How retroviruses regulate the amount of RNA genome packaged into each virion has remained a long-standing question. Our previous study showed that most HIV-1 particles contain two copies of viral RNA, indicating that the number of genomes packaged is tightly regulated. In this report, we examine the mechanism that controls the number of RNA genomes encapsidated into HIV-1 particles. We hypothesize that HIV-1 regulates genome packaging by either the mass or copy number of the viral RNA. These two distinct mechanisms predict different outcomes when the genome size deviates significantly from that of wild type. Regulation by RNA mass would result in multiple copies of a small genome or one copy of a large genome being packaged, whereas regulation by copy number would result in two copies of a genome being packaged independent of size. To distinguish between these two hypotheses, we examined the packaging of viral RNA that was larger (≈17 kb) or smaller (≈3 kb) than that of wild-type HIV-1 (≈9 kb) and found that most particles packaged two copies of the viral genome regardless of whether they were 17 kb or 3 kb. Therefore, HIV-1 regulates RNA genome encapsidation not by the mass of RNA but by packaging two copies of RNA. To further explore the mechanism that governs this regulation, we examined the packaging of viral RNAs containing two packaging signals that can form intermolecular dimers or intramolecular dimers (self-dimers) and found that one self-dimer is packaged. Therefore, HIV-1 recognizes one dimeric RNA instead of two copies of RNA. Our findings reveal that dimeric RNA recognition is the key mechanism that regulates HIV-1 genome encapsidation and provide insights into a critical step in the generation of infectious viruses.
Viruses must package their genomes in particles to pass their genetic information to the next generation. Although many aspects of RNA packaging are well-studied, how retroviruses regulate the number of genomes in the particle is currently unknown. Based on the dimeric nature of retroviral genomes in particles, it was often assumed that two copies of RNA were packaged into one particle. This assumption was validated recently when we demonstrated that most HIV-1 particles contain two copies of viral RNA, which revealed that the number of genomes packaged is tightly controlled. In this report, we examined the mechanism that regulates the amount of RNAs encapsidated into HIV-1 particles. Our results showed that RNA packaging is not regulated by the mass of the viral RNA as two copies of small or large genomes are packaged. However, packaging of two copies of RNA can be perturbed; HIV-1 can encapsidate one copy of its genome when the RNA contains two packaging/dimerization signals that allow for intramolecular dimer (self-dimer) formation. These studies revealed that HIV-1 regulates genome packaging by recognizing the dimeric RNA structure, and suggest that the interaction of viral protein Gag and dimeric RNA serves as the nucleation point of virus assembly.
DEB025 (alisporivir) is a synthetic cyclosporine with inhibitory activity against human immunodeficiency virus type-1 (HIV-1) and hepatitis C virus (HCV). It binds to cyclophilin A (CypA) and blocks essential functions of CypA in the viral replication cycles of both viruses. DEB025 inhibits clinical HIV-1 isolates in vitro and decreases HIV-1 virus load in the majority of patients. HIV-1 isolates being naturally resistant to DEB025 have been detected in vitro and in nonresponder patients. By sequence analysis of their capsid protein (CA) region, two amino acid polymorphisms that correlated with DEB025 resistance were identified: H87Q and I91N, both located in the CypA-binding loop of the CA protein of HIV-1. The H87Q change was by far more abundant than I91N. Additional polymorphisms in the CypA-binding loop (positions 86, 91 and 96), as well as in the N-terminal loop of CA were detected in resistant isolates and are assumed to contribute to the degree of resistance. These amino acid changes may modulate the conformation of the CypA-binding loop of CA in such a way that binding and/or isomerase function of CypA are no longer necessary for virus replication. The resistant HIV-1 isolates thus are CypA-independent.
DEB025; alisporivir; cyclophilin inhibitors; cyclosporines; HIV/retroviruses; HIV capsid; natural resistance
Non-peptide inhibition of fusion remains an important goal in anti-HIV research, due to its potential for low cost prophylaxis or prevention of cell–cell transmission of the virus. We report here on a series of indole compounds that have been identified as fusion inhibitors of gp41 through a structure-based drug design approach. Experimental binding affinities of the compounds for the hydrophobic pocket were strongly correlated to fusion inhibitory data (R2 = 0.91), and corresponding inhibition of viral replication confirmed the hydrophobic pocket as a valid target for low molecular weight fusion inhibitors. The most active compound bound to the hydrophobic pocket and inhibited cell-cell fusion and viral replication at sub-µM levels. A common binding mode for the inhibitors in this series was established by carrying out docking studies using structures of gp41 in the Protein Data Bank. The molecules were flexible enough to conform to the contours of the pocket, and the most active compound was able to adopt a structure mimicking the hydrophobic contacts of the D-peptide PIE7. The results enhance our understanding of indole compounds as inhibitors of gp41.
