Yosuke Maeda

Identification of Critical Amino Acid Residues in Human Immunodeficiency Virus Type 1 IN Required for Efficient Proviral DNA Formation at Steps prior to Integration in Dividing and Nondividing Cells

Human immunodeficiency virus type 1 integrase (HIV-1 IN) is thought to have several putative roles at steps prior to integration, such as reverse transcription and nuclear transport of the preintegration complex (PIC). Here, we investigated new functional aspects of HIV-1 IN in the context of the viral replication cycle through point mutagenesis of Ser, Thr, Tyr, Lys, and Arg residues conserved in IN, some of which are located at possible phosphorylation sites. Our results showed that mutations of these Ser or Thr residues had no effect on reverse transcription and nuclear transport of PIC but had a slight effect on integration. Of note, mutations in the conserved KRK motif (amino acids 186 to 189), proposed previously as a putative nuclear localization signal (NLS) of HIV-1 IN, did not affect the karyophilic property of HIV-1 IN as shown by using a green fluorescent protein fusion protein expression system. Instead, these KRK mutations resulted in an almost complete lack of viral gene expression due to the failure to complete reverse transcription. This defect was complemented by supplying wild-type IN in trans, suggesting a trans-acting function of the KRK motif of IN in reverse transcription. Mutation at the conserved Tyr 143 (Y143G) resulted in partial impairment of completion of reverse transcription in monocyte-derived macrophages (MDM) but not in rhabdomyosarcoma cells. Similar effects were obtained by introducing a stop codon in the vpr gene (DeltaVpr), and additive effects of both mutations (Y143G plus DeltaVpr) were observed. In addition, these mutants did not produce two-long terminal repeat DNA, a surrogate marker for nuclear entry, in MDM. Thus, the possible impairment of Y143G might occur during the nuclear transport of the PIC. Taken together, our results identified new functional aspects of the conserved residues in HIV-1 IN: i) the KRK motif might have a role in efficient reverse transcription in both dividing and nondividing cells but not in the NLS function; ii) Y143 might be an important residue for maintaining efficient proviral DNA formation in nondividing cells.

Preliminary evidence for inhibitory effect of glycyrrhizin on HIV replication in patients with AIDS.

Glycyrrhizin (GL) at a dose of 400-1600 mg/day (7.2-30.8 mg/kg/day) was administered intravenously for a period of more than a month, on 6 separate occasions, to 3 hemophiliacs with acquired immune deficiency syndrome (AIDS). Human immunodeficiency virus type 1 (HIV-1) p24 antigen was detected at the beginning of 5 of the 6 treatment courses. Viral antigen was not detected at the end of or during 3 of the 5 treatment courses and decreased to a low level following the 2 other courses. These findings suggest that GL might inhibit HIV-1 replication in vivo.

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

ABSTRACT 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)-1α-resistant variants were isolated from CCR5-tropic (R5) HIV-1 in vitro. The selected variants displayed reduced sensitivities to MIP-1α (fourfold) through CCR5-expressing CD4-HeLa/long terminal repeat–β-galactosidase (MAGI/CCR5) cells. The variants were also resistant to other natural ligands for CCR5, namely, MIP-1β (>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-1α. 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-1α, MIP-1β, 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.

Isolation of TAK-779-resistant HIV-1 from an R5 HIV-1 GP120 V3 Loop Library

The human immunodeficiency virus (HIV-1) envelope glycoprotein (GP) 120 interacts with CD4 and the CCR5 coreceptor for viral entry. The V3 loop in GP120 is a crucial region for determining coreceptor usage during viral entry, and a variety of amino acid substitutions has been observed in clinical isolates. To construct an HIV-1 V3 loop library, we chose 10 amino acid positions in the V3 loop and incorporated random combinations (27,648 possibilities) of the amino acid substitutions derived from 31 R5 viruses into the V3 loop of HIV-1JR-FL proviral DNA. The constructed HIV-1 library contained 6.6 × 106 independent clones containing a set of 0–10 amino acid substitutions in the V3 loop. To address whether restricted steric alteration in the V3 loop could confer resistance to an entry inhibitor, TAK-779, we selected entry inhibitor-resistant HIV-1 by increasing the concentration of TAK-779 from 0.10 to 0.30 μm in PM1-CCR5 cells with high expression of CCR5. The selected viruses at passage 8 contained five amino acid substitutions in the V3 loop without any other mutations in GP120 and showed 15-fold resistance compared with the parental virus. These results indicated that a certain structure of the V3 loop containing amino acid substitutions derived from 31 R5 viruses can contribute to the acquisition of resistance to entry inhibitors binding to CCR5. Taken together, this type of HIV-1 V3 loop library is useful for isolating and analyzing the specific biological features of HIV-1 with respect to alterations of the V3 loop structure.

