CCR3, CCR5, or CXCR4, we have tested chemokine receptor specificity for a panel of genetically diverse envelope glycoprotein genes cloned from primary HIV-1 isolates and have found that receptor usage was closely associated with the biological phenotype of the virus isolate but not the genetic subtype. We have also analyzed a panel of 36 well-characterized primary HIV-1 isolates for syncytium induction and replication in the same series of cell lines. Infection by slow/low viruses was restricted to cells expressing CCR5, whereas rapid/high viruses could use a variety of chemokine receptors. In addition to the regular use of CXCR4, many rapid/high viruses used CCR5 and some also used CCR3 and CCR2b. Progressive HIV-1 infection is characterized by the emergence of viruses resistant to inhibition by beta-chemokines, which corresponded to changes in coreceptor usage. The broadening of the host range may even enable the use of uncharacterized coreceptors, in that two isolates from immunodeficient patients infected the parental U87.CD4 cell line lacking any engineered coreceptor. Two primary isolates with multiple coreceptor usage were shown to consist of mixed populations, one with a narrow host range using CCR5 only and the other with a broad host range using CCR3, CCR5, or CXCR4, similar to the original population. The results show that all 36 primary HIV-1 isolates induce syncytia, provided that target cells carry the particular coreceptor required by the virus.

Abstract: The NS5B protein of the hepatitis C virus (HCV) is an RNA-dependent RNA polymerase (RdRp) (S.-E. Behrens, L. Tomei, and R. De Francesco, EMBO J. 15:12-22, 1996) that is assumed to be required for replication of the viral genome. To further study the biochemical and structural properties of this enzyme, an NS5B-hexahistidine fusion protein was expressed with recombinant baculoviruses in insect cells and purified to near homogeneity. The enzyme was found to have a primer-dependent RdRp activity that was able to copy a complete in vitro-transcribed HCV genome in the absence of additional viral or cellular factors. Filter binding assays and competition experiments showed that the purified enzyme binds RNA with no clear preference for HCV 3'-end sequences. Binding to homopolymeric RNAs was also examined, and the following order of specificity was observed: poly(U) > poly(G) > poly(A) > poly(C). An inverse order was found for the RdRp activity, which used poly(C) most efficiently as a template but was inactive on poly(U) and poly(G), suggesting that a high binding affinity between polymerase and template interferes with processivity. By using a mutational analysis, four amino acid sequence motifs crucial for RdRp activity were identified. While most substitutions of conserved residues within these motifs severely reduced the enzymatic activities, a single substitution in motif D which enhanced the RdRp activity by about 50% was found. Deletion studies indicate that amino acid residues at the very termini, in particular the amino terminus, are important for RdRp activity but not for RNA binding. Finally, we found a terminal transferase activity associated with the purified enzyme. However, this activity was also detected with NS5B proteins with an inactive RdRp, with an NS4B protein purified in the same way, and with wild-type baculovirus, suggesting that it is not an inherent activity of NS5B.

Abstract: Flaviviruses are assembled intracellularly in an immature form containing heterodimers of two envelope proteins, E and prM. Shortly before the virion exits the cell, prM is cleaved by a cellular enzyme, and this processing step can be blocked by treatment with agents that raise the pH of exocytic compartments. We carried out in vivo and in vitro studies with tick-borne encephalitis (TBE) virus to investigate the possible role of furin in this process as well as the functional consequences of prM cleavage. We found that prM in immature virions can be correctly cleaved in vitro by recombinant bovine furin but that efficient cleavage occurs only after exposure of the virion to mildly acidic pH. The data suggest that exposure to an acidic environment induces an irreversible structural change that renders the cleavage site accessible to the enzyme. Cleavage by furin in vitro resulted in biological activation, as shown by a 100-fold increase in specific infectivity, the acquisition of membrane fusion and hemagglutination activity, and the ability of the envelope proteins to undergo low-pH-induced structural rearrangements characteristic of mature virions. In vivo, prM cleavage was blocked by a furin inhibitor, and infection of the furin-deficient cell line LoVo yielded only immature virions, suggesting that furin is essential for cleavage activation of flaviviruses.