gp41; small molecule; fusion inhibitor; docking; indole
The elicitation of broadly cross-reactive HIV-1 neutralizing antibodies in humans remains a major challenge in developing a viable AIDS vaccine. We hypothesized that prolonged exposure to candidate vaccine immunogens could enhance the elicitation of such antibodies. In an attempt to develop HIV-1 vaccine immunogens with prolonged half-lives and increased stability, we constructed a fusion protein, gp41Fc, in which a truncated HIV-1 gp4189.6 was fused to a human IgG1 Fc. Gp41Fc is stable in solution, retains its antigenic structure and is highly immunogenic in rabbits. The serum titers reached 1:102,400 for the gp41Fc and 1:5,120 for gp14089.6. Rabbit IgG neutralized diverse HIV-1 isolates and HIV-2, and the neutralization activity was attributed to gp41-specific IgG. The concentration of the gp41Fc in the serum correlated with the neutralization activity of rabbit IgG which recognized mostly conformation-independent epitopes on gp41 and predominantly bound to peptides derived from the gp41 immunodominant loop region. These results suggest that the prolonged half-life of gp41Fc in the serum may enhance the generation of cross-reactive neutralizing antibodies. Further research is needed to confirm and extend these results which may have implications for the development of vaccine immunogens with enhanced capability to elicit cross-reactive HIV-1-neutralizing antibodies.
HIV/AIDS; Vaccines; Gp41
Human immunodeficiency virus type 2 (HIV-2) has been reported to have a distinct RNA packaging mechanism, referred to as cis packaging, in which Gag proteins package the RNA from which they were translated. We examined the progeny generated from dually infected cell lines that contain two HIV-2 proviruses, one with a wild-type gag/gag-pol and the other with a mutant gag that cannot express functional Gag/Gag-Pol. Viral titers and RNA analyses revealed that mutant viral RNAs can be packaged at efficiencies comparable to that of viral RNA from which wild-type Gag/Gag-Pol is translated. These results do not support the cis-packaging hypothesis but instead indicate that trans packaging is the major mechanism of HIV-2 RNA packaging. To further characterize the mechanisms of HIV-2 RNA packaging, we visualized HIV-2 RNA in individual particles by using fluorescent protein-tagged RNA-binding proteins that specifically recognize stem-loop motifs in the viral genomes, an assay termed single virion analysis. These studies revealed that >90% of the HIV-2 particles contained viral RNAs and that RNAs derived from different viruses were copackaged frequently. Furthermore, the frequencies of heterozygous particles in the viral population could be altered by changing a 6-nucleotide palindromic sequence at the 5′-untranslated region of the HIV-2 genome. This finding indicates that selection of copackaging RNA partners occurs prior to encapsidation and that HIV-2 Gag proteins primarily package one dimeric RNA rather than two monomeric RNAs. Additionally, single virion analyses demonstrated a similar RNA distribution in viral particles regardless of whether both viruses had a functional gag or one of the viruses had a nonfunctional gag, providing further support for the trans-packaging hypothesis. Together, these results revealed mechanisms of HIV-2 RNA packaging that are, contrary to previous studies, in many respects surprisingly similar to those of HIV-1.
The development of an anti-HIV microbicide is critical in the fight against the spread of HIV. It is shown here that the covalent linking of compounds that bind gp120 with compounds that bind gp41 can inhibit HIV entry even more potently than individual inhibitors or noncovalent combinations. The most striking example involves griffithsin, a potent HIV inhibitor that binds to the surface of HIV gp120. While griffithsin inhibits HIV Env-mediated fusion in a CCR5-tropic cell-cell fusion assay with a 50% inhibitory concentration (IC50) of 1.31 ± 0.87 nM and the gp41-binding peptide C37 shows an IC50 of 18.2 ± 7.6 nM, the covalently linked combination of griffithsin with C37 (Griff37) has an IC50 of 0.15 ± 0.05 nM, exhibiting a potency 8.7-fold greater than that of griffithsin alone. Similarly, in CXCR4-tropic cell-cell fusion assays, Griff37 is 5.2-fold more potent than griffithsin alone. In viral assays, both griffithsin and Griff37 inhibit HIV replication at midpicomolar levels, but the linked compound Griff37 is severalfold more potent than griffithsin alone against both CCR5- and CXCR4-tropic virus strains. Another example of this strategy is the covalently linked combination of peptide C37 with a variant of the gp120-binding peptide CD4M33 (L. Martin et al., Nat. Biotechnol. 21:71-76, 2003). Also, nuclear magnetic resonance (NMR) spectra for several of these compounds are shown, including, to our knowledge, the first published NMR spectrum for griffithsin.
