Emi E. Nakayama

A Specific Region of 37 Amino Acid Residues in the SPRY (B30.2) Domain of African Green Monkey TRIM5α Determines Species-Specific Restriction of Simian Immunodeficiency Virus SIVmac Infection

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) efficiently enters cells of Old World monkeys but encounters a block before reverse transcription. This restriction is mediated by a dominant repressive factor. Recently, a member of the tripartite motif (TRIM) family proteins, TRIM5α, was identified as a blocking factor in a rhesus macaque cDNA library. Among Old World monkey cell lines, the African green monkey kidney cell line CV1 is highly resistant to not only HIV-1 but also simian immunodeficiency virus SIVmac infection. We analyzed TRIM5α of CV1 cells and HSC-F cells, a T-cell line from a cynomolgus monkey, and found that both CV1- and HSC-F-TRIM5αs could inhibit CD4-dependent HIV-1 infection, as well as vesicular stomatitis virus glycoprotein-mediated infection. CV1-TRIM5α could also inhibit SIVmac infection, whereas HSC-F-TRIM5α could not. In the SPRY (B30.2) domain of CV1-TRIM5α, there was a 20-amino-acid duplication that was not present in HSC-F-TRIM5α. A chimeric TRIM5α containing 37 amino acid residues from CV1-TRIM5α, which spanned the 20-amino-acid duplication, in the background of HSC-F-TRIM5α fully gained the ability to inhibit SIVmac infection. Conversely, the mutant CV1-TRIM5α lacking the 20-amino-acid duplication completely lost the ability to restrict SIVmac infection. These findings clearly indicated that a specific region of 37 amino acid residues in the SPRY domain of CV1-TRIM5α contained a determinant of species-specific restriction of SIVmac.

Polymorphism in the Interleukin-4 Promoter Affects Acquisition of Human Immunodeficiency Virus Type 1 Syncytium-Inducing Phenotype

ABSTRACT The emergence of syncytium-inducing (SI) variants of human immunodeficiency virus type 1 (HIV-1) in infected individuals is an indicator of poor prognosis and is often correlated with faster CD4+ cell depletion and rapid disease progression. Interleukin-4 (IL-4) is a pleiotropic cytokine with various immune-modulating functions including induction of immunoglobulin E (IgE) production in B cells, down-regulation of CCR5 (a coreceptor for HIV-1 non-SI [NSI] strains), and up-regulation of CXCR4 (a coreceptor for HIV-1 SI variants). Here we show that homozygosity of a polymorphism in the IL-4 promoter region, IL-4 −589T, is correlated with increased rates of SI variant acquisition in HIV-1-infected individuals in Japan. This mutation was also shown to be associated with elevated serum IgE levels in HIV-1-infected individuals, especially in those at advanced stages of disease. In contrast, neither a triallele polymorphism in IL-10, another Th2 cytokine, nor a biallele polymorphism in the RANTES promoter affected acquisition of the SI phenotype. This finding suggested that IL-4-589T increases IL-4 production in the human body and thus accelerates the phenotypic switch of HIV-1 from NSI to SI and possibly disease progression of AIDS.

A CCR2-V64I polymorphism affects stability of CCR2A isoform.

Objective: A valine to isoleucine substitution at position 64 of CCR2 (CCR2-64I) is associated with a delay in progression to AIDS in HIV-1-infected individuals. The aim of the present study is to elucidate the molecular mechanism underlying the effect of this allele. Design: We analysed the effect of the 64I substitution on levels of expression of CCR2A and CCR2B, two CCR2 isoforms produced by alternative splicing. Methods: Sendai virus vector was used to express CCR2 molecules. Results: While CCR2B trafficked well to the cell surface, CCR2A, which differs from CCR2B only by the sequence of its C-terminal cytoplasmic tail, was detected predominantly in the cytoplasm. The level of expression of CCR2A-64I was significantly higher than that of CCR2A without the substitution. On the other hand, the 64I substitution did not affect levels of CCR2B expression. Pulse–chase experiments revealed that the 64I substitution increased the half-life of CCR2A in cells. When co-expressed with CCR5, CCR2A-64I interfered more severely with cell surface expression of CCR5 than did wild-type CCR2A. Furthermore, immunoprecipitation experiments showed that CCR2A co-precipitated with an immature form of CCR5. Conclusion: These results suggest that CCR2A binds to CCR5 in the cytoplasm and down-modulates its surface expression. We propose that the increased ability of CCR2A-64I to down-modulate CCR5 expression might be a possible cause of a delay in HIV-1 disease progression in patients with this allele.

