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Self-binding peptides containing zipper-like sequences, such as the Leu/Ile zipper sequence within the coiled coil regions of proteins and the cross-β spine steric zippers within the amyloid-like fibrils, could bind to the protein-of-origin through homophilic sequence-specific zipper motifs. These self-binding sequences represent opportunities for the development of biochemical tools and/or therapeutics. Here, we report on the identification of a putative self-binding β-zipper-forming peptide within the severe acute respiratory syndrome-associated coronavirus spike (S) protein and its application in viral detection. Peptide array scanning of overlapping peptides covering the entire length of S protein identified 34 putative self-binding peptides of six clusters, five of which contained octapeptide core consensus sequences. The Cluster I consensus octapeptide sequence GINITNFR was predicted by the Eisenberg's 3D profile method to have high amyloid-like fibrillation potential through steric β-zipper formation. Peptide C6 containing the Cluster I consensus sequence was shown to oligomerize and form amyloid-like fibrils. Taking advantage of this, C6 was further applied to detect the S protein expression in vitro by fluorescence staining. Meanwhile, the coiled-coil-forming Leu/Ile heptad repeat sequences within the S protein were under-represented during peptide array scanning, in agreement with that long peptide lengths were required to attain high helix-mediated interaction avidity. The data suggest that short β-zipper-like self-binding peptides within the S protein could be identified through combining the peptide scanning and predictive methods, and could be exploited as biochemical detection reagents for viral infection. MOE (Min. of Education, S’pore)

The Global Virus Network (GVN) was established in 2011 to strengthen research and responses to emerging viral causes of human disease and to prepare against new viral pandemics. There are now 52 GVN Centers of Excellence and 9 Affiliate laboratories in 32 countries. The 11th International GVN meeting was held from June 9–11, 2019 in Barcelona, Spain and was jointly organized with the Spanish Society of Virology. A common theme throughout the meeting was globalization and climate change. This report highlights the recent accomplishments of GVN researchers in several important areas of medical virology, including severe virus epidemics, anticipation and preparedness for changing disease dynamics, host-pathogen interactions, zoonotic virus infections, ethical preparedness for epidemics and pandemics, one health and antivirals., Instituto de Biotecnologia y Biologia Molecular

The Global Virus Network (GVN) was established in 2011 in order to strengthen research and responses to current viral causes of human disease and to prepare against new viral pandemic threats. There are now 38 GVN Centers of Excellence and 6 Affiliate laboratories in 24 countries. GVN scientists meet annually to learn about each other's current research, address collaborative priorities and plan future programs. The 2016 meeting was held from October 23–25 in Hokkaido, Japan, in partnership with the Japanese Society for Virology, the National Institute of Infectious Diseases of Japan and the Research Center for Zoonosis Control of Hokkaido University. This report highlights the accomplishments of GVN researchers in many priority areas of medical virology, including the current Zika epidemic, infections by human papillomavirus, influenza, Ebola, Lassa, dengue, HIV, hepatitis C, and chikungunya viruses, and the development of improved diagnostics and new vaccines., Highlights • The GVN is an international research network comprised of 38 Centers of Excellence and 6 Affiliates in 24 countries. • The 2016 Global Virus Network (GVN) Meeting was held in Sapporo, Japan from October 23–25. • New data were presented on all various aspects of medical virology, therapy, and vaccines were presented. • International collaboration is needed to develop effective viral vaccines and therapeutics. • The 2017 international GVN meeting will be held on September 25–28 in Melbourne, Australia.

OBJECTIVE To study the dose-dependent manner of HIV-1 Tat-induced effects on viral replication, internalization and spread, and to directly observe these effects on soluble Env immunogens and virus-like particles. DESIGN In order to determine the manner through which Tat affects viral replication, we incubated cells, virions and soluble Env spikes with Tat at different concentrations, and directly visualized the effects of such incubation. METHODS Cell-based infectivity assays were carried out to assay Tat dose-dependency of viral infectivity. Transmission electron microscopy of virus-like particles and soluble gp140 immunogens incubated with Tat at various concentrations was performed to directly observe Tat-induced effects. RESULTS Treating virus with exogenous Tat increased infectivity in a dose-dependent manner. In the presence of anti-Tat antibodies, virus replication and spread were repressed, postulating Tat contributions to disease progression. When CXCR4 coreceptors were blocked, Tat treatment overcame the inhibition relative to absence of Tat treatment. Similarly, syncytium formation between chronically infected and uninfected target cells was also increased by exogenous Tat treatment. Inhibiting the CD4 receptor for virus entry abolished syncytium formation and Tat treatment was unable to overcome CD4 dependency. We show that Tat reduces virus infectivity at higher Tat concentrations through Env interactions resulting in viral aggregation. CONCLUSION Treating virions or chronically infected cells with exogenous Tat could enhance virus infectivity and spread through coreceptor tropism switch or through another undetermined mechanism. The aggregation potential of Tat suggests a mechanism of negative-feedback regulation of viral replication, providing another regulative function to control viral replication.

Objective: To study the complex formed between Tat protein and Env soluble trimeric immunogen, and compare with previously determined structures of Env native trimers and Env–CD4m complexes. Design: The soluble Env trimer was used to mimic the spike glycoprotein on the virus surface for the study. To overcome limitations of other structural determination methods, cryoelectron microscopy was employed to image the complex, and single particle reconstruction was utilized to reconstruct the structure of the complex from collected micrographs. Molecular modeling of gp120–Tat was performed to provide atomic coordinates for docking. Methods: Images were preprocessed by multivariate statistical analysis to identify principal components of variation then submitted for reconstruction. Reconstructed structures were docked with modeled gp120–Tat atomic coordinates to study the positions of crucial epitopes. Results: Analysis of the Env–Tat complex demonstrated an intermediate structure between Env native trimers and Env–CD4m structures. Docking results indicate that the CD4-binding site and the V3 loop are exposed in the Env–Tat complex. The integrin-binding sequence in Tat was also exposed in Env–Tat docking. Conclusion: The intermediate structure induced by Tat-interaction with Env could potentially provide an explanation for increased virus infection in the presence of Tat protein. Consequently, exposure of CD4-binding sites and a putative integrin-binding sequence on Tat in the complex may provide a new avenue for rational design of an effective HIV vaccine.