Several human monoclonal antibodies (hmAbs) exhibit relatively potent and broad neutralizing activity against HIV-1, but there has not been much success in using them as potential therapeutics. We have previously hypothesized and demonstrated that small engineered antibodies can target highly conserved epitopes that are not accessible by full-size antibodies. However, their potency has not been comparatively evaluated with known HIV-1-neutralizing hmAbs against large panels of primary isolates. We report here the inhibitory activity of an engineered single chain antibody fragment (scFv), m9, against several panels of primary HIV-1 isolates from group M (clades A–G) using cell-free and cell-associated virus in cell line-based assays. M9 was much more potent than scFv 17b, and more potent than or comparable to the best-characterized broadly neutralizing hmAbs IgG1 b12, 2G12, 2F5 and 4e10. It also inhibited cell-to-cell transmission of HIV-1 with higher potency than enfuvirtide (t-20, Fuzeon). M9 competed with a sulfated CCR5 N-terminal peptide for binding to gp120-CD4 complex, suggesting an overlapping epitope with the coreceptor binding site. M9 did not react with phosphatidylserine (pS) and cardiolipin (CL), nor did it react with a panel of autoantigens in an antinuclear autoantibody (ANA) assay. We further found that escape mutants resistant to m9 did not emerge in an immune selection assay. these results suggest that m9 is a novel anti-HIV-1 candidate with potential therapeutic or prophylactic properties, and its epitope is a new target for drug or vaccine development.
HIV; AIDS; antibodies; scFv; microbicides; therapeutics; vaccines
CMX157 is a lipid (1-0-hexadecyloxypropyl) conjugate of the acyclic nucleotide analog tenofovir (TFV) with activity against both wild-type and antiretroviral drug-resistant HIV strains, including multidrug nucleoside/nucleotide analog-resistant viruses. CMX157 was consistently >300-fold more active than tenofovir against multiple viruses in several different cell systems. CMX157 was active against all major subtypes of HIV-1 and HIV-2 in fresh human peripheral blood mononuclear cells (PBMCs) and against all HIV-1 strains evaluated in monocyte-derived macrophages, with 50% effective concentrations (EC50s) ranging between 0.20 and 7.2 nM. The lower CMX157 EC50s can be attributed to better cellular uptake of CMX157, resulting in higher intracellular levels of the active antiviral anabolite, TFV-diphosphate (TFV-PP), inside target cells. CMX157 produced >30-fold higher levels of TFV-PP in human PBMCs exposed to physiologically relevant concentrations of the compounds than did TFV. Unlike conventional prodrugs, including TFV disoproxil fumarate (Viread), CMX157 remains intact in plasma, facilitating uptake by target cells and decreasing relative systemic exposure to TFV. There was no detectable antagonism with CMX157 in combination with any marketed antiretroviral drug, and it possessed an excellent in vitro cytotoxicity profile. CMX157 is a promising clinical candidate to treat wild-type and antiretroviral drug-resistant HIV, including strains that fail to respond to all currently available nucleoside/nucleotide reverse transcriptase inhibitors.
