Single-round Replication Assay Research Articles

4-amino-1-hydroxy-2-oxo-1,8-naphthyridine-containing compounds having high potency against raltegravir-resistant integrase mutants of HIV-1.

There are currently three HIV-1 integrase (IN) strand transfer inhibitors (INSTIs) approved by the FDA for the treatment of AIDS. However, the emergence of drug-resistant mutants emphasizes the need to develop additional agents that have improved efficacies against the existent resistant mutants. As reported herein, we modified our recently disclosed 1-hydroxy-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamides IN inhibitors to develop compounds that have improved efficacies against recombinant IN in biochemical assays. These new compounds show single-digit nanomolar antiviral potencies against HIV vectors that carry wild-type (WT) IN in a single round replication assay and have improved potency against vectors harboring the major forms of drug resistant IN mutants. These compounds also have low toxicity for cultured cells, which in several cases, results in selectivity indices (CC50/EC50) of greater than 10000. The compounds have the potential, with additional structural modifications, to yield clinical agents that are effective against the known strains of resistant viruses.

Decreased Infectivity of a Neutralization-Resistant Equine Infectious Anemia Virus Variant Can Be Overcome by Efficient Cell-to-Cell Spread

Two variants of equine infectious anemia virus (EIAV) that differed in sensitivity to broadly neutralizing antibody were tested in direct competition assays. No differences were observed in the growth curves and relative fitness scores of EIAVs of principal neutralizing domain variants of groups 1 (EIAV(PND-1)) and 5 (EIAV(PND-5)), respectively; however, the neutralization-resistant EIAV(PND-5) variant was less infectious in single-round replication assays. Infectious center assays indicated similar rates of cell-to-cell spread, which was approximately 1,000-fold more efficient than cell-free infectivity. These data indicate that efficient cell-to-cell spread can overcome the decreased infectivity that may accompany immune escape and should be considered in studies assessing the relative levels of fitness among lentivirus variants, including HIV-1.

Retrocyclin RC-101 overcomes cationic mutations on the heptad repeat 2 region of HIV-1 gp41

Retrocyclin RC-101, a theta-defensin with lectin-like properties, potently inhibits infection by many HIV-1 subtypes by binding to the heptad repeat 2 (HR2) region of glycoprotein 41 (gp41) and preventing six-helix bundle formation. In the present study, we used in silico computational exploration to identify residues of HR2 that interacted with RC-101, and then analyzed the HIV-1 sequence database at Los Alamos National Laboratory (New Mexico, USA) for residue variations in the heptad repeat 1 (HR1) and HR2 segments that could plausibly impart in vivo resistance. Docking RC-101 to gp41 peptides in silico confirmed its strong preference for HR2 over HR1, and implicated residues crucial for its ability to bind HR2. We mutagenized these residues in pseudotyped HIV-1 JR.FL reporter viruses, and subjected them to single-round replication assays in the presence of 1.25-10 microg x mL(-1) RC-101. Apart from one mutant that was partially resistant to RC-101, the other pseudotyped viruses with single-site cationic mutations in HR2 manifested absent or impaired infectivity or retained wild-type susceptibility to RC-101. Overall, these data suggest that most mutations capable of rendering HIV-1 resistant to RC-101 will also exert deleterious effects on the ability of HIV-1 to initiate infections - an interesting and novel property for a potential topical microbicide.

Resistance profile of a neutralizing anti-HIV monoclonal antibody, KD-247, that shows favourable synergism with anti-CCR5 inhibitors

The high-affinity humanized monoclonal antibody (MAb) KD-247 reacts with a tip region in gp120-V3 and cross-neutralizes primary isolates with a matching neutralization sequence motif. We induced an HIV-1 variant that was resistant to KD-247 by exposing the JR-FL virus to increasing concentrations of KD-247 in PM1/CCR5 cells, which expressed high levels of CCR5 in vitro. We determined the amino acid sequence of the gp120-encoding region of the JR-FL escape mutant from KD-247. To confirm that this substitution was responsible for the KD-247-resistance, a single-round replication assay was performed. We further evaluated the anti-HIV-1 interactions between KD-247 and various CCR5 inhibitors in vitro. At passage 8 of the culture in the presence of 1000 mug/ml KD-247, one amino acid substitution, Gly to Glu at position 314 (G314E), was identified in the V3-tip of gp120. A pseudotyped virus with the G314E mutation was highly resistant to KD-247. Unexpectedly, this mutant virus was sensitive to CCR5 inhibitors, RANTES, recombinant human soluble CD4 (rsCD4) and an anti-CCR5 MAb, but resistant to an anti-CD4 MAb, compared with the wild-type virus. We also found that combinations of KD-247 and CCR5 inhibitors were highly synergistic. The present data suggest that KD-247 has certain advantages for possible passive immunotherapy. They are: high concentrations of KD-247 are needed for viral acquisition of KD-247 resistance; the escape variants are more sensitive to CCR5 inhibitors and rsCD4; and there are high levels of synergism between KD-247 and CCR5 inhibitors at all concentrations tested.