Abstract: We examined intratype human papillomavirus type 16 (HPV-16) sequence variation in tumor samples that were collected and analyzed in an international study of invasive cervical cancer. The collection included tumors from 22 countries in five continents. Using our recently developed E6 and L1 PCR-based hybridization systems to distinguish HPV-16 variant lineages, we analyzed material from tumors previously found to contain HPV-16 DNA. Of 408 specimens analyzed in the E6 hybridization assay, 376 (92.2%) belonged to previously reported HPV-16 variant lineages. The remaining 32 specimens (7.8%) harbored HPV-16 variants with novel hybridization patterns, novel nucleotide changes, or both. Nucleotide sequences (1,203 bp) were determined for the E6, the MY09/11 region of L1, and the long control region of each novel variant and representative specimens from each hybridization pattern observed. Based on E6 hybridization patterns, most of the variants from European and North American samples were phylogenetically classified as European prototype (E) while samples from Africa contained primarily African 1 (Af1) or African 2 (Af2) variants. The majority of Asian (As) variants were observed in Southeast Asia, and almost all Asian American (AA) variants were from Central and South America or Spain. A single North American 1 (NA1) variant was detected in a tumor from Argentina. Nucleotide changes previously shown to covary between the MY09/11 region of L1 and the E6 coding region were examined in a subset of 249 specimens. We observed 22 combined E6-L1 hybridization patterns, of which 11 (in 21 samples) were novel. No unanticipated nucleotide covariation was observed between the E class and the AA-Af1-Af2-NA1 classes, suggesting the absence or rarity of genomic recombination between HPV-16 lineages. This extensive description of HPV-16 variants forms a basis for further examining the relationship between intratype variation and basic functional differences in biological activities. HPV-16 variants may prove important for the determination of the risk of cervical neoplasia and for the design of HPV-16 vaccine strategies.

Abstract: Systemic application of first-generation adenovirus induces pathogenic effects in the liver. To begin unraveling the mechanisms underlying early liver toxicity after adenovirus infusion, particularly the role of macrophage activation and expression of viral genes in transduced target cells, first-generation adenovirus or adenovirus vectors that lacked most early and late gene expression were administered to C3H/HeJ mice after transient depletion of Kupffer cells by gadolinium chloride treatment. Activation of NF-kappaB, and the serum levels of the proinflammatory cytokines tumor necrosis factor (TNF) and interleukin-6 (IL-6) were studied in correlation with liver damage, apoptosis, and hepatocellular DNA synthesis. While Kupffer cell depletion nearly eliminated adenovirus-induced TNF release, it resulted in a more robust IL-6 release. These responses were greatly reduced in animals receiving the deleted adenovirus. Although there were quantitative differences, NF-kappaB activation was observed within minutes of first-generation or deleted adenovirus vector administration regardless of the status of the Kupffer cells, suggesting that the induction is related to a direct effect of the virus particle on the hepatocyte. Early liver toxicity as determined by serum glutamic-pyruvic transaminase elevation and inflammatory cell infiltrates appeared to be dependent on adenovirus-mediated early gene expression and intact Kupffer cell function. Kupffer cell depletion had little effect on adenovirus-mediated hepatocyte apoptosis but did increase hepatocellular DNA synthesis. Finally, Kupffer cell depletion decreased the persistence of transgene (human alpha1-antitrypsin [hAAT]) expression that was associated with a more pronounced humoral immune response against hAAT. The elucidation of these events occurring after intravenous adenovirus injection will be important in developing new vectors and transfer techniques with reduced toxicity.

Abstract: Mutational analysis of the nonstructural protein 1 (NS1) of yellow fever virus (YF) has implicated it in viral RNA replication. To further explore this observation, we sought a method for uncoupling NS1 function from NS1 expression and processing as part of the large YF polyprotein. Here we describe a strategy for providing NS1 in trans, utilizing a noncytopathic Sindbis virus vector. Replication of a defective YF genome containing a large in-frame deletion of NS1 was dependent on functional expression of NS1. Recovered mutant virus was shown to contain the deletion and was neutralized by YF-specific antiserum. Complemented mutant virus increased in titer with kinetics similar to those of parental YF 17D but peaked at lower titers. trans-complementation has allowed us to derive high-titer, helper-free stocks of YF defective in NS1 with which to further characterize the role of this gene product in RNA replication. The first cycles of RNA replication were analyzed by using a sensitive strand-specific RNase protection assay. We document these events for mutant and wild-type viruses in the presence or absence of complementation. These data strongly suggest a role for NS1 prior to or at initial minus-strand synthesis.