Triciribine (TCN) is a tricyclic nucleoside that inhibits human immunodeficiency virus type 1 (HIV-1) replication by a unique mechanism not involving the inhibition of enzymes directly involved in viral replication. This activity requires the phosphorylation of TCN to its 5′ monophosphate by intracellular adenosine kinase. New testing with a panel of HIV and simian immunodeficiency virus isolates, including low-passage-number clinical isolates and selected subgroups of HIV-1, multidrug resistant HIV-1, and HIV-2, has demonstrated that TCN has broad antiretroviral activity. It was active in cell lines chronically infected with HIV-1 in which the provirus was integrated into chromosomal DNA, thereby indicating that TCN inhibits a late process in virus replication. The selection of TCN-resistant HIV-1 isolates resulted in up to a 750-fold increase in the level of resistance to the drug. DNA sequence analysis of highly resistant isolate HIV-1H10 found five point mutations in the HIV-1 gene nef, resulting in five different amino acid changes. DNA sequencing of the other TCN-resistant isolates identified at least one and up to three of the same mutations observed in isolate HIV-1H10. Transfer of the mutations from TCN-resistant isolate HIV-1H10 to wild-type virus and subsequent viral growth experiments with increasing concentrations of TCN demonstrated resistance to the drug. We conclude that TCN is a late-phase inhibitor of HIV-1 replication and that mutations in nef are necessary and sufficient for TCN resistance.
Although many interactions between HIV-1 and human proteins have been reported in the scientific literature, no publicly accessible source for efficiently reviewing this information was available. Therefore, a project was initiated in an attempt to catalogue all published interactions between HIV-1 and human proteins. HIV-related articles in PubMed were used to develop a database containing names, Entrez GeneIDs, and RefSeq protein accession numbers of interacting proteins. Furthermore, brief descriptions of the interactions, PubMed identification numbers of articles describing the interactions, and keywords for searching the interactions were incorporated. Over 100,000 articles were reviewed, resulting in the identification of 1448 human proteins that interact with HIV-1 comprising 2589 unique HIV-1-to-human protein interactions. Preliminary analysis of the extracted data indicates 32% were direct physical interactions (e.g., binding) and 68% were indirect interactions (e.g., up-regulation through activation of signaling pathways). Interestingly, 37% of human proteins in the database were found to interact with more than one HIV-1 protein. For example, the signaling protein mitogen-activated protein kinase 1 has a surprising range of interactions with 10 different HIV-1 proteins. Moreover, large numbers of interactions were published for the HIV-1 regulatory protein Tat and envelope proteins: 30% and 33% of total interactions identified, respectively. The database is accessible at http://www.ncbi.nlm.nih.gov/RefSeq/HIVInteractions/ and is cross-linked to other National Center for Biotechnology Information databases and programs via Entrez Gene. This database represents a unique and continuously updated scientific resource for understanding HIV-1 replication and pathogenesis to assist in accelerating the development of effective therapeutic and vaccine interventions.
Protease activity within nascently released human immunodeficiency virus type 1 (HIV-1) particles is responsible for the cleavage of the viral polyproteins Gag and Gag-Pol into their constituent parts, which results in the subsequent condensation of the mature conical core surrounding the viral genomic RNA. Concomitant with viral maturation is a conformational change in the packaged viral RNA from a loosely associated dimer into a more thermodynamically stable form. In this study we used suboptimal concentrations of two protease inhibitors, lopinavir and atazanavir, to study their effects on Gag polyprotein processing and on the properties of the RNA in treated virions. Analysis of the treated virions demonstrated that even with high levels of inhibition of viral infectivity (IC90), most of the Gag and Gag-Pol polyproteins were processed, although slight but significant increases in processing intermediates of Gag were detected. Drug treatments also caused a significant increase in the proportion of viruses displaying either immature or aberrant mature morphologies. The aberrant mature particles were characterized by an electron-dense region at the viral periphery and an electron-lucent core structure in the viral center, possibly indicating exclusion of the genomic RNA from these viral cores. Intriguingly, drug treatments caused only a slight decrease in overall thermodynamic stability of the viral RNA dimer, suggesting that the dimeric viral RNA was able to mature in the absence of correct core condensation.
HIV-1; protease inhibitor; virus maturation; RNA dimer; virion morphology
Entry inhibitors of human immunodeficiency virus, type 1 (HIV-1) have been
the focus of much recent research. C34, a potent fusion inhibitor derived from
the HR2 region of gp41, was engineered into a 1:1 human serum albumin
conjugate through stable covalent attachment of a maleimido-C34 analog onto
cysteine 34 of albumin. This bioconjugate, PC-1505, was designed to require
less frequent dosing and less peptide than T-20 and was assessed for its
antifusogenic activity both in vitro and in vivo in the
SCID-hu Thy/Liv mouse model. PC-1505 was essentially equipotent to the
original C34 peptide and to T-20 in vitro. In HIV-1-infected SCID-hu
Thy/Liv mice, T-20 lost activity with infrequent dosing, whereas the antiviral
potency of PC-1505 was sustained, and PC-1505 was active against
T-20-resistant (“DIV”) virus with a G36D substitution in gp41. The
in vivo results are the direct result of a significantly improved
pharmacokinetic profile for the C34 peptide following albumin conjugation.