A second-site suppressor significantly improves the defective phenotype imposed by mutation of an aromatic residue in the N-terminal domain of the HIV-1 capsid protein

The HIV-1 capsid (CA) protein plays an important role in virus assembly and infectivity. Previously, we showed that Ala substitutions in the N-terminal residues Trp23 and Phe40 cause a severely defective phenotype. In searching for mutations at these positions that result in a non-lethal phenotype, we identified one candidate, W23F. Mutant virions contained aberrant cores, but unlike W23A, also displayed some infectivity in a single-round replication assay and delayed replication kinetics in MT-4 cells. Following long-term passage in MT-4 cells, two second-site mutations were isolated. In particular, the W23F/V26I mutation partially restored the wild-type phenotype, including production of particles with conical cores and wild-type replication kinetics in MT-4 cells. A structural model is proposed to explain the suppressor phenotype. These findings describe a novel occurrence, namely suppression of a mutation in a hydrophobic residue that is critical for maintaining the structural integrity of CA and proper core assembly.

Intravirion display of a peptide corresponding to the dimer structure of protease attenuates HIV-1 replication.

Current treatment of HIV-1-infected individuals involves the administration of several drugs, all of which target either the reverse transcriptase or the protease activity of the virus. Unfortunately, the benefits of such treatments are compromised by the emergence of viruses exhibiting resistance to the drugs. This situation warrants new approaches for interfering with virus replication. Considering the activation of protease in the virus particles, a novel strategy to inhibit HIV-1 replication was tested targeting the dimerization domain of the protease. To test this idea, we have selected four residues from the C terminus of HIV-1 protease that map to the dimer interface region of the enzyme. We have exploited Vpr to display the peptides in the virus particles. The chimeric Vpr exhibited expression and virion incorporation similar to wildtype Vpr. The virus derived from the HIV-1 proviral DNA containing chimeric Vpr sequences registered a reduced level of replication in CEM and CEM X 174 cells in comparison with viruses containing wildtype Vpr. Similar results were observed in a single-round replication assay. These results suggest that the intravirion display of peptides targeting viral proteins is a powerful approach for developing antiviral agents and for dissecting the dynamic interactions between structural proteins during virus assembly and disassembly.

Involvement of both the V2 and V3 Regions of the CCR5-Tropic Human Immunodeficiency Virus Type 1 Envelope in Reduced Sensitivity to Macrophage Inflammatory Protein 1α

To determine whether C-C chemokines play an important role in the phenotype switch of human immunodeficiency virus (HIV) from CCR5 to CXCR4 usage during the course of an infection in vivo, macrophage inflammatory protein (MIP)-1alpha-resistant variants were isolated from CCR5-tropic (R5) HIV-1 in vitro. The selected variants displayed reduced sensitivities to MIP-1alpha (fourfold) through CCR5-expressing CD4-HeLa/long terminal repeat-beta-galactosidase (MAGI/CCR5) cells. The variants were also resistant to other natural ligands for CCR5, namely, MIP-1beta (>4-fold) and RANTES (regulated upon activation, normal T-cell expressed and secreted) (6-fold). The env sequence analyses revealed that the variants had amino acid substitutions in V2 (valine 166 to methionine) and V3 (serine 303 to glycine), although the same V3 substitution appeared in virus passaged without MIP-1alpha. A single-round replication assay using a luciferase reporter HIV-1 strain pseudotyped with mutant envelopes confirmed that mutations in both V2 and V3 were necessary to confer the reduced sensitivity to MIP-1alpha, MIP-1beta, and RANTES. However, the double mutant did not switch its chemokine receptor usage from CCR5 to CXCR4, indicating the altered recognition of CCR5 by this mutant. These results indicated that V2 combined with the V3 region of the CCR5-tropic HIV-1 envelope modulates the sensitivity of HIV-1 to C-C chemokines without altering the ability to use chemokine receptors.

Marked increase in anti-HIV activity, as well as inhibitory activity against HIV entry mediated by CXCR4, linked to enhancement of the binding ability of tachyplesin analogs to CXCR4.