Abstract: Although CD8+ lymphocytes in human immunodeficiency virus type 1 (HIV-1)-infected individuals have been demonstrated to suppress viral replication, the mechanisms of inhibition have not been defined precisely. A large body of evidence indicates that these cells act via soluble inhibitory factors, but the potential role of HLA class I-restricted cytolysis has remained controversial. Here we demonstrate that HIV-1-specific cytotoxic T lymphocytes (CTL) mediate antiviral suppression by both cytolytic and noncytolytic mechanisms. The predominant mechanism requires direct contact of CTL with the infected cells, is HLA class I restricted, and can achieve complete elimination of detectable virus in infected cell cultures. Inhibition occurs even at high multiplicities of infection or at ratios of CTL to CD4 cells as low as 1:1,000. The other mechanism is mediated by soluble inhibitory factors which are triggered in an antigen-specific and HLA-restricted fashion but then act without HLA restriction. These include MIP-1alpha, MIP-1beta, and RANTES, as well as a distinct factor(s) capable of inhibiting HIV-1 strains insensitive to these chemokines. These data indicate that HIV-1-specific CTL are potent mediators of HIV-1 suppression at cell ratios existing in vivo and demonstrate an antigen-specific trigger for CD8+ cell-derived soluble inhibitory factors. These results suggest that CTL play an important role in the observed antiviral activity of CD8+ cells from infected individuals.

Abstract: Two different responses to the therapy were observed in a group of patients receiving the protease inhibitor indinavir. In one, suppression of virus replication occurred and has persisted for 90 weeks (bDNA, < 500 human immunodeficiency virus type 1 [HIV-1] RNA copies/ml). In the second group, a rebound in virus levels in plasma followed the initial sharp decline observed at the start of therapy. This was associated with the emergence of drug-resistant variants. Sequence analysis of the protease gene during the course of therapy revealed that in this second group there was a sequential acquisition of protease mutations at amino acids 46, 82, 54, 71, 89, and 90. In the six patients in this group, there was also an identical mutation in the gag p7/p1 gag protease cleavage site. In three of the patients, this change was seen as early as 6 to 10 weeks after the start of therapy. In one patient, a second mutation occurred at the gag p1/p6 cleavage site, but it appeared 18 weeks after the time of appearance of the p7/p1 mutation. Recombinant HIV-1 variants containing two or three mutations in the protease gene were constructed either with mutations at the p7/p1 cleavage site or with wild-type (WT) gag sequences. When recombinant HIV-1-containing protease mutations at 46 and 82 was grown in MT2 cells, there was a 68% reduction in its rate of replication compared to the WT virus. Introduction of an additional mutation at the gag p7/p1 protease cleavage site compensated for the partially defective protease gene. Similarly, rates of replication of viruses with mutations M46L/I, I54V, and V82A in protease were enhanced both in the presence and in the absence of Indinavir when combined with mutations in the gag p7/p1 and the gag p1/p6 cleavage sites. Optimal rates of virus replication require protease cleavage of precursor polyproteins. A mutation in the cleavage site that enhanced the availability of a protein that was rate limiting for virus maturation would confer on that virus a significant growth advantage and may explain the uniform emergence of viruses with alterations at the p7/p1 cleavage site. This is the first report of the emergence of mutations in the gag p7/p1 protease cleavage sites in patients receiving protease therapy and identifies this change as an important determinant of HIV-1 resistance to protease inhibitors in patient populations.

Abstract: The hepatitis C virus (HCV) glycoproteins (E1 and E2) interact to form a heterodimeric complex, which has been proposed as a functional subunit of the HCV virion envelope. As examined in cell culture transient-expression assays, the formation of properly folded, noncovalently associated E1E2 complexes is a slow and inefficient process. Due to lack of appropriate immunological reagents, it has been difficult to distinguish between glycoprotein molecules that undergo productive folding and assembly from those which follow a nonproductive pathway leading to misfolding and aggregation. Here we report the isolation and characterization of a conformation-sensitive E2-reactive monoclonal antibody (H2). The H2 monoclonal antibody selectively recognizes slowly maturing E1E2 heterodimers which are noncovalently linked, protease resistant, and no longer associated with the endoplasmic reticulum chaperone calnexin. This complex probably represents the native prebudding form of the HCV glycoprotein heterodimer. Besides providing a novel reagent for basic studies on HCV virion assembly and entry, this monoclonal antibody should be useful for optimizing production and isolation of native HCV glycoprotein complexes for serodiagnostic and vaccine applications.