In vitro studies on HIV (HIV-1) replication and neutralization are usually performed in human cell cultures supplemented with FBS instead of human serum (HS). Here we show that in contrast to FBS, addition of increasing amounts of human serum from noninfected donors to the cell culture directly correlates with an increase in HIV-1 replication in vitro. This effect is independent of cell line, virus strain, or batch of pooled human serum used. We found that human serum affects viral transcription in a dose-dependent manner by activating the activator protein-1 (AP-1) member proteins c-FOS, JunD, and JunB in TZM-bl cells. Analysis of the human serum component responsible for this effect indicates that it is a protein having a molecular mass between 250 and 300 kDa. This serum protein, HIV-1 enhancing serum protein (HESP), might promote viral transcription in vivo and consequently play a role in disease progression.

Human immunodeficiency virus type 1 (HIV-1) is dependent on its envelope glycoprotein (Env) to bind, fuse, and subsequently infect a cell. We show here that treatment of HIV-1-infected cells with glycyl-prolyl-glycine amide (GPG-NH2), dramatically reduced the infectivity of the released viral particles by decreasing their Env incorporation. The mechanism of GPG-NH2 was uncovered by examining Env expression and maturation in treated cells. GPG-NH2 treatment was found to affect Env by significantly decreasing its steady-state levels, its processing into gp120/gp41, and its mass by inducing glycan removal in a manner dependent on its native signal sequence and the proteasome. Therefore, GPG-NH2 negatively impacts Env maturation, facilitating its targeting for endoplasmic reticulum-associated protein degradation, where Env is deglycosylated en route to its degradation. These findings illustrate that nontoxic drugs such as GPG-NH2, which can selectively target glycoproteins to existing cellular degradation pathways, may be useful for pathogen therapy. The endoplasmic reticulum (ER) contains a number of molecular chaperones and folding factors that aid in the maturation of proteins that traverse the secretory pathway. This process is strictly monitored by the ER quality control system, which selects properly folded proteins for export to the Golgi (16) and targets misfolded proteins for destruction through the ER-associated protein degradation pathway (ERAD) (4, 28). Once an ER protein is selected as a substrate for ERAD, it is translocated from the ER lumen to the cytosol through an ER translocon. This retrotranslocation process is thought to be driven by either the cytosolic AAA-ATPase p97 (39) or the 19S proteasome cap (23).

The membrane proximal external region (MPER) is a highly conserved membrane-active region located at the juxtamembrane positions within class I viral fusion glycoproteins and essential for membrane fusion events during viral entry. The MPER in the human immunodeficiency virus type I (HIV-1) envelope protein (Env) interacts with the lipid bilayers through a cluster of tryptophan (Trp) residues and a C-terminal cholesterol-interacting motif. The inclusion of the MPER N-terminal sequence contributes to the membrane reactivity and anti-viral efficacy of the first two anti-HIV peptidyl fusion inhibitors T20 and T1249. As a type I transmembrane protein, Env also interacts with the cellular membranes during its biosynthesis and trafficking. Here we investigated the roles of MPER membrane-active sequences during both viral entry and assembly, specifically, their roles in the design of peptidyl fusion inhibitors and the biosynthesis of viral structural proteins. We found that elimination of the membrane-active elements in MPER peptides, namely, penta Trp→alanine (Ala) substitutions and the disruption of the C-terminal cholesterol-interacting motif through deletion inhibited the anti-viral effect against the pseudotyped HIV-1. Furthermore, as compared to C-terminal dimerization, N-terminal dimerization of MPER peptides and N-terminal extension with five helix-forming residues enhanced their anti-viral efficacy substantially. The secondary structure study revealed that the penta-Trp→Ala substitutions also increased the helical content in the MPER sequence, which prompted us to study the biological relevance of such mutations in pre-fusion Env. We observed that Ala mutations of Trp664, Trp668 and Trp670 in MPER moderately lowered the intracellular and intraviral contents of Env while significantly elevating the content of another viral structural protein, p55/Gag and its derivative p24/capsid. The data suggest a role of the gp41 MPER in the membrane-reactive events during both viral entry and budding, and provide insights into the future development of anti-viral therapeutics. NRF (Natl Research Foundation, S’pore) Accepted version

Hypervariable loops of HIV-1 Env protein gp120 are speculated to play roles in the conformational transition of Env to the receptor binding-induced metastable state. Structural analysis of full-length Env-based immunogens, containing the entire V2 loop, displayed tighter association between gp120 subunits, resulting in a smaller trimeric diameter than constructs lacking V2. A prominent basal quaternary location of V2 and V3′ that challenges previous reports would facilitate gp41-independent gp120-gp120 interactions and suggests a quaternary mechanism of epitope occlusion facilitated by hypervariable loops. Deletion of V2 resulted in dramatic exposure of basal, membrane-proximal gp41 epitopes, consistent with its predicted basal location. The structural features of HIV-1 Env characterized here provide grounds for a paradigm shift in loop exposure and epitope occlusion, while providing substantive rationale for epitope display required for elicitation of broadly neutralizing antibodies, as well as substantiating previous pertinent literature disregarded in recent reports.

The capsid protein (CA or p24) of human immunodeficiency virus type 1 (HIV-1) plays a major role both early and late in the virus replication cycle. Many studies have suggested that the C-terminal domain of this protein is involved in dimerization and proper assembly of the viral core. Point mutations were introduced in two conserved sites of this region and their effects on viral protein expression, particle assembly and infectivity were studied. Eight different mutants (L205A+P207A, L205A, P207A, 223GPG225AAA, G223A, P224A, G225A and V221G) of the infectious clone pNL4-3 were constructed. Most substitutions had no substantial effect on HIV-1 protein synthesis, yet they impaired viral infectivity and particle production. The two mutants P207A and V221G also had a profound effect on Gag–Pol protein processing in HeLa–tat cells. However, these results were cell line-specific and Gag–Pol processing of P207A was not affected in 293T cells. In HeLa–tat cells, no virus particles were detected with the P207A mutation, whereas the other mutant virus particles were heterogeneous in size and morphology. None of the mutants showed normal, mature, conical core structures in HeLa–tat cells. These results indicate that the two conserved sequences in the C-terminal CA domain are essential for proper morphogenesis and infectivity of HIV-1 particles.

