Masako Nomaguchi

Vpx Is Critical for Reverse Transcription of the Human Immunodeficiency Virus Type 2 Genome in Macrophages

ABSTRACT The abilities of wild-type and vpx-defective human immunodeficiency virus type 2 (HIV-2) clones to synthesize viral DNA in human monocyte-derived macrophages (MDMs) and lymphocytic cells were comparatively and quantitatively evaluated. While the vpx-defective mutant directed the synthesis of viral DNA comparably to the wild-type virus and normally in lymphocytic cells, no appreciable viral DNA was detected in MDMs infected with the mutant. To substantiate this finding and to determine whether there is some specific region(s) in Vpx crucial for viral DNA synthesis in MDMs, we generated a series of site-specific point mutants of vpx and examined their phenotypes. The resultant five mutants, with no infectivity for MDMs, showed, without exception, the same defect as the vpx-defective mutant. Our results here clearly demonstrated that the entire Vpx protein is critical for reverse transcription of the HIV-2 genome in human MDMs.

Role of HIV-1 Vpu protein for virus spread and pathogenesis.

Vpu is an accessory viral protein almost unique to HIV-1 among primate immunodeficiency viruses, and has two major functions: degradation of the CD4 molecule in endoplasmic reticulum and enhancement of virion release from cells. Recent identification of a novel host restriction factor, tetherin, as a Vpu-antagonist suggests that Vpu contributes to virus spread by facilitating progeny virion production. This review focuses on the two distinct functions of Vpu and summarizes current knowledge on its virological role in the HIV-1 life cycle.

Identification of amino acid residues in HIV-1 Vif critical for binding and exclusion of APOBEC3G/F.

To define a region(s) in human immunodeficiency virus type 1 (HIV-1) Vif that involves binding to its target APOBEC3G (A3G), we have generated a series of site-specific proviral vif mutants. Of 30 mutants examined, 15 did not grow at all or grew more poorly than wild-type virus in non-permissive cells. Eight clones with N-terminal mutations located outside of the HCCH motif and BC-box, which are known to be directly crucial for the degradation of A3G, were chosen from these growth-defective mutants and mainly analyzed in detail for functional activity of their mutant Vif proteins. By single-cycle replication and immunoprecipitation/immunoblotting analyses, mutants designated W21A, S32A, W38A, Y40A, and H43A were demonstrated to hardly or poorly bind to and neutralize A3G. Upon transfection, these mutants produced progeny virions containing much more A3G than wild-type clone. Interestingly, while mutants designated E76A and W79A acted normally to inactivate A3G, they were found to exhibit a Vif-defective phenotype against A3F. Another unique mutant designated Y69A incompetent against both of A3G/F was also identified. Our results here have indicated that at least two distinct regions in the N-terminal half of HIV-1 Vif are critical for binding and exclusion of A3G/F.

Requirements for West Nile Virus (–)- and (+)-Strand Subgenomic RNA Synthesis in Vitro by the Viral RNA-dependent RNA Polymerase Expressed in Escherichia coli

RNA-dependent RNA polymerases (RdRPs) of the Flaviviridae family catalyze replication of positive (+)- strand viral RNA through synthesis of minus (–)-and progeny (+)-strand RNAs. West Nile virus (WNV), a mosquito-borne member, is a rapidly re-emerging human pathogen in the United States since its first outbreak in 1999. To study the replication of the WNV RNA in vitro, an assay is described here that utilizes the WNV RdRP and subgenomic (–)- and (+)-strand template RNAs containing 5′- and 3′-terminal regions (TR) with the conserved sequence elements. Our results show that both 5′- and 3′-TRs of the (+)-strand RNA template including the wild type cyclization (CYC) motifs are important for RNA synthesis. However, the 3′-TR of the (–)-strand RNA template alone is sufficient for RNA synthesis. Mutational analysis of the CYC motifs revealed that the (+)-strand 5′-CYC motif is critical for (–)-strand RNA synthesis but neither the (–)-strand 5′- nor 3′-CYC motif is important for the (+)-strand RNA synthesis. Moreover, the 5′-cap inhibits the (–)-strand RNA synthesis from the 3′ fold-back structure of (+)-strand RNA template without affecting the de novo synthesis of RNA. These results support a model that “cyclization” of the viral RNA play a role for (–)-strand RNA synthesis but not for (+)-strand RNA synthesis.

Requirements for West Nile Virus Minus- and Plus-Strand Subgenomic RNA Synthesis in vitro by the viral RNA- dependent RNA Polymerase Expressed in E. coli

RNA-dependent RNA polymerases (RdRPs) of the Flaviviridae family catalyze replication of positive (+)- strand viral RNA through synthesis of minus (–)-and progeny (+)-strand RNAs. West Nile virus (WNV), a mosquito-borne member, is a rapidly re-emerging human pathogen in the United States since its first outbreak in 1999. To study the replication of the WNV RNA in vitro, an assay is described here that utilizes the WNV RdRP and subgenomic (–)- and (+)-strand template RNAs containing 5′- and 3′-terminal regions (TR) with the conserved sequence elements. Our results show that both 5′- and 3′-TRs of the (+)-strand RNA template including the wild type cyclization (CYC) motifs are important for RNA synthesis. However, the 3′-TR of the (–)-strand RNA template alone is sufficient for RNA synthesis. Mutational analysis of the CYC motifs revealed that the (+)-strand 5′-CYC motif is critical for (–)-strand RNA synthesis but neither the (–)-strand 5′- nor 3′-CYC motif is important for the (+)-strand RNA synthesis. Moreover, the 5′-cap inhibits the (–)-strand RNA synthesis from the 3′ fold-back structure of (+)-strand RNA template without affecting the de novo synthesis of RNA. These results support a model that “cyclization” of the viral RNA play a role for (–)-strand RNA synthesis but not for (+)-strand RNA synthesis.