Abstract: In acute hepatitis C virus infection, 50 to 70% of patients develop chronic disease. Considering the low rate of spontaneous viral clearance during chronic hepatitis C infection, the first few months of interaction between the patient’s immune system and the viral population seem to be crucial in determining the outcome of infection. We previously reported the association between a strong and sustained CD4 1 T-cell response to nonstructural protein 3 (NS3) of the hepatitis C virus and a self-limited course of acute hepatitis C infection. In this study, we identify an immunodominant CD4 1 T-cell epitope (amino acids 1248 to 1261) that was recognized by the majority (14 of 23) of NS3-specific CD4 1 T-cell clones from four of five patients with acute hepatitis C infection. This epitope can be presented to CD4 1 T cells by HLA-DR4, -DR11, -DR12, -DR13, and -DR16. HLA-binding studies revealed a high binding affinity for 10 of 13 common HLA-DR alleles. Two additional CD4 1 T-cell epitopes, amino acids 1388 to 1407 and amino acids 1450 to 1469, showed a very narrow pattern of binding to individual HLA-DR alleles. Our data suggest that the NS3-specific CD4 1 T-cell response in acute hepatitis C infection is dominated by a single, promiscuous peptide epitope which could become a promising candidate for the development of a CD4 1 T-cell vaccine. Hepatitis C virus (HCV) infection has an estimated worldwide prevalence of 0.3 to 1.5% and is a leading cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma (1, 15). More than 50% of acute infections lead to chronic disease (2), and once chronic infection is established, spontaneous recovery is exceptional. Therefore, characterization of the antiviral immune response during the first few weeks of acute hepatitis C infection in patients with self-limited disease as opposed to those developing chronic hepatitis C may allow the identification of successful antiviral immune strategies. In two recent studies of patients with acute hepatitis C infection, a strong association between a vigorous and sustained HCVspecific CD4 1 T-cell response and a self-limited course of acute hepatitis C infection could be demonstrated (5, 16). Although the CD4 1 T-cell response was directed against several HCV antigens (core, E2, nonstructural protein 3 [NS3], NS4, and NS5), in the majority of patients with self-limited disease, the response to NS3 was frequently strongest and was detected most consistently. In this study, we identify one immunodominant CD4 1 T-cell epitope within the NS3 protein that is recognized by the majority of patients with self-limited acute hepatitis C infection and which binds promiscuously to the most common HLA-DR alleles.

Abstract: Hepatitis E virus (HEV) is a pathogenic agent that causes fecally-orally transmitted acute hepatitis. The genome, a single-stranded positive-sense RNA, encodes three forward open reading frames (ORFs), in which an approximately 2-kb structural protein is located in the 3' end. To produce HEV-like particles the structural protein, with its N terminus truncated (amino acid residues 112 to 660 of ORF2), was expressed in insect Tn5 cells by a recombinant baculovirus. In addition to the primary translation product with a molecular mass of 58 kDa, a large amount of a further-processed molecule with a molecular mass of 50 kDa was generated and efficiently released into the culture medium. Electron microscopic observation of the culture medium revealed that the 50-kDa protein self-assembled to form empty virus-like particles (VLPs). The buoyant density of the VLPs in CsCl was 1.285 g/cm3 and their diameter was 23.7 nm, a little smaller than the 27 nm of native HEV particles secreted into the bile or stools of experimentally infected monkeys. The yield of the VLPs was 1 mg per 10(7) cells as a purified form. The particles possess antigenicity similar to that of authentic HEV particles and, consequently, they appear to be a good antigen for the sensitive detection of HEV-specific immunoglobulin G (IgG) and IgM antibodies. Furthermore, the VLP may be the most promising candidate yet for an HEV vaccine, owing to its potent immunogenicity.

Abstract: Infection of B lymphocytes by Epstein-Barr virus (EBV) requires attachment of virus via binding of viral glycoprotein gp350 to CD21 on the cell surface. Penetration of the cell membrane additionally involves a complex of three glycoproteins, gH, gL, and gp42. Glycoprotein gp42 binds to HLA-DR. Interference with this interaction with a soluble form of gp42, with a monoclonal antibody (MAb) to gp42, or with a MAb to HLA-DR inhibited virus infection. It was not possible to superinfect cells that failed to express HLA-DR unless expression was restored by transfection or creation of hybrid cell lines with complementing deficiencies in expression of HLA class II. HLA class II molecules thus serve as cofactors for infection of human B cells.

