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Natural single-nucleotide polymorphisms in the 3' region of the HIV-1 pol gene modulate viral replication ability.

ABSTRACT: We previously showed that prototype macaque-tropic human immunodeficiency virus type 1 (HIV-1) acquired nonsynonymous growth-enhancing mutations within a narrow genomic region during the adaptation process in macaque cells. These adaptive mutations were clustered in the 3' region of the pol gene, encoding a small portion of the C-terminal domain of integrase (IN). Mutations in HIV-1 IN have been reported to have pleiotropic effects on both the early and late phases in viral replication. cis-acting functions in the IN-coding sequence for viral gene expression have also been reported. We here demonstrated that the adaptive mutations promoted viral growth by increasing virion production with no positive effects on the early replication phase. Synonymous codon alterations in one of the adaptive mutations influenced virion production levels, which suggested nucleotide-dependent regulation. Indeed, when the single-nucleotide natural polymorphisms observed in the 3' regions of 196 HIV-1/simian immunodeficiency virus (SIVcpz) pol genes (nucleotides [nt] 4895 to 4929 for HIV-1 NL4-3) were introduced into macaque- and human-tropic HIV-1 clones, more than half exhibited altered replication potentials. Moreover, single-nucleotide mutations caused parallel increases or decreases in the expression levels of viral late proteins and viral replication potentials. We also showed that the overall expression profiles of viral mRNAs were markedly changed by single-nucleotide mutations. These results demonstrate that the 3' region of the HIV-1 pol gene (nt 4895 to 4929) can alter viral replication potential by modulating the expression pattern of viral mRNAs in a nucleotide-dependent manner.Viruses have the plasticity to adapt themselves under various constraints. HIV-1 can mutate and evolve in growth-restrictive cells by acquiring adaptive changes in its genome. We have previously identified some growth-enhancing mutations in a narrow region of the IN-coding sequence, in which a number of cis-acting elements are located. We now focus on the virological significance of this pol gene region and the mechanistic basis underlying its effects on viral replication. We have found several naturally occurring synonymous mutations within this region that alter viral replication potentials. The effects caused by these natural single-nucleotide polymorphisms are linked to the definite expression patterns of viral mRNAs. We show here that the nucleotide sequence of the pol gene (nucleotides 4895 to 4929 for HIV-1 NL4-3) plays an important role in HIV-1 replication by modulating viral gene expression.

SUBMITTER: Nomaguchi M

PROVIDER: S-EPMC3993728 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Natural single-nucleotide polymorphisms in the 3' region of the HIV-1 pol gene modulate viral replication ability.

Nomaguchi Masako M Miyake Ariko A Doi Naoya N Fujiwara Sachi S Miyazaki Yasuyuki Y Tsunetsugu-Yokota Yasuko Y Yokoyama Masaru M Sato Hironori H Masuda Takao T Adachi Akio A

Journal of virology 20140129 8

<h4>Unlabelled</h4>We previously showed that prototype macaque-tropic human immunodeficiency virus type 1 (HIV-1) acquired nonsynonymous growth-enhancing mutations within a narrow genomic region during the adaptation process in macaque cells. These adaptive mutations were clustered in the 3' region of the pol gene, encoding a small portion of the C-terminal domain of integrase (IN). Mutations in HIV-1 IN have been reported to have pleiotropic effects on both the early and late phases in viral re ...[more]