Capsid assembly during virus replication is a potential target for antiviral therapy. The Gag polyprotein is the main structural component of retroviral particles, and in human immunodeficiency virus type 1 (HIV-1), it contains the sequences for the matrix, capsid, nucleocapsid, and several small polypeptides. Here, we report that at a concentration of 100 μM, 7 of 83 tripeptide amides from the carboxyl-terminal sequence of the HIV-1 capsid protein p24 suppressed HIV-1 replication (>80%). The three most potent tripeptides, glycyl-prolyl-glycine-amide (GPG-NH 2 ), alanyl-leucyl-glycine-amide (ALG-NH 2 ), and arginyl-glutaminyl-glycine-amide (RQG-NH 2 ), were found to interact with p24. With electron microscopy, disarranged core structures of HIV-1 progeny were extensively observed when the cells were treated with GPG-NH 2 and ALG-NH 2 . Furthermore, nodular structures of approximately the same size as the broad end of HIV-1 conical capsids were observed at the plasma membranes of treated cells only, possibly indicating an arrest of the budding process. Corresponding tripeptides with nonamidated carboxyl termini were not biologically active and did not interact with p24.

Human immunodeficiency virus type 1 (HIV-1) is classified into subtypes on the basis of phylogenetic analysis of sequence differences. Inter- and intra-subtype polymorphism extends throughout the genome, including the long terminal repeat (LTR). In this study, the importance of the upstream stimulating factor (USF)-binding site (E-box) in the core-negative regulatory element (NRE) of the LTR of HIV-1 subtypes A, B, C, D, E and G was investigated.In vivo, USF was found to repress transcription directed from representative HIV-1 LTR sequences of all the subtypes tested in an epithelial cell line, yet activate the same transcription in a T-cell line. Mutation of the core-NRE USF site of the representative subtype B LTR did not affect the cell-specific, subtype-independent, dual role of USF.In vitrobinding assays showed that recombinant USF43interacts with the core-NRE from subtypes B and C, but not A, D, E or G. Thus, USF affects LTR-directed transcription in a cell-specific manner, independently of both the HIV-1 subtype from which the LTR was derived and the core-NRE USF site sequences.

Background. Real-time monitoring of cytomegalovirus (CMV) infections in transplant patients demands a rapid and high-throughput CMV DNA quantification method. Methods. TaqMan polymerase chain reaction assays based on CMV immediate early protein exon 4 and glycoprotein B were developed and were compared with a COBAS AMPLICOR CMV MONITOR (CMM) test for quantifying CMV DNA in peripheral blood leukocytes from seven stem cell transplant patients having received antiviral treatment. Results. There was a good correlation between the TaqMan assays and the CMM test for CMV DNA quantification. The throughput of the TaqMan assays was, however, about 3 times higher than that of the CMM test. The CMV DNA dynamics patterns determined by the TaqMan polymerase chain reaction were well in line with the outcome of the antiviral therapy. Conclusions. The TaqMan assays may potentially serve as a useful tool for rapid quantification of CMV infections in stem cell transplant patients. Quantification of cytomegalovirus (CMV) DNA has been suggested to be important for monitoring CMV disease progression and for assessing the efficacy of antiviral therapy for stem cell and solid organ transplant patients (1‐3). Although different DNA quantification methods have been used, the COBAS AMPLICOR CMV MONITOR (CMM) test is, at present, the only available commercial assay. This method has only been validated for quantifying CMV DNA in plasma. Previous studies have shown that CMV DNA in peripheral blood leukocytes (PBL) can be detected earlier than in plasma in stem cell transplant (SCT) patients (2). Therefore, it is of clinical importance to quantify CMV DNA in PBL, especially from SCT patients receiving antiviral therapy. Another major problem of the currently used quantification assays is that the capacity of those methods for processing large number of samples is limited. This has made it difficult to perform real-time monitoring of CMV infections in vivo. Recently, a novel TaqMan technique for high-throughput quantification has been developed (4). This assay is based on 59 nuclease assay chemistry and Perkin-Elmer 7700 sequence detection system. The close tube configuration and real-time detection feature make this method very attractive in terms of its reliability and high throughput. Indeed, this assay has been applied for quantifying a variety of microorganisms. The purpose of the present study was to develop TaqMan polymerase chain reaction (PCR) assay for quantification of CMV DNA from PBL samples. The clinical utility of the developed assay was evaluated by comparison with CMM test for quantifying CMV DNA in PBL samples from SCT patients having received antiviral therapy.

Transcription of the HIV-1 provirus genome is regulated by a complex interplay between viral regulatory proteins and cellular transcription factors that interact with the viral long terminal repeat (LTR) region of HIV-1. However, several cellular transcription factors have been identified that can interact with the HIV-1 LTR; the significance of all of these factors is not clearly understood. In this study we have characterized the LTR region of different subtypes of HIV-1 with regard to nucleotide sequence and promoter activity. The LTR regions of HIV-1 from peripheral blood mononuclear cells of 29 infected individuals originating from 10 different geographical regions were sequenced and further analyzed for promoter/enhancer activity in transient transfection of HeLa cells, in the context of a reporter gene and in the context of the complete virus genome. We found several subtype-specific LTR sequences of the various HIV-1 strains, such as an insertion that created a potential third NF-kappaB site in the LTR of the subtype C strains. The USF-binding site in the NRE also contained subtype-specific sequences. Interestingly, the promoter/enhancer activities of the subtype C LTRs were higher than the activities of the other subtypes analyzed here (subtypes A, B, D, E, and G), suggesting that the potential third NF-kappaB site may confer higher LTR activity or that the subtype C NRE may be less potent. Thus, our data suggest that genetic diversity of the LTR may result in HIV-1 subtypes with different replicative properties.