Abstract: Human immunodeficiency virus type 1 (HIV-1) requires both CD4 and a coreceptor to infect cells. Macrophage-tropic (M-tropic) HIV-1 strains utilize the chemokine receptor CCR5 in conjunction with CD4 to infect cells, while T-cell-tropic (T-tropic) strains generally utilize CXCR4 as a coreceptor. Some viruses can use both CCR5 and CXCR4 for virus entry (i.e., are dual-tropic), while other chemokine receptors can be used by a subset of virus strains. Due to the genetic diversity of HIV-1, HIV-2, and simian immunodeficiency virus (SIV) and the potential for chemokine receptors other than CCR5 or CXCR4 to influence viral pathogenesis, we tested a panel of 28 HIV-1, HIV-2, and SIV envelope (Env) proteins for the ability to utilize chemokine receptors, orphan receptors, and herpesvirus-encoded chemokine receptor homologs by membrane fusion and virus infection assays. While all Env proteins used either CCR5 or CXCR4 or both, several also used CCR3. Use of CCR3 was strongly dependent on its surface expression levels, with a larger number of viral Env proteins being able to utilize this coreceptor at the higher levels of surface expression. ChemR1, an orphan receptor recently shown to bind the CC chemokine I309 (and therefore renamed CCR8), was expressed in monocyte and lymphocyte cell populations and functioned as a coreceptor for diverse HIV-1, HIV-2, and SIV Env proteins. Use of ChemR1/CCR8 by SIV strains was dependent in part on V3 loop sequences. The orphan receptor V28 supported Env-mediated cell-cell fusion by four T- or dual-tropic HIV-1 and HIV-2 strains. Three additional orphan receptors failed to function for any of the 28 Env proteins tested. Likewise, five of six seven-transmembrane-domain receptors encoded by herpesviruses did not support Env-mediated membrane fusion. However, the chemokine receptor US28, encoded by cytomegalovirus, did support inefficient infection by two HIV-1 strains. These findings indicate that additional chemokine receptors can function as HIV and SIV coreceptors and that surface expression levels can strongly influence coreceptor use.

Abstract: Several Kunjin virus (KUN) subgenomic replicons containing large deletions in the structural region (C-prM-E) and in the 3' untranslated region (3'UTR) of the genome have been constructed. Replicon RNA deltaME with 1,987 nucleotides deleted (from nucleotide 417 [in codon 108] in the C gene to nucleotide 2403 near the carboxy terminus of the E gene, inclusive) and replicon RNA C20rep with 2,247 nucleotides deleted (from nucleotide 157 [in codon 20] in C to nucleotide 2403) replicated efficiently in electroporated BHK21 cells. A further deletion from C20rep of 53 nucleotides, reducing the coding sequence in core protein to two codons (C2rep RNA), resulted in abolishment of RNA replication. Replicon deltaME/76 with a deletion of 76 nucleotides in the 3'UTR of deltaME RNA (nucleotides 10423 to 10498) replicated efficiently, whereas replicon deltaME/352 with a larger deletion of 352 nucleotides (nucleotides 10423 to 10774), including two conserved sequences RCS3 and CS3, was significantly inhibited in RNA replication. To explore the possibility of using a reporter gene assay to monitor synthesis of the positive strand and the negative strand of KUN RNA, we inserted a chloramphenicol acetyltransferase (CAT) gene into the 3'UTR of deltaME/76 RNA under control of the internal ribosomal entry site (IRES) of encephalomyelocarditis virus RNA in both plus (deltaME/76CAT[+])- and minus (deltaME/76CAT[-])-sense orientations. Although insertion of the IRES-CAT cassette in the plus-sense orientation resulted in a significant (10- to 20-fold) reduction of RNA replication compared to that of the parental deltaME/76 RNA, CAT expression was readily detected in electroporated BHK cells. No CAT expression was detected after electroporation of RNA containing the IRES-CAT cassette inserted in the minus-sense orientation despite its apparently more efficient replication (similar to that of deltaME/76 RNA); this result indicated that KUN negative-strand RNA was probably not released from its template after synthesis. Replacement of the CAT gene in the deltaME/76CAT(+) RNA with the neomycin gene (Neo) enabled selection and recovery of a BHK cell culture in which the majority of cells were continuously expressing the replicon RNA for 41 days (nine passages) without apparent cytopathic effect. The constructed KUN replicons should provide valuable tools to study flavivirus RNA replication as well as providing possible vectors for a long-lasting and noncytopathic RNA virus expression system.

Abstract: Epstein-Barr (EBV) is a powerful immortalizing virus for human B lymphocytes in vitro and is associated with several human neoplasias in vivo. Previously, we have shown that the majority of EBV-infected cells in the peripheral blood of healthy, persistently infected individuals do not express the activated phenotype, e.g., high levels of cell surface CD23 and CD80 (B7), characteristically expressed on in vitro-immortalized cells. Here, we show that > or = 90% of the CD23-, virus-infected cells in the peripheral blood are in G0 and therefore resting. The remaining cells may be G1 arrested, but we were unable to detect a significant number of cells traversing the S-G2-M stages of the cell cycle. The mRNA for LMP2A, but not EBNA1 originating from Qp, was readily detected in this population, and these cells appear competent in the processing and presentation of antigen by class I major histocompatibility complex. We propose that these resting B cells are the site of long-term latent persistence for EBV. We further propose that the persistence of the virus in a resting B7- B cell provides an important mechanism to escape immunosurveillance. The demonstration that EBV can persist latently in a resting B cell means that the immortalizing functions of EBV can be down regulated in a normal B cell. This conclusion has important implications for understanding and controlling EBV-associated neoplasia.

