Retroelements versus APOBEC3 family members: No great escape from the magnificent seven.

Retroelements comprise a large and successful family of transposable genetic elements that, through intensive infiltration, have shaped the genomes of humans and other mammals over millions of years. In fact, retrotransposons now account for approximately 45% of the human genome. Because of their genomic mobility called retrotransposition, some retroelements can cause genetic diseases; such retrotransposition events occur not only in germ cells but also in somatic cells, posing a threat to genomic stability throughout all cellular populations. In response, mammals have developed intrinsic immunity mechanisms that provide resistance against the deleterious effects of retrotransposition. Among these, seven members of the APOBEC3 (A3) family of cytidine deaminases serve as highly active, intrinsic, antiretroviral host factors. Certain A3 proteins effectively counteract infections of retroviruses such as HIV-1, as well as those of other virus families, while also blocking the transposition of retroelements. Based on their preferential expression in the germ cells, in which retrotransposons may be active, it is likely that A3 proteins were acquired through mammalian evolution primarily to inhibit retrotransposition and thereby maintain genomic stability in these cells. This review summarizes the recent advances in our understanding of the interplay between the retroelements currently active in the human genome and the anti-retroelement A3 proteins.

Epigenetic displacement of HP1 from heterochromatin by HIV-1 Vpr causes premature sister chromatid separation.

Although pericentromeric heterochromatin is essential for chromosome segregation, its role in humans remains controversial. Dissecting the function of HIV-1-encoded Vpr, we unraveled important properties of heterochromatin during chromosome segregation. In Vpr-expressing cells, hRad21, hSgo1, and hMis12, which are crucial for proper chromosome segregation, were displaced from the centromeres of mitotic chromosomes, resulting in premature chromatid separation (PCS). Interestingly, Vpr displaced heterochromatin protein 1-α (HP1-α) and HP1-γ from chromatin. RNA interference (RNAi) experiments revealed that down-regulation of HP1-α and/or HP1-γ induced PCS, concomitant with the displacement of hRad21. Notably, Vpr stimulated the acetylation of histone H3, whereas p300 RNAi attenuated the Vpr-induced displacement of HP1-α and PCS. Furthermore, Vpr bound to p300 that was present in insoluble regions of the nucleus, suggesting that Vpr aberrantly recruits the histone acetyltransferase activity of p300 to chromatin, displaces HP1-α, and causes chromatid cohesion defects. Our study reveals for the first time centromere cohesion impairment resulting from epigenetic disruption of higher-order structures of heterochromatin by a viral pathogen.

Differential anti-APOBEC3G activity of HIV-1 Vif proteins derived from different subtypes.

Antiretroviral cytidine deaminase APOBEC3G, which is abundantly expressed in peripheral blood lymphocytes and macrophages, strongly protects these cells against HIV-1 infection. The HIV-1 Vif protein overcomes this antiviral effect by enhancing proteasome-mediated APOBEC3G degradation and is key for maintaining viral infectivity. The 579-bp-long vif gene displays high genetic diversity among HIV-1 subtypes. Therefore, it is intriguing to address whether Vif proteins derived from different subtypes differ in their viral defense activity against APOBEC3G. Expression plasmids encoding Vif proteins derived from subtypes A, B, C, CRF01_AE, and CRF02_AG isolates were created, and their anti-APOBEC3G activities were compared. Viruses produced from cells expressing APOBEC3G and Vif proteins from different subtypes showed relatively different viral infectivities. Notably, subtype C-derived Vif proteins tested had the highest activity against APOBEC3G that was ascribed to its increased binding activity, for which the N-terminal domain of the Vif protein sequences was responsible. These results suggest that the biological differences of Vif proteins belonging to different subtypes might affect viral fitness and quasispecies in vivo.

HIV-1 accessory protein Vpu internalizes cell-surface BST-2/tetherin through transmembrane interactions leading to lysosomes.