Contrary to previous reports that the gp41 NHR trimer is poorly accessible to
C34 fused to protein cargoes of increasing size (Hamburger, A. E., Kim, S.,
Welch, B. D., and Kay, M. S. (2005) J. Biol. Chem. 280,
12567–12572), these results are the first demonstration of the capacity
for a large, endogenous serum protein to gain unobstructed access to the
transient gp41 intermediates that exist during the HIV fusion process, and it
supports further development of albumin conjugation as a promising approach to
inhibit HIV-1 entry.
Topical microbicides are self-administered, prophylactic products for protection against sexually transmitted pathogens. A large number of compounds with known anti-human immunodeficiency virus type 1 (HIV-1) inhibitory activity have been proposed as candidate topical microbicides. To identify potential leads, an in vitro screening algorithm was developed to evaluate candidate microbicides in assays that assess inhibition of cell-associated and cell-free HIV-1 transmission, entry, and fusion. The algorithm advances compounds by evaluation in a series of defined assays that generate measurements of relative antiviral potency to determine advancement or failure. Initial testing consists of a dual determination of inhibitory activity in the CD4-dependent CCR5-tropic cell-associated transmission inhibition assay and in the CD4/CCR5-mediated HIV-1 entry assay. The activity is confirmed by repeat testing, and identified actives are advanced to secondary screens to determine their effect on transmission of CXCR4-tropic viruses in the presence or absence of CD4 and their ability to inhibit CXCR4- and CCR5-tropic envelope-mediated cell-to-cell fusion. In addition, confirmed active compounds are also evaluated in the presence of human seminal plasma, in assays incorporating a pH 4 to 7 transition, and for growth inhibition of relevant strains of lactobacilli. Leads may then be advanced for specialized testing, including determinations in human cervical explants and in peripheral blood mononuclear cells against primary HIV subtypes, combination testing with other inhibitors, and additional cytotoxicity assays. PRO 2000 and SPL7013 (the active component of VivaGel), two microbicide products currently being evaluated in human clinical trials, were tested in this in vitro algorithm and were shown to be highly active against CCR5- and CXCR4-tropic HIV-1 infection.
The ‘Human Immunodeficiency Virus Type 1 (HIV-1), Human Protein Interaction Database’, available through the National Library of Medicine at www.ncbi.nlm.nih.gov/RefSeq/HIVInteractions, was created to catalog all interactions between HIV-1 and human proteins published in the peer-reviewed literature. The database serves the scientific community exploring the discovery of novel HIV vaccine candidates and therapeutic targets. To facilitate this discovery approach, the following information for each HIV-1 human protein interaction is provided and can be retrieved without restriction by web-based downloads and ftp protocols: Reference Sequence (RefSeq) protein accession numbers, Entrez Gene identification numbers, brief descriptions of the interactions, searchable keywords for interactions and PubMed identification numbers (PMIDs) of journal articles describing the interactions. Currently, 2589 unique HIV-1 to human protein interactions and 5135 brief descriptions of the interactions, with a total of 14 312 PMID references to the original articles reporting the interactions, are stored in this growing database. In addition, all protein–protein interactions documented in the database are integrated into Entrez Gene records and listed in the ‘HIV-1 protein interactions’ section of Entrez Gene reports. The database is also tightly linked to other databases through Entrez Gene, enabling users to search for an abundance of information related to HIV pathogenesis and replication.