485 THE RAPID ACCUMULATION of mutations and recombination occurs by processes that actively contribute to the evolution of HIV-1 and its genetic diversity. At least 10 distinct genetic subtypes of HIV-1 have been identified on the basis of the phylogenetic analysis of nucleotide sequences in the envV3 and gagp17 regions.1,2 According to an analysis of the 1993 Los Alamos HIV Database, 10% of the reported sequences were V3/p17 recombinants. Intersubtype mosaic forms of HIV-1 have been reported in geographic areas where multiple HIV-1 subtypes cocirculate. While many studies have been focused on phylogenetic analysis of the structural genes,1,2,5 relatively little attention has been paid to the genetic variation in the HIV1 long terminal repeat (LTR) region. Here we have characterized a collection of the LTR sequences from seven different HIV-1 subtypes and intersubtype recombinants representing different geographic origins. HIV-1 DNA was extracted from peripheral blood mononuclear cells (PBMCs) of 28 randomly chosen HIV-1-infected patients (21 African and 7 Swedish patients), as previously described. HIV-1 was subtyped by direct sequencing of the V3 and p17 regions of all of the patients, followed by phylogenetic analyses, as previously described. Full-length HIV genomic sequences (subtypes A±H), as presented in the 1997 Los Alamos HIV Database, were used as reference sequences in the alignments for V3 and p17 regions. For some subtypes unpublished full-length sequences (subtype H: VI991-3, VI997-2; subtype F: BZ126, F9363, ITM850; subtype A: SE8538, SE8131-3, SE8891, SE7253, SE7535; and subtype J: SE91733, SE9280-9) were available. The LTR was amplified by a nested polymerase chain reaction (PCR), using the previously described primers BJLTR1, BJLTR3, and BJLTR4.8 After cloning of the PCR fragments into the pCR2.1 vector (Invitrogen, San Diego, CA) the double-stranded (ds) DNA was used as a template for sequencing. The sequencing reactions were primed with CY5-labeled universal (or reverse) primers, provided in the Sequenase version 2.0 kit (United States Biochemical, Cleveland, OH), according to the protocol of the manufacturer. Multiple LTR clones (3±10 clones per patient) for each of the 28 HIV-1 strains were sequenced from both directions. The LTR sequences (nucleotides 2 382 to 1 113 relative to transcription start) were then aligned using Omiga 1.01 (ClustalW Oxford Molecular, Ltd., Oxford, UK) and DNASIS sequence analysis software (Hitachi Software Engineering America, San Bruno, CA). The LTR sequences presented in this study were aligned with the various subtype LTR sequences from the Los Alamos HIV Database of 1997 (data not shown). The alignment was optimized manually. On the basis of the LTR, p17, and V3 phylogenetic analyses 1 of the samples was classified as subtype A, 10 as B, 6 as C, and 1 as G (Fig. 1A, B, and C, respectively2,9). A discrepant topology was observed in the V3, p17, or LTR phylogenetic tree in 5 of the 28 HIV-1 strains (marked with asterisks in Fig. 1). The HIV-1 strains that gave discrepant clustering in the phylogenetic trees were therefore putatively classified as recombinant strains (SO5549, A/C; GM6139, A/G; IC5381, A/G; GM6452, E/A; and TH6098, E/A). On the basis of the LTR analysis five of the strains were classified as subtype D (UG6476, UG6083, UG4696, UG6357, and UG5609); however, a clear subtype classification based on p17 and V3 (except for patient UG6476) could not be obtained for those strains (marked with double asterisks in Fig. 1B and C). A possible explanation may be that in those strains these regions contain a recombination point so that one part of the sequence belong to one subtype and one part to another, leading to unclear classification of those isolates. Interestingly, all of these strains are from Uganda, where a high frequency of HIV-1 hybrid genomes can be expected owing to the cocirculation of multiple divergent HIV-1 strains, mainly subtypes A and D.1 The LTR analysis revealed a distinction between the LTR region of subtype C and that of all the other subtypes studied (A, B, D, E, F, and G), in that subtype C isolates (except the sample SO5549) contained an additional potential NFk B-binding site (NFk B1) (GGGCGKTCY). Such a unique pattern for the LTR region of HIV-1 clade C has previously been reported from Ethiopia

We have analyzed the relation between intrapatient variabilities of the p17 gene and the location of known host p17 cytotoxic T lymphocyte (CTL) epitopes in five patients infected with human immunodeficiency virus type 1 (HIV-1). All patients were typed with respect to the human leukocyte antigen (HLA) class I type. One to seven previously fine-mapped p17 CTL epitopes corresponded to the HLA class I restriction elements of each patient. An average of 28+/-16% of the p17 gene of each patient encoded CTL epitopes corresponding to the HLA restriction elements of the host. Twenty full-length p17 gene clones were sequenced from each patient. The intrapatient homology between the p17 sequences ranged from 96.4 to 98.9%. The interpatient homology between the consensus sequences of each patient ranged from 83.1 to 91.6%. A total of 246 nucleotide differences within the 100 p17 clones was noted. Fifteen (16%) of 96 synonymous substitutions were found within host CTL epitopes, whereas 72 (48%) of 150 nonsynonymous nucleotide changes were found within CTL epitopes corresponding to the HLA restriction elements of the host (p 20% of the clones) were determined to be more common at positions contained within these CTL epitopes (p < 0.01). The present data suggest that the evolution of the p17 gene is influenced by contact areas with the host HLA class I molecules.

Objectives Alpha-hydroxy glycineamide (αHGA) is the active antiviral metabolite of tri-peptide glycyl-prolyl-glycine-amide (GPG-NH2). αHGA inhibits the replication of HIV-1 in vitro by interfering with the capsid formation. It has also an effect on viral gp160 envelope protein. Since drug transport is an important aspect of drug function, we investigated the mechanism of [14C] αHGA uptake by a human T cell line. Methods H9 cells were incubated with defined amounts of radiolabelled αHGA for definite time durations. After harvesting the cells and removal of radiolabelled material, the radioactivity associated with the cells was assayed. Experiments were also designed to address the effect of metabolic inhibitors, temperature and extra unlabelled compound as potential competitor on the cellular uptake of αHGA. Key findings Uptake of αHGA into H9 cells was time- and dose-dependent. The uptake properties showed a low temperature dependency (Q10 < 2). Moreover the uptake was not inhibited by increasing concentrations of cold competitors. There was no effect on cellular uptake of αHGA by known metabolic inhibitors, NaN3 and NaF. Conclusions Kinetic analysis of compound uptake, metabolic inhibition studies, saturation studies and the Q10 value of αHGA uptake indicate that the compound enters H9 cells by a mechanism of passive diffusion.

Clearance of infections caused by the hepatitis C virus (HCV) correlates with HCV-specific T cell function. We therefore evaluated therapeutic vaccination in 12 patients with chronic HCV infection. Eight patients also underwent a subsequent standard-of-care (SOC) therapy with pegylated interferon (IFN) and ribavirin. The phase I/IIa clinical trial was performed in treatment naive HCV genotype 1 patients, receiving four monthly vaccinations in the deltoid muscles with 167, 500, or 1,500 μg codon-optimized HCV nonstructural (NS) 3/4A-expressing DNA vaccine delivered by in vivo electroporation (EP). Enrollment was done with 2 weeks interval between patients for safety reasons. Treatment was safe and well tolerated. The vaccinations significantly improved IFN-γ–producing responses to HCV NS3 during the first 6 weeks of therapy. Five patients experienced 2–10 weeks 0.6–2.4 log10 reduction in serum HCV RNA. Six out of eight patients starting SOC therapy within 1–30 months after the last vaccine dose were cured. This first-in-man therapeutic HCV DNA vaccine study with the vaccine delivered by in vivo EP shows transient effects in patients with chronic HCV genotype 1 infection. The interesting result noted after SOC therapy suggests that therapeutic vaccination can be explored in a combination with SOC treatment.