Bone marrow stromal antigen 2 (BST-2, also known as tetherin) is a recently identified interferon-inducible host restriction factor that can block the production of enveloped viruses by trapping virus particles at the cell surface. This antiviral effect is counteracted by the human immunodeficiency virus type 1 (HIV-1) accessory protein viral protein U (Vpu). Here we show that HIV-1 Vpu physically interacts with BST-2 through their mutual transmembrane domains and leads to the degradation of this host factor via a lysosomal, not proteasomal, pathway. The degradation is partially controlled by a cellular protein, beta-transducin repeat-containing protein (betaTrCP), which is known to be required for the Vpu-induced degradation of CD4. Importantly, targeting of BST-2 by Vpu occurs at the plasma membrane followed by the active internalization of this host protein by Vpu independently of constitutive endocytosis. Thus, the primary site of action of Vpu is the plasma membrane, where Vpu targets and internalizes cell-surface BST-2 through transmembrane interactions, leading to lysosomal degradation, partially in a betaTrCP-dependent manner. Also, we propose the following configuration of BST-2 in tethering virions to the cell surface; each of the dimerized BST-2 molecules acts as a bridge between viral and cell membranes.

All APOBEC3 family proteins differentially inhibit LINE-1 retrotransposition.

Approximately 17% of the human genome is comprised of long interspersed nuclear element 1 (LINE-1, L1) non-LTR retrotransposons. L1 retrotransposition is known to be the cause of several genetic diseases, such as hemophilia A, Duchene muscular dystrophy, and so on. The L1 retroelements are also able to cause colon cancer, suggesting that L1 transposition could occur not only in germ cells, but also in somatic cells if innate immunity would not function appropriately. The mechanisms of L1 transposition restriction in the normal cells, however, are not fully defined. We here show that antiretroviral innate proteins, human APOBEC3 (hA3) family members, from hA3A to hA3H, differentially reduce the level of L1 retrotransposition that does not correlate either with antiviral activity against Vif-deficient HIV-1 and murine leukemia virus, or with patterns of subcellular localization. Importantly, hA3G protein inhibits L1 retrotransposition, in striking contrast to the recent reports. Inhibitory effect of hA3 family members on L1 transposition might not be due to deaminase activity, but due to novel mechanism(s). Thus, we conclude that all hA3 proteins act to differentially suppress uncontrolled transposition of L1 elements.

HIV-1 proteases from drug-naive West African patients are differentially less susceptible to protease inhibitors.

BACKGROUND: Now that highly active antiretroviral therapy (HAART) is being initiated on a large scale in West Africa, it remains controversial whether protease inhibitors (PIs), originally designed and tested against human immunodeficiency virus type 1 (HIV-1) subtype B, are equally effective against the non-B subtypes that are prevalent in West African countries. In this study, we investigated whether Ghanaian HIV-1 isolates, as representatives of West African isolates, are susceptible to PIs. METHODS: We first generated an HIV-1 protease cassette vector proviral DNA carrying a luciferase gene, which allows patient-derived HIV-1 proteases to be inserted and to be subjected to both genotypic and phenotypic assays. HIV-1 protease genes derived from 39 treatment-naive Ghanaian patients were used in this experiment as representatives of West African strains. The cloned patient-derived HIV-1 protease genes were first sequenced and then genetically compared. Phenotypic analysis was performed with Ghanaian HIV-1 protease-chimeric viruses in the presence of 6 different PIs. Structural models of HIV-1 protease homodimers were constructed by the molecular modeling software. RESULTS: Genetic analysis of cloned patient-derived HIV-1 protease genes indicated that most of the Ghanaian HIV-1 proteases are placed as subtype CRF02_AG strains, which are phylogenetically distant from subtype B strains, and that Ghanaian HIV-1 proteases do not harbor known major mutations influencing drug resistance but commonly carry 2-3 minor mutations. Phenotypic analysis performed with HIV-1 protease-recombinant viruses in the presence of 6 different PIs revealed that Ghanaian HIV-1 proteases are differentially less susceptible to the PIs. In support of this finding of differential susceptibility, structural analysis showed a significant distortion of nelfinavir, but not of amprenavir, in the Ghanaian protease pocket, suggesting nelfinavir might be less insertable into the Ghanaian protease than into the protease of subtype B. CONCLUSIONS: These findings provide implications for the combination of PIs during the introduction of HAART into West Africa.