The antiretroviral potency of APOBEC1 deaminase from small animal species

Although the role of the APOBEC3-dependent retroelement restriction system as an intrinsic immune defense against human immunodeficiency virus type1 (HIV-1) infection is becoming clear, only the rat ortholog of mammalian APOBEC1s (A1) thus far has been shown to possess antiviral activity. Here, we cloned A1 cDNAs from small animal species, and showed that similar to rat A1, both wild-type and Δvif HIV-1 infection was inhibited by mouse and hamster A1 (4- to 10-fold), whereas human A1 had negligible effects. Moreover, rabbit A1 significantly reduced the infectivity of both HIV-1 virions (>300-fold), as well as that of SIVmac, SIVagm, FIV and murine leukemia virus. Immunoblot analysis showed that A1s were efficiently incorporated into the HIV-1 virion, and their packaging is mediated through an interaction with the nucleocapsid Gag domain. Interestingly, there was a clear accumulation of particular C-T changes in the genomic RNAs of HIV-1 produced in their presence, with few G-A changes in the proviral DNA. Together, these data reveal that A1 may function as a defense mechanism, regulating retroelements in a wide range of mammalian species.

Promoter Targeting shRNA Suppresses HIV-1 Infection In vivo Through Transcriptional Gene Silencing

Despite prolonged and intensive application, combined antiretroviral therapy cannot eradicate human immunodeficiency virus (HIV)-1 because it is harbored as a latent infection, surviving for long periods of time. Alternative approaches are required to overcome the limitations of current therapy. We have been developing a short interfering RNA (siRNA) gene silencing approach. Certain siRNAs targeting promoter regions of genes induce transcriptional gene silencing. We previously reported substantial transcriptional gene silencing of HIV-1 replication by an siRNA targeting the HIV-1 promoter in vitro. In this study, we show that this siRNA, expressed as a short hairpin RNA (shRNA) (shPromA-JRFL) delivered by lentiviral transduction of human peripheral blood mononuclear cells (PBMCs), which are then used to reconstitute NOJ mice, is able to inhibit HIV-1 replication in vivo, whereas a three-base mismatched variant (shPromA-M2) does not. In shPromA-JRFL–treated mice, HIV-1 RNA in serum is significantly reduced, and the ratio of CD4+/CD8+ T cells is significantly elevated. Expression levels of the antisense RNA strand inversely correlates with HIV-1 RNA in serum. The silenced HIV-1 can be reactivated by T-cell activation in ex vivo cultures. HIV-1 suppression is not due to offtarget effects of shPromA-JRFL. These data provide “proof-of principle” that an shRNA targeting the HIV-1 promoter is able to suppress HIV-1 replication in vivo.

A combination of polymorphic mutations in V3 loop of HIV-1 gp120 can confer noncompetitive resistance to maraviroc

Maraviroc binds to the pocket of extracellular loops of the cell surface CCR5 and prevents R5 HIV-1 from using CCR5 as a coreceptor for entry into CD4-positive cells. To evaluate the contribution of the V3 loop structure in gp120 to maraviroc resistance, we isolated maraviroc-resistant variants from the V3 loop library virus (HIV-1(V3Lib)) containing a set of random combinations of 0-10 polymorphic mutations in vitro. HIV-1(V3Lib) at passage 17 could not be suppressed even at 10 μM (>1400-fold resistance), while HIV-1(JR-FL) at passage 17 revealed an 8-fold resistance to maraviroc. HIV-1(V3Lib-P17) contained T199K and T275M plus 5 mutations in the V3 loop, I304V/F312W/T314A/E317D/I318V. The profile of pseudotyped virus containing I304V/F312W/T314A/E317D/I318V in V3 loop alone revealed a typical noncompetitive resistance, although T199K and/or T275M could not confer noncompetitive resistance. This type of library virus is useful for isolation of escape viruses from effective entry inhibitors.