Debio-025 is a synthetic cyclosporine with no immunosuppressive capacity but a high inhibitory potency against cyclophilin A (CypA)-associated cis-trans prolyl isomerase (PPIase) activity. A lack of immunosuppressive effects compared to that of cyclosporine was demonstrated both in vitro and in vivo. For three cyclosporines, the inhibitory potential against PPIase activity was quantitatively correlated with that against human immunodeficiency virus type 1 (HIV-1) replication. Debio-025 selectively inhibited the replication of HIV-1 in a CD4+ cell line and in peripheral blood mononuclear cells: potent activity was demonstrated against clinical isolates of various HIV-1 subtypes, including isolates with multidrug resistance to reverse transcriptase and protease inhibitors. Simian immunodeficiency virus and HIV-2 strains were generally resistant to inhibition by Debio-025; however, some notable exceptions of sensitive HIV-2 clinical isolates were detected. In two-drug combination studies, additive inhibitory effects were found between Debio-025 and 19 clinically used drugs of different classes. Clinical HIV-1 isolates that are naturally resistant to Debio-025 and that do not depend on CypA for infection were identified. Comparison of the amino acid sequences of the CypA binding domain of the capsid (CA) protein from Debio-025-sensitive and -resistant HIV-1 isolates indicated that resistance was mostly associated with an H87Q/P exchange. Mechanistically, cyclosporines competitively inhibit the binding of CypA to the HIV-1 CA protein, which is an essential interaction required for early steps in HIV-1 replication. By real-time PCR we demonstrated that early reverse transcription is reduced in the presence of Debio-025 and that late reverse transcription is almost completely blocked. Thus, Debio-025 seems to interfere with the function of CypA during the progression/completion of HIV-1 reverse transcription.
Frequent human immunodeficiency virus type 1 (HIV-1) recombination occurs during DNA synthesis when portions of the two copackaged RNAs are used as templates to generate a hybrid DNA copy. Therefore, the frequency of copackaging of genomic RNAs from two different viruses (heterozygous virion formation) affects the generation of genotypically different recombinants. We hypothesized that the selection of copackaged RNA partners is largely determined by Watson-Crick pairing at the dimer initiation signal (DIS), a 6-nucleotide palindromic sequence at the terminal loop of stem-loop 1 (SL1). To test our hypothesis, we examined whether heterozygous virion formation could be encouraged by manipulation of the DIS. Three pairs of viruses were generated with compensatory DIS mutations, designed so that perfect DIS base pairing could only occur between RNAs derived from different viruses, not between RNAs from the same virus. We observed that vector pairs with compensatory DIS mutations had an almost twofold increase in recombination rates compared with wild-type viruses. These data suggest that heterozygous virion formation was enhanced in viruses with compensatory DIS mutations (from 50% to more than 90% in some viral pairings). The role of the SL1 stem in heterozygous virion formation was also tested; our results indicated that the intermolecular base pairing of the stem sequences does not affect RNA partner selection. In summary, our results demonstrate that the Watson-Crick pairing of the DIS is a major determinant in the selection of the copackaged RNA partner, and altering the base pairing of the DIS can change the proportion of heterozygous viruses in a viral population. These results also strongly support the hypothesis that HIV-1 RNA dimers are formed prior to encapsidation.
The benzimidazole nucleosides 2-bromo-5,6-dichloro-1-(β-d-ribofuranosyl)benzimidazole (BDCRB) and 2-isopropylamino-5,6-dichloro-1-(β-l-ribofuranosyl)benzimidazole (1263W94, or maribavir) are potent and selective inhibitors of human cytomegalovirus (HCMV) replication. These inhibitors act by two different mechanisms: BDCRB blocks the processing and maturation of viral DNA, whereas maribavir prevents viral DNA synthesis and capsid nuclear egress. In order to determine by which of these two mechanisms other benzimidazole nucleosides acted, we performed time-of-addition studies and other experiments with selected new analogs. We found that the erythrofuranosyl analog and the α-lyxofuranosyl analog acted late in the viral replication cycle, similar to BDCRB. In marked contrast, the α-5′-deoxylyxofuranosyl analog of 2,5,6-trichloro-1-(β-d-ribofuranosyl)benzimidazole (compound UMJD1311) acted early in the replication cycle, too early to be consistent with either mechanism. Similar to other reports on early acting inhibitors of herpesviruses, compound 1311 was multiplicity of infection dependent, an observation that could not be reproduced with UV-inactivated virus. HCMV isolates resistant to BDCRB and maribavir were sensitive to compound 1311, as were viruses resistant to ganciclovir, cidofovir, and foscarnet. The preincubation of host cells with compound 1311 and removal prior to the addition of HCMV did not produce an antiviral cellular response. We conclude that this newly discovered early mode of action occurs at a stage of viral replication after entry to cells but prior to viral DNA synthesis, thereby strongly suggesting that the trisubstituted benzimidazole nucleoside series possesses three distinct biochemical modes of action for inhibition of HCMV replication.