PMID: 24478432

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Project description:UnlabelledWe previously found that natural single-nucleotide variations located within a proximal region of splicing acceptor 1 (SA1prox) in the HIV-1 genome could alter the viral replication potential and mRNA expression pattern, especially the vif mRNA level. Here, we studied the virological and molecular basis of nucleotide sequence variations in SA1prox for alterations of viral replication ability. Consistent with our previous findings, variant clones indeed expressed Vif at different levels and grew distinctively in cells with various APOBEC3G expression levels. Similar effects were observed for natural variations found in HIV-2 SA1prox, suggesting the importance of the SA1prox sequence. To define nucleotides critical for the regulation of HIV-1 Vif expression, effects of natural SA1prox variations newly found in the HIV Sequence Compendium database on vif mRNA/Vif protein levels were examined. Seven out of nine variations were found to produce Vif at lower, higher, or more excessive levels than wild-type NL4-3. Combination experiments of variations giving distinct Vif levels suggested that the variations mutually affected vif transcript production. While low and high producers of Vif grew in an APOBEC3G-dependent manner, excessive expressers always showed an impeded growth phenotype due to defects in single-cycle infectivity and/or virion production levels. The phenotype of excessive expressers was not due primarily to inadequate expression of Tat or Rev, although SA1prox variations altered the overall HIV-1 mRNA expression pattern. Collectively, our results demonstrate that HIV SA1prox regulates Vif expression levels and suggest a relationship between SA1prox and viral adaptation/evolution given that variations occurred naturally.ImportanceWhile human cells possess restriction factors to inhibit HIV-1 replication, HIV-1 encodes antagonists to overcome these barriers. Conflicts between host restriction factors and viral counterparts are critical driving forces behind mutual evolution. The interplay of cellular APOBEC3G and viral Vif proteins is a typical example. Here, we demonstrate that naturally occurring single-nucleotide variations in the proximal region of splicing acceptor 1 (SA1prox) of the HIV-1 genome frequently alter Vif expression levels, thereby modulating viral replication potential in cells with various ABOBEC3G levels. The results of the present study reveal a previously unidentified and important way for HIV-1 to compete with APOBEC3G restriction by regulating its Vif expression levels. We propose that SA1prox plays a regulatory role in Vif counteraction against APOBEC3G in order to contribute to HIV-1 replication and evolution, and this may be applicable to other primate lentiviruses.

Project description:Recently, a genome-wide association study using plasma HIV RNA from antiretroviral therapy-naive patients reported that 14 naturally occurring nonsynonymous single-nucleotide polymorphisms (SNPs) in HIV derived from antiretrovirus drug-naive patients were associated with virus load (VL). Those SNPs were detected in reverse transcriptase, RNase H, integrase, envelope, and Nef. However, the impact of each mutation on viral fitness was not investigated. Here, we constructed a series of HIV variants encoding each SNP and examined their replicative abilities. An HIV variant containing a Met-to-Ile change at codon 50 in integrase [HIV(IN:M50I)] was found as an impaired virus. Despite the mutation being in integrase, the virus release was significantly suppressed (P < 0.001). Transmission electron microscopy analysis revealed that abnormal bud accumulation on the plasma membrane and the released virus particles retained immature forms. Western blot analysis demonstrated a defect in autoprocessing of GagPol and Gag polyproteins' autoprocessing in the HIV(IN:M50I) particles, although Förster resonance energy transfer (FRET) assay displayed that GagPol containing IN:M50I forms a homodimer with a similar efficiency with GagPol (wild type). The impaired maturation and replication were rescued by two other VL-associated SNPs, Ser-to-Asn change at codon 17 of integrase and Asn-to-Ser change at codon 79 of RNase H. These data demonstrate that Gag and GagPol assembly, virus release, and autoprocessing are regulated by not only integrase but also RNase H. IMPORTANCE Nascent HIV-1 is a noninfectious viral particle. Cleaving Gag and GagPol polyproteins in the particle by mature HIV protease (PR), the nascent virus becomes an infectious virus. PR is initially translated as an inactive embedded enzyme in a GagPol polyprotein. The embedded PR in homodimerized GagPol polyproteins catalyzes a proteolytic reaction to release the mature PR. This excision step by self-cleavage is called autoprocessing. Here, during the evaluation of the roles of naturally emerging nonsynonymous SNPs in HIV RNA, we found that autoprocessing is inhibited by Met-to-Ile change at codon 50 in integrase GagPol. Other coexisting SNPs, Ser-to-Asn change at codon 17 in integrase or Asn-to-Ser mutation at codon 79 in RNase H, recovered this defect, suggesting that autoprocessing is regulated by not only integrase but also RNase H in GagPol polyprotein.