The molecular basis for cross-reactive antibody binding to human T cell leukemia virus type I (HTLV-I) p19 core protein and human thymic epithelium has been defined with two monoclonal antibodies (mAbs), 12/1-2 and 13B12, raised to HTLV-I p19. The mAb 12/1-2 has previously been shown to react with HTLV-I p19, HTLV-II p22, and antigens of normal human thymic epithelium, placenta, and foreskin, whereas mAb 13B12 binds only to the carboxyl terminus of HTLV-I p19. In the present study, mAb 12/1-2 bound to a subset of Triton X-100-insoluble intermediate filaments in human thymic epithelium also recognized by antikeratin antibodies AE1 and AE3. The mAb 12/1-2 also reacted in Western blot assays with proteins of 54, 46, and 40 kDa present in extracts of human thymic epithelium and with hexameric peptides containing overlapping sequences of HTLV-I p19 with the amino acids IPP (amino acids 117-119). In contrast, the HTLV-I-specific mAb 13B12 did not bind to human thymic epithelium and reacted with a single hexameric peptide containing the carboxy-terminal HTLV-I p19 sequence IPPPYV (amino acids 117-122). Binding of mAb 12/1-2 to thymic epithelium could be inhibited by adsorption with peptide SP-79 containing a C-terminal sequence (amino acids 112-125) of p19. The crossreactive IPP site is within a region of p19 that has been previously shown to be highly immunogenic in HTLV-I-infected individuals and that is also encoded by genes or mRNA of human cytokeratin 17, keratin 4, epidermal cytokeratin 2, and 50-kDa type I epidermal keratin. Thus, our studies define the sequence of a cross-reactive antigen on HTLV-I p19 that is also associated with keratin intermediate filaments from human thymic epithelium and other normal human tissues and that could serve as a focus of an autoimmune response during HTLV-I infection.

The G-->A mutation of nucleotide (nt) 1896 in the precore region of hepatitis B virus (HBV) prevents production of hepatitis B e antigen (HBeAg) by creating a TAG stop codon. This mutation is often found in anti-HBe-positive patients with productive HBV infection and has been associated with severe chronic and fulminant hepatitis in some geographic areas. Emergence of the TAG mutation during HBe seroconversion was studied as was its relationship with nt 1858, which forms a base pair with nt 1896 in the pregenomic RNA loop. A TAG mutant evolved in 18 (72%) of 25 patients with a T-1858 strain but in only 1 (8%) of 13 with a C-1858 strain. Viremia with C-1858 strains was, despite their limited ability to develop the TAG mutation, as persistent as with T-1858 strains. Generally, T-1858-infected patients in whom a TAG mutant did not emerge were HBV DNA-negative by polymerase chain reaction on follow-up, whereas patients who developed the TAG mutation had prolonged viremia.

Serum specimens (n = 1899) were assayed for infections with HTLV-I, HTLV-II, and HIV-1 in seven classified groups of normal healthy controls, children, pregnant women, prostitutes, intravenous drug abusers, patients under going hemodialysis, and hemophiliacs in South and North Vietnam. Surprisingly, 125 of 954 samples from South Vietnam exhibited seropositivity for HTLV-II and 119 of these belonged to the group of IVDAs (n = 200). The remaining six positives were a healthy control, a prostitute, two children, and two patients under going hemodialysis. Two IVDAs who were seropositive for HTLV-I and 10 of 15 seropositives for HIV-1 were also positive for HTLV-II in this population. In contrast, no seropositives to any of the viruses were detected in the North Vietnamese samples (0 of 945). The HTLV-II-seropositive IVDAs exhibited increased seropositivity with age compared with HIV-1 seropositivity in the population, and there was no statistical relation between seropositivity for HTLV-II and HIV-1. The HTLV-IIs in South Vietnam IVDAs appeared, by subtype-specific peptide ELISA, to be a mixture of both subtypes a and b, with subtype a predominant. It seems possible that HTLV-II may have been introduced into this population from IVDAs from the United States during the Vietnam conflict, but in a period prior to, or early in, the introduction of HIV-1 to IVDAs.

Objectives To measure the prevalence of HIV-1 infection in different subgroups of blood donors and to identify groups at high risk of acquiring HIV-1. Methods Between March 1988 and April 1991 all blood donors at Ilembula Lutheran Hospital, Tanzania were asked about their age, marital status, home village and occupation, and tested for the presence of HIV antibodies using a first generation, whole virus lysate enzyme-linked immunosorbent assay (ELISA). Some negative (n = 265) and nearly all positive samples (439 out of 485) were subjected to confirmatory testing including recombinant and peptide-based ELISA and Western blot assays. Results A total of 3474 male and 1287 female blood donors were studied. The overall HIV-1 prevalences for men and women were 6.6 and 7.0%, respectively, with a higher prevalence in urban villages (13.6 and 15.0%, respectively), an intermediate prevalence in semi-urban villages (7.2 and 7.9%, respectively), and a lower prevalence in rural villages (3.7 and 3.0%, respectively). HIV-1 infection occurred mostly in men aged 20-44 and women aged 15-34 years. Urban donors, but not semi-urban and rural donors from the highlands, had a higher HIV-1 prevalence (21.4%) than the corresponding group from the lowlands (10.2%). Apart from area of residence, HIV-1 infection was found to be associated with occupation and marital status. There was an increase in HIV-1 prevalence, although not statistically significant, during the period studied. None of the blood donors were positive for HIV-2. Conclusions Male and female donors from urban and semi-urban villages, non-farmers from urban villages, and unmarried donors were identified as high-risk groups, which is consistent with more extensive risk behaviour in urban communities. In addition to using an HIV test with high sensitivity, the importance of pre-donation selection of blood donors is stressed.

The human immunodeficiency virus envelope protein is the key element mediating entry into host cells. Conformational rearrangement of Env upon binding to the host CD4 receptor and chemokine coreceptor drives membrane fusion. We elucidated the quaternary arrangement of the soluble Env trimeric immunogen o-gp140ΔV2TV1, in both its native (unliganded) and CD4-induced (liganded) states by cryoelectron microscopy and molecular modeling. The liganded conformation was elicited by binding gp140 to the synthetic CD4-mimicking miniprotein CD4m. Upon CD4m binding, an outward domain shift of the three gp120 subunits diminishes gp120–gp41 interactions, whereas a “flat open” concave trimer apex is observed consequent to gp120 tilting away from threefold axis, likely juxtaposing the fusion peptide with the host membrane. Additional features observed in the liganded conformation include rotations of individual gp120 subunits that may release gp41 for N- and C-helix refolding and also may lead to optimal exposure of the elicited coreceptor binding site. Such quaternary arrangements of gp140 lead to the metastable liganded conformation, with putative locations of exposed epitopes contributing to a description of sequential events occurring prior to membrane fusion. Our observations imply a mechanism whereby a soluble Env trimeric construct, as opposed to trimers extracted from virions, may better expose crucial epitopes such as the CD4 binding site and V3, as well as epitopes in the vicinity of gp41, subsequent to conjugation with CD4m. Structural features gleaned from our studies should aid the design of Env-based immunogens for inducement of potent broadly neutralizing antibodies against exposed conformational epitopes.