Abstract: Neuronal death and vacuolation are characteristics of the CNS degeneration found in prion diseases Relatively few cultured cell lines have been identified that can be persistently infected with scrapie prions, and none of these cells show cytopathologic changes reminiscent of prion neuropathology The differentiated neuronal cell line GT1, established from gonadotropin hormone releasing-hormone neurons immortalized by genetically targeted tumorigenesis in transgenic mice (P L Mellon, JJ Windle, P C Goldsmith, C A Padula, J L Roberts, and R I Weiner, Neuron 5:1-10, 1990), was examined for its ability to support prion formation We found that GT1 cells could be persistently infected with mouse RML prions and that conditioned medium from infected cells could transfer prions to uninfected cells In many but not all experiments, a subpopulation of cells showed reduced viability, morphological signs of neurodegeneration and vacuolation, and features of apoptosis Subclones of GT1 cells that were stably transfected with the trk4 gene encoding the high-affinity nerve growth factor (NGF) receptor (GT1-trk) could also be persistently infected NGF increased the viability of the scrapie-infected GT1-trk cells and reduced the morphological and biochemical signs of vacuolation and apoptosis GT1 cells represent a novel system for studying the molecular mechanisms underlying prion infectivity and subsequent neurodegenerative changes

Abstract: Human immunodeficiency virus type 1 (HIV-1) acquires several host cell membrane proteins when it buds from infected cells. To study the effect of virally incorporated host-derived ICAM-1 glycoproteins on the biology of HIV-1, we have developed a transient expression system that has enabled us to produce virus particles differing only in the absence or the presence of virion-bound ICAM-1. By using a single-round infection assay based on an ICAM-1-negative target T-cell line stably transfected with an HIV-1 long terminal repeat driven luciferase gene construct, we have been able to demonstrate that the acquisition of host-derived ICAM-1 by HIV-1 has functional significance, since it leads to a pronounced increase in viral infectivity (4.6- to 9.8-fold) in an ICAM-1/LFA-1-dependent fashion, as shown by blocking with anti-ICAM-1 and -LFA-1 antibodies. The same potentiating effect on viral infectivity was also observed with monocytoid cells. Studies of the kinetics of infection revealed that the positive effect mediated by virally embedded host cell membrane ICAM-1 is due to an increase in the efficiency of early steps in the viral life cycle. These results provide new insights into how incorporation of host proteins can modulate the biological properties of HIV-1. Our findings have direct clinical relevance, considering that ICAM-1 is expressed on the surface of virus-infected cells and, more importantly, that host-derived ICAM-1 has been shown to be acquired by clinical HIV-1 isolates grown on primary mononuclear cells. These data justify a more complete analysis of the other putative role(s) that virally incorporated ICAM-1 may play in the life cycle of HIV-1, for example, at the level of neutralization sensitivity.

Abstract: The chemokine receptor CCR5 acts as an essential cofactor for cell entry by macrophage-tropic human immunodeficiency virus type 1 (HIV-1) strains, whereas CXCR4 acts as an essential cofactor for T-cell-line-adapted strains. We demonstrated that the specific amino acids in the V3 loop of the HIV-1 envelope protein that determine cellular tropism also regulate chemokine coreceptor preference for cell entry by the virus. Further, a strong correlation was found between HIV-1 strains classified as syncytium inducing in standard assays and those using CXCR4 as a coreceptor. These data support the hypothesis that progressive adaptation to additional coreceptors is a key molecular basis for HIV-1 phenotypic evolution in vivo.

Abstract: The small hydrophobic protein SH of human respiratory syncytial virus (RSV) is a short transmembrane surface protein of unknown function. A full-length cDNA of RSV strain A2 (subgroup A) antigenomic RNA was modified such that the entire SH gene, including the transcription signals and the complete mRNA-encoding sequence, was deleted and replaced by a synthetic intergenic region. This reduced the length of the antigenome by 398 nucleotides and ablated expression of 1 of the 10 RSV mRNAs. Recombinant virus containing this engineered deletion was recovered, and the absence of the SH gene was confirmed by reverse transcription in conjunction with PCR. Northern blot analysis of intracellular RNAs and gel electrophoresis of labeled intracellular proteins confirmed the lack of expression of the SH mRNA and protein. The absence of the SH gene did not noticeably affect RNA replication, but two effects on transcription were noted. First, synthesis of the G, F, and M2 mRNAs was increased, presumably due to their being one position closer to the promoter in the gene order. Second, transcription of genes downstream of the engineered site exhibited a steeper gradient of polarity. On monolayers of HEp-2 cells, the SH-minus virus produced syncytia which were at least equivalent in size to those of the wild type and produced plaques which were 70% larger. Furthermore, the SH-minus virus grew somewhat better (up to 12.6-fold) than wild-type recombinant RSV in certain cell lines. While the function of the SH protein remains to be determined, it seems to be completely dispensable for growth in tissue culture and fusion function. When inoculated intranasally into mice, the SH-minus virus resembled the wild-type recombinant virus in its efficiency of replication in the lungs, whereas it replicated 10-fold less efficiently in the upper respiratory tract. In mice, the SH-minus and wild-type recombinant viruses were similarly immunogenic and effective in inducing resistance to virus challenge.

