Tsuguhiro Kaneda

Selection of human immunodeficiency virus type 1 variants with an insertion mutation in the p6gag and p6pol genes under highly active antiretroviral therapy

We detected several types of human immunodeficiency virus type 1 (HIV‐1) variants with an insertion mutation in the p6gag and p6pol genes in eight of twenty‐two (36.4%) patients who possessed drug‐resistant viruses under highly active antiretroviral therapy (HAART). It was characteristic that a conserved proline‐rich motif “PTAPP” in the N‐terminus of p6gag protein was completely or partially duplicated in all cases. Five among the eight cases were retrospectively investigated in terms of the occurrence of dynamic change in the gag gene between the inserted and wild‐type HIV‐1 in the course of HAART. The longitudinal analysis revealed the following: 1) The inserted‐type viruses were selected over the wild‐type during HAART in three cases in which the both types coexisted in the beginning of the therapy. 2) In two cases in which the inserted‐type HIV‐1 alone was detected before the beginning of HAART, the inserted‐type HIV‐1 alone was continuously detected during the therapy. The inserted‐type HIV‐1 was also detected in four of thirty‐nine (10.3%) therapy‐naive patients. However, the frequency of inserted‐type HIV‐1 detection in the HAART‐receiving patients is significantly higher than that in the therapy‐naive patients (P=0.02). These results suggest that this type of insertion mutation is a polymorphism of the p6gag and p6pol genes, however, it consequently gave an advantage on proliferation and/or survival of the HIV‐1 variant under the presence of antiretroviral drugs.

Identification and expression of a sialyltransferase responsible for the synthesis of disialylgalactosylgloboside in normal and malignant kidney cells: downregulation of ST6GalNAc VI in renal cancers

Although disialyl glycosphingolipids such as GD3 and GD2 have been considered to be associated with malignant tumours, whether branched-type disialyl glycosphingolipids show such an association is not well understood. We investigated the sialyltransferases responsible for the biosynthesis of DSGG (disialylgalactosylgloboside) from MSGG (monosialylgalactosylgloboside). Among six GalNAc:alpha2,6-sialyltransferases cloned to date, we focused on ST6GalNAc III, V and VI, which utilize sialylglycolipids as substrates. In vitro enzyme analyses revealed that ST6GalNAc III and VI generated DSGG from MSGG with V(max)/K(m) values of 1.91 and 4.16 respectively. Transfection of the cDNA expression vectors for these enzymes resulted in DSGG expression in a renal cancer cell line. Although both ST6GalNAc III and VI genes were expressed in normal kidney cells, the expression profiles of ST6GalNAc VI among 20 renal cancer cell lines correlated clearly with those of DSGG, suggesting that the sialyltransferase involved in the synthesis of DSGG in the kidney is ST6GalNAc-VI. ST6GalNAc-VI and DSGG were found in proximal tubule epithelial cells in normal kidney tissues, while they were downregulated in renal cancer cell lines and cancer tissues. All these findings indicated that DSGG was suppressed during the malignant transformation of the proximal tubules as a maturation arrest of glycosylation.

Prevalence of Drug‐Resistant Human Immunodeficiency Virus Type 1 in Therapy‐Naive Patients and Usefulness of Genotype Testing

In the present study, we performed genotypic drug‐resistance testing in 116 therapy‐naive human immunodeficiency virus type 1 (HIV‐1)‐infected patients between 1999 and 2002 at Nagoya National Hospital, Japan. The prevalence of drug‐resistant HIV‐1 with one or more major mutations significantly increased from 5.3% (4/75) in 1999–2001 to 17.1% (7/41) in 2002 (P=0.05), suggesting the spread of drug‐resistant HIV‐1. We identified a patient who possessed a protease (PR) inhibitor‐resistant HIV‐1 with a major mutation consisting of L90M before the initiation of therapy. The patient was administered zidovudine, lamivudine, and efavirenz as highly active antiretroviral therapy (HAART), as PR inhibitors were excluded based on the result of the drug‐resistance testing. The treatment succeeded in strongly suppressing the proliferation of drug‐resistant HIV‐1 and concomitantly increased CD4 cell counts. Thus, we conclude that drug‐resistance testing prior to the initiation of therapy is important for therapy‐naive patients to devise the optimum therapy regimen for each individual.