Background The synthetic peptide glycyl-prolyl-glycine amide (GPG-NH2) was previously shown to abolish the ability of HIV-1 particles to fuse with the target cells, by reducing the content of the viral envelope glycoprotein (Env) in progeny HIV-1 particles. The loss of Env was found to result from GPG-NH2 targeting the Env precursor protein gp160 to the ER-associated protein degradation (ERAD) pathway during its maturation. However, the anti-viral effect of GPG-NH2 has been shown to be mediated by its metabolite α-hydroxy-glycineamide (αHGA), which is produced in the presence of fetal bovine serum, but not human serum. In accordance, we wanted to investigate whether the targeting of gp160 to the ERAD pathway by GPG-NH2 was attributed to its metabolite αHGA. Results In the presence of fetal bovine serum, GPG-NH2, its intermediary metabolite glycine amide (G-NH2), and final metabolite αHGA all induced the degradation of gp160 through the ERAD pathway. However, when fetal bovine serum was replaced with human serum only αHGA showed an effect on gp160, and this activity was further shown to be completely independent of serum. This indicated that GPG-NH2 acts as a pro-drug, which was supported by the observation that it had to be added earlier to the cell cultures than αHGA to induce the degradation of gp160. Furthermore, the substantial reduction of Env incorporation into HIV-1 particles that occurs during GPG-NH2 treatment was also achieved by treating HIV-1 infected cells with αHGA. Conclusions The previously observed specificity of GPG-NH2 towards gp160 in HIV-1 infected cells, resulting in the production of Env (gp120/gp41) deficient fusion incompetent HIV-1 particles, was most probably due to the action of the GPG-NH2 metabolite αHGA.

Background Formation of an HIV-1 particle with a conical core structure is a prerequisite for the subsequent infectivity of the virus particle. We have previously described that glycineamide (G-NH2) when added to the culture medium of infected cells induces non-infectious HIV-1 particles with aberrant core structures. Results Here we demonstrate that it is not G-NH2 itself but a metabolite thereof that displays antiviral activity. We show that conversion of G-NH2 to its antiviral metabolite is catalyzed by an enzyme present in bovine and porcine but surprisingly not in human serum. Structure determination by NMR suggested that the active G-NH2 metabolite was α-hydroxy-glycineamide (α-HGA). Chemically synthesized α-HGA inhibited HIV-1 replication to the same degree as G-NH2, unlike a number of other synthesized analogues of G-NH2 which had no effect on HIV-1 replication. Comparisons by capillary electrophoresis and HPLC of the metabolite with the chemically synthesized α-HGA further confirmed that the antiviral G-NH2-metabolite indeed was α-HGA. Conclusion α-HGA has an unusually simple structure and a novel mechanism of antiviral action. Thus, α-HGA could be a lead for new antiviral substances belonging to a new class of anti-HIV drugs, i.e. capsid assembly inhibitors.

Site-directed immunization was employed to identify sites on the envelope glycoprotein gp120 for antibody-mediated neutralization of human immunodeficiency virus type 1 (HIV-1). Antisera were raised in monkeys (Macaca fascicularis) against a series of 40 overlapping synthetic peptides covering the entire amino acid sequence of gp120 from the HTLV-IIIB strain of HIV-1. Immune sera against 12 of these peptides were reactive with gp120 by immunoblotting analysis, and antisera raised against 5 peptides, corresponding to amino acids (aa) 152-176, 193-218, 206-230, 248-269, and 307-330, were highly efficient in neutralizing HIV-1 (HTLV-IIIB) infectivity in vitro. Admixture of individual neutralizing anti-peptide monkey sera resulted in increment in neutralizing antibody titer. Antisera with reactivity to the relatively conserved regions defined by aa 152-176, 193-230, and 248-269 also neutralized to different extents the infectivity of the five Swedish clinical isolates of HIV-1 tested. Only a few HIV-1-infected people were found to make antibodies to these three conserved domains of gp120 as judged by ELISA using synthetic peptides as antigens. Three of the peptides (aa 152-176, 248-269, and 307-330) that induced neutralization antibodies also induced interleukin 2 production and lymphocyte proliferation when added to cultures of peripheral blood mononuclear cells from monkeys immunized with the corresponding peptides, indicating that these domains accommodate T-cell recognition sites. The results have obvious implications for the rational design of subunit vaccines against HIV-1 infection.

Synthetic peptides of 20-25 amino acids were employed in enzyme-linked immunosorbent assays to identify linear epitopes in the external glycoprotein gp46 and the transmembrane glycoprotein gp21 of human T-cell leukemia/lymphotropic viruses type I (HTLV-I) and II (HTLV-II). Ten linear epitopes were identified in the HTLV-I glycoproteins, seven in gp46 and three in gp21. Three major linear epitopes were identified in the gp46 of HTLV-II. Peptides representing linear epitopes of gp46 were found to be sensitive and specific for the detection of antibody and permit serological identification and differentiation of HTLV-I and HTLV-II infections.

Immunoreactive regions of human immunodeficiency virus type 1 (HIV-1) gp41 were mapped by reacting HIV-1 antibody-positive human sera with overlapping synthetic peptides which covered the transmembrane protein. Three immunoreactive domains were identified, and five different and partially overlapping epitopes recognized by HIV-1-positive human sera were found within one immunodominant region. The effect on antibody recognition after single amino acid substitutions within one defined epitope was also studied. The reactivity of various HIV-1-positive sera to synthetic peptides with amino acid substitutions representing known isolates suggests an important substitution in the major epitope of African HIV-1 strains.