Abstract: High rates of mutation and replication of human immunodeficiency virus (HIV) allow for the continuous generation of diverse genetic variants in vivo. Selective pressures within the microenvironments of different anatomic compartments result in the emergence of dominant quasispecies which can be distinguished by their envelope sequences. It is not known whether comparable tissue-specific selective pressures lead to the independent evolution of pol sequences within different tissue compartments, nor is it known how differing rates of virus turnover in tissues might affect the pace of such evolution. These issues are of importance for the formulation of a model for the emergence of drug resistance in vivo and for a general understanding of virus trafficking and virus turnover. Regions of the HIV type 1 reverse transcriptase (RT) which carry the majority of the known resistance codons to RT inhibitors (700 nucleotides from each clone) were cloned and sequenced directly from autopsied brain, spleen, and lymph node specimens from four subjects who had received zidovudine therapy. Clones from proviral DNA (143) and from viral cDNA (14) were analyzed. In three of four subjects, a discordance in distribution of resistance codons was noted. Moreover, brain-derived sequences appeared to be phylogenetically distinct from spleen- and lymph node-derived sequences even after exclusion of resistance codons from analysis. In each case, evidence for differential immune selective pressure, based on comparison of inferred amino acid sequences corresponding to known major histocompatibility complex class I cytotoxic T-lymphocyte epitopes, was found. These observations support the concept of anatomically distinct, independently evolving quasispecies (virodemes).

Abstract: Primate lentiviruses use chemokine coreceptors in addition to the CD4 receptor to initiate virus infection. Simian immunodeficiency virus (SIV) productively infects human cells expressing CD4 and the human allele of the chemokine coreceptor CCR-5 as efficiently as it infects macaque cells expressing human CD4, suggesting that SIV can function with either a simian or a human coreceptor in conjunction with human CD4. In the same macaque cells expressing human CD4, the replication of human immunodeficiency virus type 1 (HIV-1) is blocked at several stages of infection; some isolates are restricted prior to reverse transcription, while others, including some macrophage-tropic and primary isolates, are restricted at a step after reverse transcription but prior to migration of the preintegration complex to the nucleus. Both blocks in HIV-1 replication can be relieved by either expression of the appropriate human coreceptor (CCR-5 or CXCR-4) or expression of SIV gene products in cis with the HIV-1 envelope as a chimera between SIV and HIV-1 (SHIV). Thus, a virus with a SIV core and HIV-1 envelope can efficiently infect macaque cells expressing human CD4, presumably by interacting with the simian coreceptor, whereas a virus with an HIV-1 core and an HIV-1 envelope requires expression of the human allele of the coreceptor for productive infection of these cells. These studies suggest that there are interactions among the coreceptor, the viral envelope, and another viral gene product that govern postentry steps of virus replication. These data are consistent with the hypothesis that such interactions may be required for translocation of the virus core to the nucleus. Moreover, the differential abilities of SIV and HIV-1 to function in these processes with heterologous primate coreceptors may have implications for cross-species transmission.

Abstract: Two cellular proteins, NPI-1 and NPI-3, were previously identified through their interaction with the influenza virus nucleoprotein (NP) by using the yeast two-hybrid system. These proteins were then shown to act as general transport factors (karyopherin alpha) and nuclear pore-docking proteins to facilitate the transport of the NP and of viral RNA into the nucleus. The yeast two-hybrid assay has now been used to identify the specific domains on the NP that bind to the NPI proteins. Mutational analysis including alanine scanning identified the motifs SxGTKRSYxxM and TKRSxxxM, which are required for binding to NPI-1 and NPI-3, respectively. These sequences were shown to possess nuclear localization signal (NLS) activity following expression of fusion proteins in HeLa cells. These sequences represent a novel nonconventional NLS motif. Another NLS activity not mediated by the NPI binding sites is associated with noncontiguous sequences in the NP.