1-(β-d-Ribofuranosyl)-2,5,6-trichlorobenzimidazole (TCRB) and its 2-bromo analog, BDCRB, are potent and selective inhibitors of human cytomegalovirus (HCMV) DNA processing and packaging. Since they are readily metabolized in vivo, analogs were synthesized to improve biostability. One of these, 1-(β-l-ribofuranosyl)-2-isopropylamino-5,6-dichlorobenzimidazole (1263W94; maribavir), inhibits viral DNA synthesis and nuclear egress. Resistance to maribavir was mapped to UL97, and this viral kinase was shown to be a direct target of maribavir. In the present study, an HCMV strain resistant to TCRB and BDCRB was passaged in increasing concentrations of maribavir, and resistant virus was isolated. This strain (G2) grew at the same rate as the wild-type virus and was resistant to both BDCRB and maribavir. Resistance to BDCRB was expected, because the parent strain from which G2 was isolated was resistant due to known mutations in UL56 and UL89. However, no mutations were found in UL97 or other relevant open reading frames that could explain resistance to maribavir. Because sequencing of selected HCMV genes did not identify the resistance mutation, a cosmid library was made from G2, and a series of recombinant G2 wild-type viruses were constructed. Testing the recombinants for sensitivity to maribavir narrowed the locus of resistance to genes UL26 to UL32. Sequencing identified a single coding mutation in ORF UL27 (Leu335Pro) as the one responsible for resistance to maribavir. These results establish that UL27 is either directly or indirectly involved in the mechanism of action of maribavir. They also suggest that UL27 could play a role in HCMV DNA synthesis or egress of HCMV particles from the nucleus.
We have previously reported that 2,5,6-trichloro-1-(β-d-ribofuranosyl)benzimidazole (TCRB) and its 2-bromo analog (2-bromo-5,6-dichloro-1-(β-d-ribofuranosy)benzimidazole [BDCRB]) are potent and selective inhibitors of human cytomegalovirus (HCMV) replication that block viral DNA maturation via HCMV gene products UL89 and UL56. To determine if phosphorylation is required for antiviral activity, the in vitro metabolism of BDCRB was examined and the antiviral activities of nonphosphorylatable 5′-deoxy analogs were determined. Reverse-phase high-performance liquid chromatography (HPLC) analysis of extracts from uninfected and HCMV-infected cells incubated with [3H]BDCRB revealed two major metabolites. Both were less polar than naturally occurring nucleoside monophosphates, but one peak coeluted with a BDCRB-5′-monophosphate (BDCRB-5′-MP) standard. Further analysis revealed, however, that neither metabolite partitioned with BDCRB-5′-MP on anion-exchange HPLC. Their retention patterns were not affected by incubation with alkaline phosphatase, thereby establishing that the compounds were not nucleoside 5′-monophosphates. Both compounds were detected in uninfected and HCMV-infected cells and in mouse live extracts, but neither has been identified. Like TCRB and BDCRB, the nonphosphorylatable 5′-deoxy analogs were potent and selective inhibitors of HCMV replication. The 5′-deoxy analogs maintained inhibition of HCMV replication upon removal of BDCRB, whereas an inhibitor of DNA synthesis did not. Similar to TCRB, its 5′-deoxy analog (5′-dTCRB) did not affect viral DNA synthesis, but 5′-dTCRB did inhibit viral DNA maturation to genome-length units. Additionally, virus isolates resistant to TCRB were also resistant to 5′-dTCRB and the 5′-deoxy analog of BDCRB. Taken together, these results confirm that TCRB, BDCRB, and their 5′-deoxy analogs have common mechanisms of action and establish that these benzimidazole ribonucleosides, unlike other antiviral nucleosides, do not require phosphorylation at the 5′ position for antiviral activity.