Protective Effect of Interleukin-4 -589T Polymorphism on Human Immunodeficiency Virus Type 1 Disease Progression: Relationship with Virus Load

The interleukin (IL)-4 -589T allele bears a single nucleotide polymorphism at position -589 upstream from the open-reading frame of the IL-4 gene. To determine the influence of this allele on human immunodeficiency virus (HIV) type 1 disease, disease progression and serum virus load were assessed by IL-4 genotype in 427 white patients with known seroconversion dates who were followed in the French SEROCO cohort between 1988 and 1996. Serum virus load was 0.20 log lower during the 6-24-month plateau phase after seroconversion in patients with IL-4 -589T than in those without this allele (P=.02). Kaplan-Meier analysis survival curves showed a slower progression to clinical AIDS in carriers of IL-4 -589T (P=.04). Adjustment for early serum virus load greatly diminished the strength of this association. These results suggest that IL-4 -589T protects against HIV-1 disease progression by reducing virus load.

Naturally Occurring Deletional Mutation in the C-Terminal Cytoplasmic Tail of CCR5 Affects Surface Trafficking of CCR5

ABSTRACT CCR5 is an essential coreceptor for the cellular entry of R5 strains of human immunodeficiency virus type 1 (HIV-1). CCR5-893(−) is a single-nucleotide deletion mutation which is observed exclusively in Asians (M. A. Ansari-Lari, et al., Nat. Genet. 16:221–222, 1997). This mutant gene produces a CCR5 which lacks the entire C-terminal cytoplasmic tail. To assess the effect of CCR5-893(−) on HIV-1 infection, we generated a recombinant Sendai virus expressing the mutant CCR5 and compared its HIV-1 coreceptor activity with that of wild-type CCR5. Although the mutant CCR5 has intact extracellular domains, its coreceptor activity was much less than that of wild-type CCR5. Flow cytometric analyses and confocal microscopic observation of cells expressing the mutant CCR5 revealed that surface CCR5 levels were greatly reduced in these cells, while cytoplasmic CCR5 levels of the mutant CCR5 were comparable to that of the wild type. Peripheral blood CD4+ T cells obtained from individuals heterozygous for this allele expressed very low levels of CCR5. These data suggest that the CCR5-893(−) mutation affects intracellular transport of CCR5 and raise the possibility that this mutation also affects HIV-1 transmission and disease progression.

Anti‐retroviral activity of TRIM5α

Human immunodeficiency virus type 1 (HIV‐1) shows a very narrow host range limited to humans and chimpanzees. Experimentally, HIV‐1 does not infect Old World monkeys, such as rhesus (Rh) and cynomolgus (CM) monkeys, and fails to replicate in activated CD4 positive T lymphocytes obtained from these monkeys. In contrast, simian immunodeficiency virus isolated from a macaque monkey (SIVmac) can replicate well in both Rh and CM. In 2004, tripartite motif 5α (TRIM5α) was identified as a host factor which plays an important role in the restricted host range of HIV‐1. Rh and CM TRIM5α restrict HIV‐1 infection but not SIVmac, while in comparison, anti‐viral activity of human TRIM5α against those viruses is very weak. TRIM5α consists of the RING, B‐box 2, coiled‐coil and SPRY (B30.2) domains. The RING domain is frequently found in E3 ubiquitin ligase and TRIM5α is degraded via the ubiquitin‐proteasome pathway during HIV‐1 restriction. TRIM5α recognises the multimerised capsid (viral core) of an incoming virus by its α‐isoform specific SPRY domain and is believed to be involved in innate immunity to control retroviral infection. Differences in amino acid sequences in the SPRY domain of TRIM5α of different monkey species were found to affect species‐specific restriction of retrovirus infection, while differences in amino acid sequences in the viral capsid protein determine viral sensitivity to restriction. Accurate structural analysis of the binding surface between the viral capsid protein and TRIM5α SPRY is thus required for the development of new antiretroviral drugs that enhance anti‐HIV‐1 activity of human TRIM5α. Copyright

A Single Amino Acid of the Human Immunodeficiency Virus Type 2 Capsid Affects Its Replication in the Presence of Cynomolgus Monkey and Human TRIM5αs

ABSTRACT Human immunodeficiency virus type 2 (HIV-2) strains vary widely in their abilities to grow in Old World monkey (OWM) cells such as those of cynomolgus monkeys (CM). We evaluated eight HIV-2 isolates for their sensitivities to CM TRIM5α, an anti-HIV factor in OWM cells. We found that different HIV-2 isolates showed differences in their sensitivities to CM TRIM5α. Sequence analysis showed that TRIM5α-sensitive viruses had proline at the 120th position of the capsid protein (CA), whereas TRIM5α-resistant viruses had either alanine or glutamine. Mutagenesis studies indicated that the single amino acid at the 120th position indeed affected the sensitivity of the virus to CM TRIM5α.