Abstract: We have developed a system for site-specific DNA integration in human cells, mediated by the adeno-associated virus (AAV) Rep proteins. In its normal lysogenic cycle, AAV integrates at a site on human chromosome 19 termed AAVS1. We describe a rapid PCR assay for the detection of integration events at AAVS1 in whole populations of cells. Using this assay, we determined that the AAV Rep proteins, delivered in cis or trans, are required for integration at AAVS1. Only the large forms of the Rep protein, Rep78 and Rep68, promoted site-specific integration. The AAV inverted terminal repeats, present in cis, were not essential for integration at AAVS1, but in cells containing Rep, they increased the efficiency of integration. In the presence of the Rep proteins, the integration of a plasmid containing AAV inverted terminal repeats occurred at high frequency, such that clones containing the plasmid could be isolated without selection. In two of the five clones analyzed by fluorescence in situ hybridization, the plasmid DNA was integrated at AAVS1. In most of the clones, at least one copy of the entire plasmid was integrated in a tandem array. Detailed analysis of the integrated plasmid structure in one clone suggested a complex mechanism producing rearrangements of the flanking genomic DNA, similar to those observed with wild-type AAV.

Abstract: One hope to maintain the benefits of antiviral therapy against the human immunodeficiency virus type 1 (HIV-1), despite the development of resistance, is the possibility that resistant variants will show decreased viral fitness. To study this possibility, HIV-1 variants showing high-level resistance (up to 1,500-fold) to the substrate analog protease inhibitors BILA 1906 BS and BILA 2185 BS have been characterized. Active-site mutations V32I and I84V/A were consistently observed in the protease of highly resistant viruses, along with up to six other mutations. In vitro studies with recombinant mutant proteases demonstrated that these mutations resulted in up to 10(4)-fold increases in the Ki values toward BILA 1906 BS and BILA 2185 BS and a concomitant 2,200-fold decrease in catalytic efficiency of the enzymes toward a synthetic substrate. When introduced into viral molecular clones, the protease mutations impaired polyprotein processing, consistent with a decrease in enzyme activity in virions. Despite these observations, however, most mutations had little effect on viral replication except when the active-site mutations V32I and I84V/A were coexpressed in the protease. The latter combinations not only conferred a significant growth reduction of viral clones on peripheral blood mononuclear cells but also caused the complete disappearance of mutated clones when cocultured with wild-type virus on T-cell lines. Furthermore, the double nucleotide mutation I84A rapidly reverted to I84V upon drug removal, confirming its impact on viral fitness. Therefore, high-level resistance to protease inhibitors can be associated with impaired viral fitness, suggesting that antiviral therapies with such inhibitors may maintain some clinical benefits.

Abstract: A herpesvirus that is related to but distinct from the Kaposi's sarcoma-associated herpesvirus (KSHV, or human herpesvirus 8) was isolated from rhesus monkeys. The sequence of 10.6 kbp from virion DNA revealed the presence of an interleukin-6 homolog similar to what is present in KSHV and a closer relatedness of the DNA polymerase and glycoprotein B reading frames to those of KSHV than to those of any other herpesvirus. This rhesus monkey herpesvirus replicated lytically and to high titers in cultured rhesus monkey fibroblasts. Antibody testing revealed a high prevalence for at least 10 years in our rhesus monkey colony and a high prevalence in two other colonies that were tested. Thus, rhesus monkeys naturally harbor a virus related to KSHV, which we have called RRV, for rhesus monkey rhadinovirus.

Abstract: Collagenous lectins (collectins) present in mammalian serum and pulmonary fluids bind to influenza virus and display antiviral activity in vitro, but their role in vivo has yet to be determined. We have used early and late isolates of H3N2 subtype influenza viruses that differ in their degree of glycosylation to examine the relationship between sensitivity to murine serum and pulmonary lectins in vitro and the ability of a virus to replicate in the respiratory tract of mice. A marked inverse correlation was found between these two parameters. Early H3 isolates (1968 to 1972) bear 7 potential glycosylation sites on hemagglutinin (HA), whereas later strains carry 9 or 10. Late isolates were shown to be much more sensitive than early strains to neutralization by the mouse serum mannose-binding lectin (MBL) and rat lung surfactant protein D (SP-D) and bound greater levels of these lectins in enzyme-linked immunosorbent assays and Western blot analyses. They also replicated very poorly in mouse lungs compared to the earlier strains. Growth in the lungs was greatly enhanced, however, if saccharide inhibitors of the collectins were included in the virus inoculum. The level of SP-D in bronchoalveolar lavage fluids increased on influenza virus infection. MBL was absent from lavage fluids of normal mice but could be detected in fluids from mice 3 days after infection with the virulent strain A/PR/8/34 (H1N1). The results implicate SP-D and possibly MBL as important components of the innate defense of the respiratory tract against influenza virus and indicate that the degree or pattern of glycosylation of a virus can be an important factor in its virulence.