The great variability and high glycosylation of gp120 poses a great challenge for the design of a functional immune therapy. The binding region of the CD4 receptor to gp120, however, is well conserved and may constitute a target to limit viral entry and infectivity. Our strategy consists in using a preexisting pool of natural antibodies directed toward the gal(α1,3)gal disaccharide and to redirect it to HIV. We here show that using CD4-derived, gp120-binding, synthetic peptides chemically linked to gal(α1,3)gal can redirect these natural antibodies and improve the HIV-1 neutralizing activity of the CD4-derived peptides in vitro . Importantly, the binding of the CD4-gal(α1,3)gal peptides to HIV-1–infected cells conferred antibody-dependent cellular cytotoxicity after the addition of human sera. Thus, the temporary redirection of naturally occurring antibodies and their biological activities to a new antigen represents a completely new way of targeting a human disease.

Upon maturation of the human immunodeficiency virus type 1 (HIV-1) virion, proteolytic cleavage of the Gag precursor protein by the viral protease is followed by morphological changes of the capsid protein p24, which will ultimately transform the virus core from an immature spherical to a mature conical structure. Virion infectivity is critically dependent on the optimal semistability of the capsid cone structure. We have reported earlier that glycineamide (G-NH2), when added to the culture medium of infected cells, inhibits HIV-1 replication and that HIV-1 particles with aberrant core structures were formed. Here we show that it is not G-NH2itself but a metabolite thereof, α-hydroxy-glycineamide (α-HGA), that is responsible for the antiviral activity. We show that α-HGA inhibits the replication of clinical HIV-1 isolates with acquired resistance to reverse transcriptase and protease inhibitors but has no effect on the replication of any of 10 different RNA and DNA viruses. α-HGA affected the ability of the HIV-1 capsid protein to assemble into tubular or core structures in vitro and in vivo, probably by binding to the hinge region between the N- and C-terminal domains of the HIV-1 capsid protein as indicated by matrix-assisted laser desorption ionization-mass spectrometry results. As an antiviral compound, α-HGA has an unusually simple structure, a pronounced antiviral specificity, and a novel mechanism of antiviral action. As such, it might prove to be a lead compound for a new class of anti-HIV substances.

In 27 patients with a first episode of herpes simplex virus type 2 (HSV-2)-induced meningitis, confirmed by virus isolation from the cerebrospinal fluid (CSF) or seroconversion to HSV-2, initial neurologic complications were found in 10 (37%) but subsided before 6 months in all patients. Long-term complications were recurrent meningitis in 5 (19%) and periodic headache related to genital HSV recrudescences in 4 (15%). Seven additional patients had possible HSV-2-induced recurrent meningitis. In contrast to the first episode of meningitis, virus isolation, HSV antigen detection, and IgG analyses in consecutive serum samples were of no diagnostic value in episodes of HSV-2-induced recurrent meningitis. Instead, immunoblotting was used to assay intrathecally produced IgG antibodies to the HSV-2 type-specific protein gG-2 in recurrent meningitis, when CSF was collected at a minimum of 3 days after onset.

Five mouse hybridomas which produce monoclonal antibodies against the p17 core protein of HIV-1 have been isolated. Cross-competition assays and mapping with synthetic peptides demonstrate that two closely related epitopes are identified by these antibodies. Directed against two neighbouring peptides at the carboxy-terminal end of the molecule, they can be used for the selective detection of p17 polypeptide in a viral extract or in an infected cell lysate by a solid-phase sandwich enzyme immunoassay.

Background The mature HIV-1 conical core formation proceeds through highly regulated protease cleavage of the Gag precursor, which ultimately leads to substantial rearrangements of the capsid (CAp24) molecule involving both inter- and intra-molecular contacts of the CAp24 molecules. In this aspect, Asp51 which is located in the N-terminal domain of HIV-1 CAp24 plays an important role by forming a salt-bridge with the free imino terminus Pro1 following proteolytic cleavage and liberation of the CAp24 protein from the Pr55Gag precursor. Thus, previous substitution mutation of Asp51 to alanine (D51A) has shown to be lethal and that this invariable residue was found essential for tube formation in vitro, virus replication and virus capsid formation. Results We extended the above investigation by introducing three different D51 substitution mutations (D51N, D51E, and D51Q) into both prokaryotic and eukaryotic expression systems and studied their effects on in vitro capsid assembly and virus infectivity. Two substitution mutations (D51E and D51N) had no substantial effect on in vitro capsid assembly, yet they impaired viral infectivity and particle production. In contrast, the D51Q mutant was defective both for in vitro capsid assembly and for virus replication in cell culture. Conclusion These results show that substitutions of D51 with glutamate, glutamine, or asparagine, three amino acid residues that are structurally related to aspartate, could partially rescue both in vitro capsid assembly and intra-cellular CAp24 production but not replication of the virus in cultured cells.

We have studied the effects associated with two single amino acid substitution mutations in HIV-1 capsid (CA), the E98A and E187G. Both amino acids are well conserved among all major HIV-1 subtypes. HIV-1 infectivity is critically dependent on proper CA cone formation and mutations in CA are lethal when they inhibit CA assembly by destabilizing the intra and/or inter molecular CA contacts, which ultimately abrogate viral replication. Glu98, which is located on a surface of a flexible cyclophilin A binding loop is not involved in any intra-molecular contacts with other CA residues. In contrast, Glu187 has extensive intra-molecular contacts with eight other CA residues. Additionally, Glu187 has been shown to form a salt-bridge with Arg18 of another N-terminal CA monomer in a N-C dimer. However, despite proper virus release, glycoprotein incorporation and Gag processing, electron microscopy analysis revealed that, in contrast to the E187G mutant, only the E98A particles had aberrant core morphology that resulted in loss of infectivity.

Background. To determine the concentration of cytokines having an immunomodulating effect in the first trimester in women who subsequently developed preeclampsia.Methods. The serum concentrations of IL-10, TNFα, IL-6 and IL-2R were determined in ten women who later developed severe preeclampsia and in ten healthy controls. The groups were compared with the Mann-Whitney U test.Results. The IL-2R concentration was significantly higher in the women who later developed preeclampsia than in normal patients (p = 0.028). No significant differences were detected between the groups with respect to the other evaluated cytokines.Conclusion. Elevated IL-2R concentrations in maternal serum as early as in the first trimester may be a sign of immunological maladaptation and might be associated with a disturbance of trophoblastic invasion.

The levels of human immunodeficiency virus type 1 p24 antibodies and p24 antigen among 256 Ethiopians and Africans and 86 Swedes were compared. The elevated levels of total anti-p24 immunoglobulin G (IgG) and anti-p24 IgG4 among the Ethiopian and African patients suggest influences from T-helper-cell type 2-like responses.