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

Background: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. Methods: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. Results:At passage 8 of the culture in the presence of 1000 μg/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. Conclusions: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.

Structure and Dynamics of the gp120 V3 Loop That Confers Noncompetitive Resistance in R5 HIV-1JR-FL to Maraviroc

Maraviroc, an (HIV-1) entry inhibitor, binds to CCR5 and efficiently prevents R5 human immunodeficiency virus type 1 (HIV-1) from using CCR5 as a coreceptor for entry into CD4+ cells. However, HIV-1 can elude maraviroc by using the drug-bound form of CCR5 as a coreceptor. This property is known as noncompetitive resistance. HIV-1V3-M5 derived from HIV-1JR-FLan is a noncompetitive-resistant virus that contains five mutations (I304V/F312W/T314A/E317D/I318V) in the gp120 V3 loop alone. To obtain genetic and structural insights into maraviroc resistance in HIV-1, we performed here mutagenesis and computer-assisted structural study. A series of site-directed mutagenesis experiments demonstrated that combinations of V3 mutations are required for HIV-1JR-FLan to replicate in the presence of 1 µM maraviroc, and that a T199K mutation in the C2 region increases viral fitness in combination with V3 mutations. Molecular dynamic (MD) simulations of the gp120 outer domain V3 loop with or without the five mutations showed that the V3 mutations induced (i) changes in V3 configuration on the gp120 outer domain, (ii) reduction of an anti-parallel β-sheet in the V3 stem region, (iii) reduction in fluctuations of the V3 tip and stem regions, and (iv) a shift of the fluctuation site at the V3 base region. These results suggest that the HIV-1 gp120 V3 mutations that confer maraviroc resistance alter structure and dynamics of the V3 loop on the gp120 outer domain, and enable interactions between gp120 and the drug-bound form of CCR5.

Role of V3 Independent Domains on a Dualtropic Human Immunodeficiency Virus Type 1 (HIV-1) Envelope gp120 in CCR5 Coreceptor Utilization and Viral Infectivity

The molecular mechanism of human immunodeficiency virus type 1 (HIV‐1) entry into cells involves specific interactions between the viral envelope glycoprotein gp120 and two target cell proteins, CD4 and either CCR5 or CXCR4 chemokine receptors. In order to delineate the functional role of HIV‐1 gp120 subdomains of dualtropic strains in CCR5 coreceptor usage, we used a panel of chimeric viruses in which the V1/V2 and V3 domains of gp120 from the dualtropic HIV‐1KMT isolate were introduced either alone or in combination into the T‐tropic HIV‐1NL4–3 background. These chimeric constructs were employed in cell‐cell fusion and cell‐free virus infectivity assays using cell lines expressing CD4 and the CCR5 chemokine receptor. In both assays, the V3 domain of HIV‐1KMT but not the V1/V2 domain proved to be the principal determinant of CCR5 coreceptor usage. However, in the cell‐free viral infectivity assay although a chimeric virus with a combined V1/V2 and V3 domains of HIV‐1KMT efficiently fused with coreceptor expressing cells, yet its infectivity was markedly diminished in CCR5 as well as CXCR4 expressing cells. Restoring a comparable level of infection of such chimeric virus required the C3‐V5 domain from HIV‐1KMT to be introduced. Our present findings confirmed that the V3 domain is the major determinant of fusion activity and cellular tropism, and demonstrated a dispensable role for the V1/V2 domain. In addition the C3‐V5 domain appeared to play an important role in viral infectivity when the corresponding V1/V2 and V3 domains are present.