Amino acid 36 in the human immunodeficiency virus type 1 gp41 ectodomain controls fusogenic activity: implications for the molecular mechanism of viral escape from a fusion inhibitor.

We have previously described a human immunodeficiency virus type 1 (HIV-1) proviral clone, pL2, derived from defective viral particles with higher fusogenicity than the prototypic NL4-3 virus. In this study, we attempted to determine the region that confers the enhanced fusion activity by creating envelope recombinants between pL2 and pNL4-3, as well as point mutants based on pNL4-3. The results indicate that amino acid 36 of gp41 is key for the fusogenic activity and infectivity enhancement and that glycine 36 (36G) of gp41 in pL2 is conserved in nearly all HIV-1 isolates except for pNL4-3. The mutation 36G-->D in a primary-isolate-derived Env decreased syncytium-forming activity and infectivity. The assays for cell-cell fusion and viral binding suggested that the enhanced fusion mediated by the 36D-->G mutation is not due to increased binding efficiency but is directly due to actual enhancement of viral fusion activity. Interestingly, this amino acid position is exactly equivalent to that at which the mutation of HIV-1 isolates that have escaped from a fusion inhibitor, enfuvirtide (T-20), has been frequently observed. The correlation between these previous findings and our findings was suggested by structural analysis. Our finding, therefore, has implications for a molecular basis of the viral escape from this drug.

Interleukin-4 up-regulates T-tropic human immunodeficiency virus type 1 transcription in primary CD4+ CD38+ T-lymphocyte subset.

The capacity of human immunodeficiency virus type 1 (HIV-1) to infect resting cells and to produce progeny particles may contribute significantly to its pathogenicity in vivo. We previously reported that primary culture of resting CD4(+) CD38(+) T-lymphocyte subset had higher production rate of CXCR4-using (X4) HIV-1 than CD4(+) CD38(-) subset. Interleukin (IL)-4 highly contributed to the up-regulation of the X4 virus production in the CD38(+) subset. Here, we show evidences that IL-4 treatment of both resting CD38(+) and CD38(-) subsets allowed the adsorption, entry, and integration of X4 virus at similar rates, while the following viral transcription rate was significantly lower in the CD38(-) than CD38(+) subset. Treatment of the CD38 subsets with IL-4 or phytohemagglutinin revealed no association of X4 virus replication ability in the subsets with classic T-cell activation or proliferation. Interestingly, the activator protein (AP)-1 was significantly activated in the CD38(+) subset after IL-4 treatment, while both nuclear factor (NF)-kappaB and signal transducers and activator of transcription (STAT)-6 were activated in the IL-4-treated CD38(-) and CD38(-) subsets at similar levels. Thus, IL-4-dependent X4 HIV-1 transcription occurs efficiently in the CD38(+) but not CD38(-) subset of CD4(+) population and AP-1 could play a significant role on viral transcription, leading to the up-regulated X4 virus production in the CD38(+) subset.

Enhancement of human immunodeficiency virus type 1 infectivity by replacing the region including Env derived from defective particles with an ability to form particle-mediated syncytia in CD4+T cells.

The infection and subsequent replication rates of human immunodeficiency virus type 1 (HIV-1) affect the pathogenicity. The initial stage of HIV-1 infection is largely regulated by viral envelope sequence. We previously reported that the defective doughnut-shaped particles produced from a persistently infected cell clone, named L-2, obtained from human CD4+ T-cell line MT-4 that was persistently infected with HIV-1 LAI strain, efficiently form particle-mediated syncytia with uninfected human CD4+ T-cell line, MOLT-4. Here, we prepared a molecular clone (pL2) containing the L-2 provirus to characterize the viral genetic region contributing to this activity to form particle-mediated syncytia. Several recombinants were constructed with pNL4-3 by replacing the pL2-derived region including full-length env. Characterization of the particles obtained by transfection with these recombinant clones confirmed that pL2-derived env carried the particle-mediated syncytia formation activity. It is noteworthy that the pL2-derived env region could also contribute to enhancement of infectivity in CD4+ T-cell lines as well as primary peripheral blood mononuclear cells (PBMCs). Thus, the HIV-1 particle-mediated syncytium formation activity could also contribute to the enhancement of HIV-1 infectivity.