T22 ([Tyr5,12, Lys7]-polyphemusin II) is a strong anti-HIV compound. Six analogs of T22 and two natural forms were synthesized. Of them, all downsized peptides (14 residues; TW70, T131, T134, and T140) showed a higher selectivity index than did other, 17- or 18-residue peptides. In particular, T134 and T140 showed both lower cytotoxicity and higher antiviral activity than did T22 against HIV infection of MT-4 cells, an HTLV-I-bearing T cell line. To clarify the inhibitory mode of T22 and its analogs, we used a single-round replication assay (luciferase assay), in which different envelope-bearing pseudotypes were used to infect CXCR4- or CCR5-bearing U87 cells via CD4. All of the analogs inhibited T cell line-tropic strain HXB-2 (X4) and dual-tropic strain 89.6 (R5X4) HIV infections mediated by CXCR4, but had no effect on macrophage-tropic strain ADA (R5) or 89.6 HIV infections mediated by CCR5. The inhibition by T134 (IC50 of 2.70 nM) and T140 (IC50 of 0.432 nM) was also stronger than that by T22 (IC50 of 5.05 nM). The binding of anti-CXCR4 monoclonal antibody 12G5 to lymphoma-derived T cell line Sup-T1 was more efficiently blocked by T134 and T140 than by T22. Taken together, T22 and its analogs T134 and T140 exerted their inhibition by specific binding to CXCR4. The marked increase in the anti-HIV activity of T134 and T140 was ascribed to an enhancement in their ability to bind to CXCR4.

HIV-1 Gag shares a signature motif with annexin (Anx7), which is required for virus replication.

Genetic and biochemical analyses of the Gag protein of HIV-1 indicate a crucial role for this protein in several functions related to viral replication, including viral assembly. It has been suggested that Gag may fulfill some of the functions by recruiting host cellular protein(s). In our effort to identify structural and functional homologies between Gag and cellular cytoskeletal and secretory proteins involved in transport, we observed that HIV-1 Gag contains a unique PGQM motif in the capsid region. This motif was initially noted in the regulatory domain of synexin the membrane fusion protein of Xenopus laevis. To evaluate the functional significance of the highly conserved PGQM motif, we introduced alanine (A) in place of individual residues of the PGQM and deleted the motif altogether in a Gag expression plasmid and in an HIV-1 proviral DNA. The proviral DNA containing mutations in the PGQM motif showed altered expression, assembly, and release of viral particles in comparison to parental (NL4-3) DNA. When tested in multiple- and single-round replication assays, the mutant viruses exhibited distinct replication phenotypes; the viruses containing the A for the G and Q residues failed to replicate, whereas A in place of the P and M residues did not inhibit viral replication. Deletion of the tetrapeptide also resulted in the inhibition of replication. These results suggest that the PGQM motif may play an important role in the infection process of HIV-1 by facilitating protein-protein interactions between viral and/or viral and cellular proteins.

Virion incorporation of human immunodeficiency virus type 1 Nef is mediated by a bipartite membrane-targeting signal: analysis of its role in enhancement of viral infectivity.

The nef gene of primate immunodeficiency viruses is essential for high-titer virus replication and AIDS pathogenesis in vivo. In tissue culture, Nef is not required for human immunodeficiency virus (HIV) infection but enhances viral infectivity. We and others have shown that Nef is incorporated into HIV-1 particles and cleaved by the viral proteinase. To determine the signal for Nef incorporation and to analyze whether virion-associated Nef is responsible for enhancement of infectivity, we generated a panel of nef mutants and analyzed them for virion incorporation of Nef and for their relative infectivities. We report that N-terminal truncations of Nef abolished its incorporation into HIV particles. Incorporation was reconstituted by targeting the respective proteins to the plasma membrane by using a heterologous signal. Mutational analysis revealed that both myristoylation and an N-terminal cluster of basic amino acids were required for virion incorporation and for plasma membrane targeting of Nef. Grafting the N-terminal anchor domain of Nef onto the green fluorescent protein led to membrane targeting and virion incorporation of the resulting fusion protein. These results indicate that Nef incorporation into HIV-1 particles is mediated by plasma membrane targeting via an N-terminal bipartite signal which is reminiscent of a Src homology region 4. Virion incorporation of Nef correlated with enhanced infectivity of the respective viruses in a single-round replication assay. However, the phenotypes of HIV mutants with reduced Nef incorporation only partly correlated with their ability to replicate in primary lymphocytes, indicating that additional or different mechanisms may be involved in this system.

Enhancement of human immunodeficiency virus type 1 infectivity by Nef is producer cell-dependent.