Trend of Drug-Resistant HIV Type 1 Emergence among Therapy-Naive Patients in Nagoya, Japan: An 8-Year Surveillance from 1999 to 2006

We studied the emergence of drug-resistant human immunodeficiency virus type 1 (HIV-1) with major amino acid mutations in 402 therapy-naive patients at Nagoya Medical Center, Japan, between 1999 and 2006. The mean prevalence of drug-resistant HIV-1 was 6.7% (range, 2.3-10.0%; n = 27). HIV-1 variants with protease inhibitor (PI)-resistant mutations alone were most frequently found (3.5%, n = 14), followed by those with nonnucleoside reverse transcriptase inhibitor (NNRTI)-resistant mutations alone (1.7%, n = 7). Variants with nucleoside reverse transcriptase inhibitor (NRTI)-resistant mutations alone were sporadically found (1.0%, n = 4). A variant possessing both NRTI- and PI-resistant mutations was detected in one patient (0.2%) and a variant possessing both NNRTI- and PI-resistant mutations was identified in another patient (0.2%). In addition, another 17 variants (4.2%, n = 17) with only 215-revertant mutations (T215C/D/G/L/S) that can easily reconvert to the nucleoside analogue-associated mutation of T215Y/F were found. The 402 viruses were phylogenetically analyzed, revealing three independent clusters comprising PI-resistant variants with the M46I or L90M mutation, NNRTI-resistant variants with the K103N mutation, and 215-revertant variants. The PI-resistant and 215-revertant strains have been spreading since 2000, and the NNRTI-resistant strain has started spreading since 2003. The nature of the epidemic and information for successfully blocking the spread of drug-resistant HIV-1 were clarified in this study.

Prevalence of infection and genotypes of GBV-C/HGV among homosexual men

Since the discovery of GB virus‐C (GBV‐C) and hepatitis G virus (HGV), many studies have been performed. These viruses are now known to be parenterally, as well as sexually transmitted. A phylogenetic analysis also revealed that GBV‐C has five major genotypes: type 1 predominates in West Africa, type 2 in Europe and the United States, type 3 in parts of Asia, type 4 in Southeast Asia, and type 5 in South Africa. Despite the number of reports so far, there have been few large‐scale surveys of homosexual men to determine the prevalence of the GBV‐C/HGV infections. We examined the levels of GBV‐C/HGV viremia in 297 homosexual men who attended the Nagoya Lesbian and Gay Revolution held in Nagoya, Japan. Reverse transcription‐polymerase chain reaction (RT‐PCR)/nested PCR of the GBV‐C/HGV 5′‐non‐coding region (NCR), and base sequence analyses showed that the infection rate was 12.5%, and genotypes in this population were classified into type 2 (32%) and type 3 (68%). None were classified as types 1, 4, or 5 in this study. Our results indicate that the GBV‐C/HGV type 2 seen mainly in Europe and the US is spreading widely in Japan, especially in the Nagoya district.

Cellular HIV-1 DNA levels in patients receiving antiretroviral therapy strongly correlate with therapy initiation timing but not with therapy duration.

BackgroundViral reservoir size refers to cellular human immunodeficiency virus-1 (HIV-1) DNA levels in CD4+ T lymphocytes of peripheral blood obtained from patients with plasma HIV-1-RNA levels (viral load, VL) maintained below the detection limit by antiretroviral therapy (ART). We measured HIV-1 DNA levels in CD4+ lymphocytes in such patients to investigate their clinical significance.MethodsCD4+ T lymphocytes were isolated from the peripheral blood of 61 patients with a VL maintained at less than 50 copies/ml for at least 4 months by ART and total DNA was purified. HIV-1 DNA was quantified by nested PCR to calculate the copy number per 1 million CD4+ lymphocytes (relative amount) and the copy number in 1 ml of blood (absolute amount). For statistical analysis, the Spearman rank or Wilcoxon signed-rank test was used, with a significance level of 5%.ResultsCD4 cell counts at the time of sampling negatively correlated with the relative amount of HIV-1 DNA (median = 33 copies/million CD4+ lymphocytes; interquartile range [IQR] = 7-123 copies/million CD4+ lymphocytes), but were not correlated with the absolute amounts (median = 17 copies/ml; IQR = 5-67 copies/ml). Both absolute and relative amounts of HIV-1 DNA were significantly lower in six patients in whom ART was initiated before positive seroconversion than in 55 patients in whom ART was initiated in the chronic phase, as shown by Western blotting. CD4 cell counts before ART introduction were also negatively correlated with both the relative and absolute amounts of HIV-1 DNA. Only the relative amounts of HIV-1 DNA negatively correlated with the duration of VL maintenance below the detection limit, while the absolute amounts were not significantly correlated with this period.ConclusionsThe amounts of cellular HIV-1 DNA in patients with VLs maintained below the detection limit by the introduction of ART correlated with the timing of ART initiation but not with the duration of ART. In addition, CD4+ T lymphocytes, which were newly generated by ART, diluted latently infected cells, indicating that measurements of the relative amounts of cellular HIV-1 DNA might be underestimated.