Project description:RNA viruses have been used as model systems to understand the patterns and processes of molecular evolution because they have high mutation rates and are genetically diverse. Human immunodeficiency virus 1 (HIV-1), the etiological agent of acquired immune deficiency syndrome, is highly genetically diverse, and is classified into several groups and subtypes. However, it has been difficult to use its diverse sequences to establish the overall phylogenetic relationships of different strains or the trends in sequence conservation with the construction of phylogenetic trees. Our aims were to systematically characterize HIV-1 subtype evolution and to identify the regions responsible for HIV-1 subtype differentiation at the amino acid level in the Pol protein, which is often used to classify the HIV-1 subtypes. In this study, we systematically characterized the mutation sites in 2,052 Pol proteins from HIV-1 group M (144 subtype A; 1,528 subtype B; 380 subtype C), using sequence similarity networks. We also used spectral clustering to group the sequences based on the network graph structures. A stepwise analysis of the cluster hierarchies allowed us to estimate a possible evolutionary pathway for the Pol proteins. The subtype A sequences also clustered according to when and where the viruses were isolated, whereas both the subtype B and C sequences remained as single clusters. Because the Pol protein has several functional domains, we identified the regions that are discriminative by comparing the structures of the domain-based networks. Our results suggest that sequence changes in the RNase H domain and the reverse transcriptase (RT) connection domain are responsible for the subtype classification. By analyzing the different amino acid compositions at each site in both domain sequences, we found that a few specific amino acid residues (i.e., M357 in the RT connection domain and Q480, Y483, and L491 in the RNase H domain) represent the differences among the subtypes. These residues were located on the surface of the RT structure and in the vicinity of the amino acid sites responsible for RT enzymatic activity or function.

Project description:A subset of HIV-infected individuals termed elite controllers (ECs) maintain CD4+ T cell counts and control viral replication in the absence of antiretroviral therapy (ART). Systemic cytokine responses may differentiate ECs from subjects with uncontrolled viral replication or from those who require ART to suppress viral replication. We measured 87 cytokines in four groups of women: 73 ECs, 42 with pharmacologically suppressed viremia (ART), 42 with uncontrolled viral replication (noncontrollers [NCs]), and 48 HIV-uninfected (NEG) subjects. Four cytokines were elevated in ECs but not NCs or ART subjects: CCL14, CCL21, CCL27, and XCL1. In addition, median stromal cell-derived factor-1 (SDF-1) levels were 43% higher in ECs than in NCs. The combination of the five cytokines suppressed R5 and X4 virus replication in resting CD4+ T cells, and individually SDF-1β, CCL14, and CCL27 suppressed R5 virus replication, while SDF-1β, CCL21, and CCL14 suppressed X4 virus replication. Functional studies revealed that the combination of the five cytokines upregulated CD69 and CCR5 and downregulated CXCR4 and CCR7 on CD4+ T cells. The CD69 and CXCR4 effects were driven by SDF-1, while CCL21 downregulated CCR7. The combination of the EC-associated cytokines induced expression of the anti-HIV host restriction factors IFITM1 and IFITM2 and suppressed expression of RNase L and SAMHD1. These results identify a set of cytokines that are elevated in ECs and define their effects on cellular activation, HIV coreceptor expression, and innate restriction factor expression. This cytokine pattern may be a signature characteristic of HIV-1 elite control, potentially important for HIV therapeutic and curative strategies.IMPORTANCE Approximately 1% of people infected with HIV control virus replication without taking antiviral medications. These subjects, termed elite controllers (ECs), are known to have stronger immune responses targeting HIV than the typical HIV-infected subject, but the exact mechanisms of how their immune responses control infection are not known. In this study, we identified five soluble immune signaling molecules (cytokines) in the blood that were higher in ECs than in subjects with typical chronic HIV infection. We demonstrated that these cytokines can activate CD4+ T cells, the target cells for HIV infection. Furthermore, these five EC-associated cytokines could change expression levels of intrinsic resistance factors, or molecules inside the target cell that fight HIV infection. This study is significant in that it identified cytokines elevated in subjects with a good immune response against HIV and defined potential mechanisms as to how these cytokines could induce resistance to the virus in target cells.

Dataset Information

Natural single-nucleotide polymorphisms in the 3' region of the HIV-1 pol gene modulate viral replication ability.

Publications

Natural single-nucleotide polymorphisms in the 3' region of the HIV-1 pol gene modulate viral replication ability.

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