Ability to induce p53 and caspase-mediated apoptosis in primary CD4+ T cells is variable among primary isolates of human immunodeficiency virus type 1.

Infection with human immunodeficiency virus type 1 (HIV-1) is associated with dramatic depletion of CD4(+) T cells, the major HIV-1-induced pathogenesis. Apoptosis has been suggested to play an important role for the T cell depletion and a number of mechanisms have been proposed for the apoptosis in T cells. Here, we compared the levels for apoptosis induction in primary peripheral blood mononuclear cells (PBMCs) among several laboratory strains and primary isolates of the HIV-1 subtypes B and E. The results showed that apoptosis in infected PBMCs, preferentially in CD4+ T cell population, became detectable around the time for virus production by flow cytometric terminal transferase dUTP nick end labeling (TUNEL) technique and staining with the nuclear dye Hoechst 33342. The abilities to induce apoptosis in PBMCs were highly variable in individual isolates. The increase of p53 protein in infected PBMCs, which was initiated before virus production, was observed in infected PBMCs and the levels of p53 protein were almost proportional to the rates of the isolates to induced apoptosis. The cells infected and cultured in the presence of Z-VAD-FMK had significantly decreased cell mortalities, indicating that activated caspases also played a significant role in the apoptosis. Thus, HIV-1-induced apoptosis in primary T cells was accompanied by the p53 protein and caspase activation at varied levels in primary isolates.

Resting CD4(+) T cells with CD38(+)CD62L(+) produce interleukin-4 which contributes to enhanced replication of T-tropic human immunodeficiency virus type 1.

A significant increase in the CD38(+) population among T lymphocytes has been observed in human immunodeficiency virus type 1 (HIV-1)-infected carriers. We previously reported a higher replication rate of T-tropic HIV-1 in the CD4(+)CD38(+)CD62L(+) than CD38(-) subset under conditions of mitogen stimulation after infection. Here, we revealed a similarly high susceptibility in the CD38(+) subset on culture with conditioned medium containing Th2 cytokine, interleukin (IL)-4 that was produced endogenously from this subset on stimulation with mitogen or anti-CD3 antibody for 3 days. The contribution of IL-4 to the upregulated production of virus in the CD38(+) subset was confirmed by culture of this subset with recombinant human IL-4. In contrast, the rate of replication in the CD38(-) subset was not augmented in the conditioned medium from either subset or with IL-4. However, there were no differences in the surface expression of IL-4 receptor or HIV-1 receptors CD4 and CXCR4 between the two subsets. Thus, the CD4(+)CD38(+)CD62L(+) subset comprises a specific cell population secreting endogenous Th2 cytokine that contributes to the efficient production of T-tropic HIV-1 through upregulation at a certain stage of the viral life cycle, probably after the adsorption step.

Construction and in vitro characterization of a molecularly cloned human immunodeficiency virus type 1 library.

Development of a safe and effective vaccine against human immunodeficiency virus (HIV) is urgent, but many concerns regarding the safety and efficacy of the currently developing vaccines remain. A major hindrance in HIV vaccine development is the genetic diversity, a hallmark of HIV biology, and a poor understanding of how HIV vaccine prevents the emergence of escape variants during infection and progression of AIDS. Here, we developed a method to construct a molecularly cloned viral library. This technique employs a long-range polymerase chain reaction (PCR) to amplify a virtually full-length HIV type 1 (HIV-1) provirus genome from peripheral blood mononuclear cells (PBMCs) infected with CRF01_AE (subtype E) Thai primary isolate. Among randomly selected 93 clones, 41 with a full-length sequence were able to replicate in PBMCs, 5 of which induced strong cytopathic effects. Replication kinetics also showed that the parental virus was intermediate among the clones. Thus, the molecular library prepared by this method showed the quasi-species in infected cells and this method could provide a new possibility for the development of an order-made therapeutic vaccine against HIV-1.