Analysis of Near Full-Length Genomic Sequences of Drug-Resistant HIV-1 Spreading among Therapy-Naïve Individuals in Nagoya, Japan: Amino Acid Mutations Associated with Viral Replication Activity

We analyzed a total of 12 near full-length genomes of drug-resistant HIV-1 spreading among therapy-naïve individuals in Nagoya, Japan. Genomes comprised seven protease inhibitor (PI)-resistant viruses possessing an M46I (n = 6) or L90M mutation (n = 1) and five non-nucleoside reverse transcriptase inhibitor-resistant viruses possessing a K103N mutation. All 12 viruses conserved both an H87Q mutation in the cyclophilin A-binding site of Gag p24 (capsid) and a T23N mutation in the cysteine-rich domain of Tat protein. PI-resistant viruses commonly possessed two cleavage site mutations in the p6(Pol)/protease of Pol polyprotein (F48L in p6(Pol)) and the anchor/core domains of Nef protein (L57V). These amino acid mutations represent candidates for enhancing replication activity of drug-resistant viruses and supporting expansion of such viruses in therapy-naïve individuals.

An 11-Year Surveillance of HIV Type 1 Subtypes in Nagoya, Japan.

Abstract To monitor active HIV-1 transmission in Nagoya, Japan, we have been determining the subtypes of HIV-1 infecting therapy-naive individuals who have newly visited the Nagoya Medical Center since 1997. The subtypes were determined by phylogenetic analyses using the base sequences in three regions of the HIV-1 genes including gag p17, pol protease (PR) and reverse transcriptase (RT), and env C2V3. Almost all HIV-1 subtypes from 1997 to 2007 and 93% of all HIV-1 isolates in 2007 were subtype B. HIV-1 subtypes A, C, D, and F have been detected sporadically since 1997, almost all in Africans and South Americans. The first detected circulating recombinant form (CRF ) was CRF01_AE (11-year average annual detection rate, 7.7%). Only two cases of CRF02_AG were detected in 2006. A unique recombinant form (URF ) was first detected in 1998 and the total number of URFs reached 25 by year 2007 (average annual detection rate, 4.7%). Eleven of these 25 were detected from 2000 to 2005 and had subtypes AE/B/AE as determined by base sequencing of the gag p17, pol PR and RT, and env C2V3 genes (average annual detection rate, 3.7%). Unique subtype B has been detected in six cases since 2006. All 17 of these patients were Japanese. Other recombinant HIV-1s have been detected intermittently in eight cases since 1998. During the 11-year surveillance, most HIV-1s in Nagoya, Japan were of subtype B. We expect that subtype B HIV-1 will continue to predominate for the next several years. Active recombination between subtype B and CRF01_AE HIV-1 and its transmission were also shown.

Quantitative SNP-detection method for estimating HIV-1 replicative fitness: application to protease inhibitor-resistant viruses.

We have improved the methods for the standard competitive growth assay of human immunodeficiency virus type 1 (HIV‐1). The cloning step for the mixed viral population and subsequent genotype analysis for arbitrary numbers of clones were excluded from procedures. Instead, a single nucleotide polymorphism (SNP)‐detection step was devised for the determination of viral populations. The quantitative SNP‐detection method can rapidly estimate the proportion of wild‐type and mutant populations with high reproducibility. Consequently, this method allows manipulation of many samples within a short period. Using this new competitive growth assay, replicative fitness of drug‐resistant HIV‐1 containing an M46I amino acid mutation in the protease was assessed in the presence or absence of indinavir. Without indinavir, replicative fitness of wild‐type HIV‐1 surpassed that of M46I‐mutated HIV‐1, and the fraction of mutated virus was reduced to about 10% at passage #9. In contrast, the fraction of M46I‐mutated virus increased to >90% at passage #5 in the presence of 26.4 nM indinavir. Almost identical results were obtained for L90M‐mutated HIV‐1 with or without saquinavir. HIV‐1 can survive under indinavir pressure by acquiring M46I mutation, as with acquisition of the L90M mutation under saquinavir pressure. However, these mutations damage viral replicative fitness under natural conditions without any drugs. Subtle differences between wild‐type and mutant viruses are thus easily detected using the improved method.

PNA-In Situ Hybridization Method for Detection of HIV-1 DNA in Virus-Infected Cells and Subsequent Detection of Cellular and Viral Proteins

We describe in situ hybridization protocols using peptide nucleic acid (PNA) as a probe for detecting HIV-1 DNA in virus-infected cells and the subsequent detection of cellular and/or viral proteins. Because a PNA probe of approx 20 bases was sufficiently long to detect a specific target sequence, a conserved sequence of such a short length was easily identified. Therefore, this probe is valuable even to identify quasi-species of HIV-1. In addition, we adopted a catalyzed signal amplification method to amplify weak viral DNA signals; thus, stringent washing was crucial for eliminating false-positive signals. Our double-staining method using PNA-in situ hybridization and subsequent immunostaining enabled the active and inactive proviruses to be distinguished.