2,5,6-Trichloro-1-β-d-ribofuranosyl benzimidazole (TCRB) is a potent and selective inhibitor of human cytomegalovirus (HCMV) replication. TCRB acts via a novel mechanism involving inhibition of viral DNA processing and packaging. Resistance to the 2-bromo analog (BDCRB) has been mapped to the UL89 open reading frame (ORF), and this gene product was proposed as the viral target of the benzimidazole nucleosides. In this study, we report the independent isolation of virus that is 20- to 30-fold resistant to TCRB (isolate C4) and the characterization of the virus. The six ORFs known to be essential for viral DNA cleavage and packaging (UL51, UL52, UL56, UL77, UL89, and UL104) were sequenced from wild-type HCMV, strain Towne, and from isolate C4. Mutations were identified in UL89 (D344E) and in UL56 (Q204R). The mutation in UL89 was identical to that previously reported for virus resistant to BDCRB, but the mutation in UL56 is novel. Marker transfer analysis demonstrated that each of these mutations individually caused ∼10-fold resistance to the benzimidazoles and that the combination of both mutations caused ∼30-fold resistance. The rate and extent of replication of the mutants was the same as for wild-type virus, but the viruses were less sensitive to inhibition of DNA cleavage by TCRB. Mapping of resistance to UL56 supports and extends recent work showing that UL56 codes for a packaging motif binding protein which also has specific nuclease activity (E. Bogner et al., J. Virol. 72:2259–2264, 1998). Resistance which maps to two different genes suggests that their putative proteins interact and/or that either or both have a benzimidazole ribonucleoside binding site. The results also suggest that the gene products of UL89 and UL56 may be antiviral drug targets.
The HIV-1 capsid (CA) protein plays essential roles in both early and late stages of virl replication and has emerged as a novel drug target. We report hybrid structure-based virtual screening to identify small molecules with the potential to interact with the N-terminal domain (NTD) of HIV-1 CA and disrupt early, preintegration steps of the HIV-1 replication cycle. The small molecule 4,4′-[dibenzo[b,d]furan-2,8-diylbis(5-phenyl-1H-imidazole-4,2-diyl)]dibenzoic acid (CK026), which had anti-HIV-1 activity in single- and multiple-round infections but failed to inhibit viral replication in peripheral blood mononuclear cells (PBMCs), was identified. Three analogues of CK026 with reduced size and better drug-like properties were synthesized and assessed. Compound I-XW-053 (4-(4,5-diphenyl-1H-imidazol-2-yl)benzoic acid) retained all of the antiviral activity of the parental compound and inhibited the replication of a diverse panel of primary HIV-1 isolates in PBMCs, while displaying no appreciable cytotoxicity. This antiviral activity was specific to HIV-1, as I-XW-053 displayed no effect on the replication of SIV or against a panel of nonretroviruses. Direct interaction of I-XW-053 was quantified with wild-type and mutant CA protein using surface plasmon resonance and isothermal titration calorimetry. Mutation of Ile37 and Arg173, which are required for interaction with compound I-XW-053, crippled the virus at an early, preintegration step. Using quantitative PCR, we demonstrated that treatment with I-XW-053 inhibited HIV-1 reverse transcription in multiple cell types, indirectly pointing to dysfunction in the uncoating process. In summary, we have identified a CA-specific compound that targets and inhibits a novel region in the NTD-NTD interface, affects uncoating, and possesses broad-spectrum anti-HIV-1 activity.
Real-time PCR array for rapid detection of multiple viral pathogens should be highly useful in cases where the sample volume and the time of testing are limited, i.e. in the eligibility testing of tissue and organ donors.
We developed a real-time PCR array capable of simultaneously detecting eight human viral pathogens: human immunodeficiency virus types 1 and 2 (HIV-1 and -2), hepatitis B virus (HBV), hepatitis C virus (HCV), human T-cell leukemia virus-1 and -2 (HTLV-1 and -2), vaccinia virus (VACV) and West Nile virus (WNV). One hundred twenty (120) primers were designed using a combination of bioinformatics approaches, and, after experimental testing, 24 primer sets targeting eight viral pathogens were selected to set up the array with SYBR Green chemistry. The specificity and sensitivity of the virus-specific primer sets selected for the array were evaluated using analytical panels with known amounts of viruses spiked into human plasma. The array detected: 10 genome equivalents (geq)/ml of HIV-2 and HCV, 50 geq of HIV-1 (subtype B), HBV (genotype A) and WNV. It detected 100–1,000 geq/ml of plasma of HIV-1 subtypes (A – G), group N and CRF (AE and AG) isolates. Further evaluation with a panel consisting of 28 HIV-1 and HIV-2 clinical isolates revealed no cross-reactivity of HIV-1 or HIV-2 specific primers with another type of HIV. All 28 viral isolates were identified with specific primer sets targeting the most conserved genome areas. The PCR array correctly identified viral infections in a panel of 17 previously quantified clinical plasma samples positive for HIV-1, HCV or HBV at as low as several geq per PCR reaction.
The viral array described here demonstrated adequate performance in the testing of donors’ clinical samples. Further improvement in its sensitivity for the broad spectrum of HIV-1 subtypes is under development.