Background We have previously shown that the tripeptide glycyl-prolyl-glycine-amide (GPG-amide) inhibits HIV-1 replication in vitro by affecting proper capsid assembly of HIV-1. However, GPG-amide failed in a phase II clinical trial on HIV-infected individuals. In the search for what went wrong we have now found that the tripeptide in itself does not exert the antiviral activity but is metabolised in two steps into the active compound by serum enzymes. The first step is cleavage of GPG-amide into GP and glycineamide (G-amide) by the soluble di-peptidyl peptidase CD26. In the present study we show that G-amide is further metabolised to the actve antiviral substance by an enzyme present in foetel bovine serum but not in human serum.

The tripeptide amide glycyl-prolyl-glycinamide (GPG-amide) is a new antiretroviral drug candidate, but its absorption mechanism is unknown. In this investigation, the transport and metabolism of GPG-amide were studied in a model of the human intestinal epithelium, Caco-2 cell monolayers. The results show that when the tripeptide amide came into contact with the apical enterocyte membrane, it was degraded by CD26 (dipeptidyl peptidase IV) to glycylproline and the antiretrovirally active metabolite glycinamide. Glycinamide retained antiretroviral activity in vitro after transport through the Caco-2 cell monolayers. The transport of glycinamide across Caco-2 cell monolayers occurred via passive diffusion with an apparent permeability coefficient of about 2 × 10 −6 cm s −1 , which suggests that it is absorbed by the oral route in sufficient amounts to be considered for oral administration. In conclusion, the tripeptide GPG-amide acts as a prodrug that is activated by CD26 to release the orally active antiretroviral compound glycinamide.

The chemically modified tripeptide glycyl-prolyl-glycine-amide (GPG-NH 2 ) inhibits replication of human immunodeficiency virus (HIV) type 1 (HIV-1) in vitro, probably by interfering with capsid formation. The aim of the present study was to determine whether the metabolites glycyl-proline (GP-OH), glycine (G-OH), prolyl-glycine-amide (PG-NH 2 ), proline (P-OH), and glycine-amide (G-NH 2 ) from proteolytic cleavage may inhibit the replication of HIV-1 in vitro. PG-NH 2 has previously been shown to have a modest effect on HIV-1 replication. In the present study we show that G-NH 2 exhibits a pronounced inhibitory effect on HIV-1. This effect was not due to a decrease in cell proliferation or viability and could not be shown for herpes simplex virus type 1. The G-NH 2 concentration that inhibited virus replication by 50% (IC 50 ) was equimolar to that of GPG-NH 2 and ranged from 3 to 41 μM. Transmission electron microscopy revealed that the effect of G-NH 2 on HIV-1 morphology was equivalent to that of GPG-NH 2 and showed disarranged capsid structures, indicating interference with capsid formation. Serial passage of HIV-infected cells with G-NH 2 for more than 20 subcultivations did not decrease the susceptibility to the compound. The results from this study suggest that GPG-NH 2 might act as a prodrug and that G-NH 2 is an active antiretroviral metabolite.

GPG-NH2 and G-NH2 are highly selective antiretroviral agents in cell culture, and both compounds inhibit HIV replication in CEM cell cultures to an equal extent (50% effective concentration: approximately 30 microM). The lymphocyte surface glycoprotein marker CD26, which is identical to dipeptidyl peptidase IV, efficiently converted GPG-NH2 to G-NH2 releasing the dipeptide GP-OH. The closely related QPG-NH2 derivative was also inhibitory to HIV, presumably by the dipeptidyl peptidase IV (DPP IV)-catalyzed release of G-NH2. In contrast, the cyclic pQPG-NH2 derivative in which the glutamine at the amino terminal position of QPG-NH2 was replaced by pyroglutamine and which is resistant to cleavage by purified CD26, was devoid of antiviral activity. CD26 is abundantly expressed on a variety of HIV target cells and is also present in serum of bovine, murine and human origin. The CD26/DPP IV enzymatic activity in serum and in cell suspensions could be efficiently inhibited by the CD26/DPP IV inhibitor L-isoleucinepyrrolidine (IlePyr) with 50% inhibitory concentrations ranging between 20 and 100 microM. When combined in HIV-1-infected cell cultures, IlePyr and Diprotin A (DP-A), another CD26/DPP IV inhibitor, abrogated the antiviral activity of GPG-NH2 but not of G-NH2. Therefore, it was concluded that the anti-HIV drug GPG-NH2 is not active as such, but rather behaves as a prodrug that must be obligatorily cleaved by CD26/DPP IV to G-NH2 to exert its antiretroviral activity. This is the first demonstration of a lymphocyte activation/differentiation marker (i.e. CD26) that plays a direct regulatory and indispensable role in the eventual antiretroviral activity of small synthetic molecules such as the antiretroviral (pro)drug GPG-NH2.

The chemically modified tripeptide glycyl-prolyl-glycine-amide (GPG-NH 2 ) inhibits replication of HIV-1 in vitro, probably by interfering with capsid formation. This study was aimed at determining cross-resistance between antiretroviral drugs and GPG-NH 2 , and whether resistance to GPG-NH 2 can be induced in vitro. Fifty-five clinical HIV-1 isolates with different resistance-related mutations were tested for susceptibility to GPG-NH 2 . No correlation between NRTI-, NNRTI- or PI-resistance and efficacy of GPG-NH 2 was found, indicating the lack of cross-resistance. Serial passages were performed with GPG-NH 2 , and with lamivudine, and genotypic or phenotypic changes were determined. Resistance to lamivudine was detected after six passages. No resistance to GPG-NH 2 was generated after 30 passages in two parallel series. However, one mutation (T107I) in the p24 gene was detected in both series, but this mutation was not associated with decreased sensitivity to GPG-NH 2 .

We investigated whether the high mobility group B 1 (HMGB1), an abundant nuclear protein in all mammalian cells, affects HIV-1 transcription. Intracellular expression of human HMGB1 repressed HIV-1 gene expression in epithelial cells. This inhibitory effect of HMGB1 was caused by repression of long terminal repeat (LTR)-mediated transcription. Other viral promoters/enhancers, including simian virus 40 or cytomegalovirus, were not inhibited by HMGB1. In addition, HMGB1 inhibition of HIV-1 subtype C expression was dependent on the number of NFκB sites in the LTR region. The inhibitory effect of HMGB1 on viral gene expression observed in HeLa cells was confirmed by an upregulation of viral replication in the presence of antisense HMGB1 in monocytic cells. In contrast to what was found in HeLa cells and monocytic cells, endogenous HMGB1 expression did not affect HIV-1 replication in unstimulated Jurkat cells. Thus, intracellular HMGB1 affects HIV-1 LTR-directed transcription in a promoter- and cell-specific manner.