New Rev-transport inhibitor with anti-HIV activity from Valerianae Radix.

Bioassay-guided separation by use of the fission yeast expressing NES of Rev, a HIV-1 viral regulatory protein, resulted in isolation of valtrate (1) as a new Rev-transport inhibitor from the nucleus to cytoplasm from Valerianae Radix. Valtrate (1) also inhibited the p-24 production of HIV-1 virus without showing any cytotoxicity against the host MT-4 cells.

Protective effects of IL4-589T and RANTES-28G on HIV-1 disease progression in infected Thai females.

Objective:To evaluate the effect of polymorphisms in interleukin-4 (IL4) and RANTES promoters on disease progression in HIV-1 infected Thais. Design:Antiretroviral (ARV) drug-free HIV-1 infected females from the prospective cohort. Methods:A total of 246 DNA samples were genotyped for IL4 and RANTES promoter polymorphisms by PCR–RFLP. Associations of genotype with HIV-1 disease progression were assessed with respect to baseline clinical data including plasma HIV-1 load, CD4 cell counts, and proportion of symptomatic/AIDS, and survival status during 3 years of follow-up. Results:Patients with homozygous IL4-589T allele showed a significantly lower HIV-1 viral load (P = 0.005) and a higher CD4 cell count (P = 0.003) than the other patients with heterozygous IL4-589C/T or homozygous IL4-589C allele. Kaplan–Meier analysis demonstrated an apparent but insignificant trend towards better survival in homozygous IL4-589T patients. On the other hand, patients with RANTES-28G allele showed a significantly better survival while those with RANTES In1.1C allele without RANTES-28G showed a significantly poorer survival compared with those who did not possess either RANTES In1.1C or RANTES-28G (P = 0.02), although those polymorphisms only weakly associated with baseline viral load and CD4 cell counts. Conclusions:Our results implicate the significant protective effect of IL4-589T and RANTES-28G on HIV disease progression in Thais. In contrast, RANTES In1.1C without RANTES-28G had an accelerating effect on HIV disease progression.

Influence of Glycosylation on the Efficacy of an Env-Based Vaccine against Simian Immunodeficiency Virus SIVmac239 in a Macaque AIDS Model

ABSTRACT The envelope glycoprotein (Env) of human immunodeficiency viruses (HIVs) and simian immunodeficiency viruses (SIVs) is heavily glycosylated, and this feature has been speculated to be a reason for the insufficient immune control of these viruses by their hosts. In a macaque AIDS model, we demonstrated that quintuple deglycosylation in Env altered a pathogenic virus, SIVmac239, into a novel attenuated mutant virus (Δ5G). In Δ5G-infected animals, strong protective immunity against SIVmac239 was elicited. These HIV and SIV studies suggested that an understanding of the role of glycosylation is critical in defining not only the virological properties but also the immunogenicity of Env, suggesting that glycosylation in Env could be modified for the development of effective vaccines. To examine the effect of deglycosylation, we constructed prime-boost vaccines consisting of Env from SIVmac239 and Δ5G and compared their immunogenicities and vaccine efficacies by challenge infection with SIVmac239. Vaccination-induced immune responses differed between the two vaccine groups. Both Env-specific cellular and humoral responses were higher in wild-type (wt)-Env-immunized animals than in Δ5G Env-immunized animals. Following the challenge, viral loads in SIVmac239 Env (wt-Env)-immunized animals were significantly lower than in vector controls, with controlled viral replication in the chronic phase. Unexpectedly, viral loads in Δ5G Env-immunized animals were indistinguishable from those in vector controls. This study demonstrated that the prime-boost Env vaccine was effective against homologous SIVmac239 challenge. Changes in glycosylation affected both cell-mediated and humoral immune responses and vaccine efficacy.