The growth kinetics of wild-type and nef mutant viruses of human immunodeficiency virus type 1 were comparatively analysed in several human CD4+ cell lines. Delayed replication of nef mutant virus was observed in all cell lines examined. To determine the stage in the virus replication cycle that is affected by Nef, a single-round replication assay was performed. Initially, the expression of marker genes in transfected cells was examined in order to study the role of Nef in the late phase of infection. The results obtained indicated that Nef is dispensable during the transcription to virion production stage. Next, the effect of Nef on the early phase was investigated with a single-round infection. It was demonstrated that Nef is required in the early phase of the virus replication cycle, from virion adsorption to integration. Finally, the infectivity of virus stocks prepared from four cell lines was determined. The relative infectivity of the nef mutant from the four cell lines differed. Taken together, we conclude that Nef acts via modulation of viral particles to enhance virus infectivity in a cell-dependent manner.

Early Function of HIV-1 Gag Proteins Is Cell-Dependent

Variousgaggene mutants of human immunodeficiency virus type 1 (HIV-1) were monitored for their replication potentials and defective replication sites in various CD4-positive T-cell lines. Some matrix, capsid, and nucleocapsid mutants displayed a replication defect in a cell-dependent manner. The single-round replication assays demonstrated that these mutants were defective at an early infection phase also in a cell-dependent way. These results indicated that interaction of a cell factor(s) and Gag proteins is involved in an early process of HIV-1 replication.

Functional Domain Mapping of HIV-1 Gag Proteins

A series of human immunodeficiency virus type 1 (HIV-1) proviralgaggene mutants carrying bacterial CAT gene were constructed and monitored for the expression of reverse transcriptase and CAT in a highly sensitive single-round replication assay system to determine the defective replication phase in lymphocytic cells. All the mutants displayed no abnormality in the process of transcription and translation at late replication stage. In contrast, some matrix, capsid, and p6 mutants were defective at final phase, that is, assembly and virion release. Most of the mutants including nucleocapsid mutants, which showed normal phenotype at late stage, were defective at early replication phase. From the functional domain map thus obtained, it is evident that HIV-1 Gag proteins are required for both early and late replication phases.

HIV-1 capsid mutants inhibit the replication of wild-type virus at both early and late infection phases

In-frame mutations were introduced into various portions of the human immunodeficiency virus type 1 (HIV-1) gag gene, and potentials of the mutants to suppress the replication of wild-type HIV-1 were monitored. In contrast to results obtained with matrix and nucleocapsid mutants, almost all capsid mutants blocked HIV-1 replication completely in single-round replication assays. A capsid mutant designated C6b was demonstrated to be one of the most efficient inhibitors for HIV-1 reported to date, and to be effective at both early and late viral replication phases. T-cells, which are engineered to express the C6b Gag in response to HIV-1 infection, were perfectly resistant to HIV-1.

Cleavage of Gag precursor is required for early replication phase of HIV-1

A mutant of human immunodeficiency virus type 1 (HIV-1), which is deficient for Gag precursor cleavage and non-infectious, was characterized with respect to its defective step in the viral replication phase. Upon transfection, the mutant produced a normal level of progeny virions as monitored by electron microscopy and RNA hybridization. Single-round replication assay demonstrated, in contrast, that the mutant was defective at the early phase of the replication cycle. Furthermore, no viral DNA was detected in the cells infected with the mutant. Taken together, it is concluded that maturation of Gag precursor protein of HIV-1 is required for an early event(s) before or during a coupled process of uncoating/reverse transcription.

Regulation of HIV replication by a host transcription factor NFκB

On the basis of sequence homology, four distinct groups of primate immunodeficiency viruses are now recognized. These viruses possess a number of auxiliary genes not found in other retroviruses, i.e., tat, rev, nef, vif, vpr, vpu, and vpx genes. The general conservation of these accessory genes, within a group and in some cases among all four primate immunodeficiency viruses, argl~es for an important functional role in the virus life cycle. These genes can be further characterized as essential and non-essential. Non-essential genes include nef, vif, vpr, vpu, and vpx. Human immunodeficiency virus type I (HIV-I), a magor causative agent of human AIDS, contains these auxiliary genes except for the vpx. Although the biology and molecular biology of HIV-I have been extensively studied, molecular bases for actions of the non-essential gene products, which are unique to the primate lentivirus group, are not fully understood yet. Recent studies have d~monstrated that most of the non-essential gene products act at various stages of virus life cycle in a cell-specific manner. We also have shown t_hat some gag gene mutants can replicate in some specific cell lines. The observed cell-specificity is critical for our understanding of the virology of HIV. In this report, to study functions of various HIV proteins, a large number of mutants were constructed, and bilogically and biochemically characterized. Growth kinetics of mutant viruses in several cell lines and primary blood cell cultures were determined, and defective steps in the viral life cycle of the mutants were monitored by our single-round replication assays. Target genes for study include gag, nef, vif, vpr, vpu, and vpx. We s~mmarize our new data regarding cell-dependent requirement of various HIV proteins for viral replication.