Improved capacity of a monkey-tropic HIV-1 derivative to replicate in cynomolgus monkeys with minimal modifications

Human immunodeficiency virus type 1 (HIV-1) hardly replicates in Old World monkeys. Recently, a mutant HIV-1 clone, NL-DT5R, in which a small part of gag and the entire vif gene are replaced with SIVmac239-derived ones, was shown to be able to replicate in pigtail monkeys but not in rhesus monkeys (RM). In the present study, we found that a modified monkey-tropic HIV-1 (HIV-1mt), MN4-5S, acquired the ability to replicate efficiently in cynomolgus monkeys as compared with the NL-DT5R, while neither NL-DT5R nor MN4-5S replicated in RM cells. These results suggest that multiple determinants may be involved in the restriction of HIV-1 replication in macaques, depending on the species of macaques. The new HIV-1mt clone will be useful for studying molecular mechanisms by which anti-viral host factors regulate HIV-1 replication in macaques.

Multifaceted activity of HIV Vpr/Vpx proteins: the current view of their virological functions.

Primate immunodeficiency viruses encode viral proteins that are uniquely auxiliary to their growth in host cells. Of these accessory proteins, those designated Vpr and Vpx are least well understood with respect to their functions in the viral replication cycle. Moreover, their assigned roles based on the results in published studies remain controversial. This review summarises current knowledge on human immunodeficiency virus (HIV) Vpr/Vpx proteins, and discusses their functional activities during the viral life cycle in macrophages and T lymphocytes, the two major target cells of HIV infection. Copyright

Histamine increases the expression of LOX-1 via H2 receptor in human monocytic THP-1 cells

Lectin‐like oxidized low‐density lipoprotein receptor‐1 (LOX‐1) is a member of the scavenger receptor family, and is known to be expressed in monocytes/macrophages. We investigated the effect of histamine on the expression of LOX‐1 in cells of the human monocytic leukemia cell line THP‐1. Histamine as well as forskolin and dibutyryl cyclic AMP (Bt2‐cAMP) stimulated the THP‐1 monocytes to express the LOX‐1 gene at the transcription level. This histamine effect on LOX‐1 gene expression, via the histamine H2 receptor‐mediated cAMP signal transduction pathway, was reduced after differentiation of the cells into macrophages, even though forskolin and Bt2‐cAMP still enhanced the gene expression. The alteration of the responsiveness of LOX‐1 expression to histamine was related to suppressed expression of the H2 receptor in THP‐1 macrophages. The switch of the predominant class of histamine receptors between H1 and H2 would modulate the effects of histamine on LOX‐1 gene expression in monocytes and macrophages, and therefore, would play a certain role in the inflammatory aspects of atherogenesis.

Species barrier of HIV-1 and its jumping by virus engineering

Monkey infection models are absolutely necessary for studies of human immunodeficiency virus type 1 (HIV‐1) pathogenesis and of developing drugs/vaccines against HIV‐1. In addition, currently unknown roles of its accessory proteins for in vivo replication await elucidation by experimental approaches. Due to the fact that HIV‐1 is tropic only for chimpanzees and humans, studies of this line have been impeded for a long time, although various investigations have been carried out utilising genetically related SIV and SIV/HIV chimeric virus (SHIV) as pathogens. Recent findings of anti‐HIV‐1 innate factors such as tripartite motif protein 5α (TRIM5α) and APOBEC3G/F prompted us to re‐initiate an old and vital research project which would, as a result, confer the capability to overcome the species barrier on the HIV‐1. We currently have obtained, by virus engineering through genetic manipulation and adaptation, some new and promising HIV‐1 clones for in vivo studies in macaque monkeys as mentioned above. In this review, we summarise the past, present and future of HIV‐1/SIV chimeric viruses with special reference to relevant basic HIV‐1/SIV studies. Copyright

Role of HIV-1 Nef protein for virus replication in vitro

The Nef protein of primate lentiviruses (simian and human immunodeficiency viruses; SIV/HIVs) appears to be multi-functional and plays a pivotal role in viral persistence and pathogenesis in vivo. Of its numerous functions reported to date, the ability to enhance virion infectivity in indicator cell lines and to augment viral replication in peripheral blood mononuclear cells (PBMCs) and lymphocytes (PBLs) is very well conserved among various SIV/HIVs. This review summarizes and organizes current knowledge of HIV-1 Nef with respect to this particularly virological activity for understanding the basis of its in vivo function.