胞嘧啶脱氨酶APOBEC3G可能通过G→A突变抑制乙型肝炎病毒的复制

目的研究胞嘧啶脱氨酶APOBEC3G(A3G)对HBV复制的影响及作用机制。方法利用脂质体介导A3G和HBV的真核表达质粒瞬时共转染人肝癌细胞株HepG2,以空载体pcDNA3.1与HBV真核表达质粒共转染为对照,同时转染含增强型绿色荧光蛋白(EGFP)基因的质粒载体以判定转染效率。共转染两天后用荧光定量PCR方法定量细胞内核壳体(core)相关HBV DNA水平,用Western blot检测细胞内A3G和HBcAg的表达,并对细胞内core相关HBV DNA X基因进行PCR扩增、T-A克隆和测序,分析X基因中的碱基突变。结果经EGFP判定的转染效率平均为29%。共转染0.5μgA3G和0.5μgHBV的HepG2细胞内core相关HBV DNA量平均下降为对照的8.9%,共转染2μgA3G和0.5μgHBV的平均下降为对照的0.6%。共转染A3G和HBV表达质粒后,HepG2细胞内HBV的X基因中发生G→A突变的数目明显增多,33个克隆中有9个克隆检测到了16～37个G→A突变,突变总数达254个,而对照的33个克隆中仅有2个克隆各检测到2个G→A突变。进一步的分析发现,共转染A3G后发生的G→A突变大多集中于几个热点区域,突变靶点常在GG二核苷酸中的第一个G上。结论胞嘧啶脱氨酶APOBEC3G可能通过诱导。HBV DNA的X基因发生G→A超突变,抑制HBV在人肝癌细胞HepG2中的复制。     

Distinct viral determinants for the packaging of human cytidine deaminases APOBEC3G and APOBEC3C

Human APOBEC3G and other APOBEC3 cytidine deaminases inhibit a variety of retroviruses, including Vif-deficient HIV-1. These host proteins are packaged into viral particles and inhibit the replication of virus in new target cells. A3G and A3F are known to be efficiently packaged into HIV-1 virions by binding to 7SL RNA through the Gag NC domain; however, the packaging mechanisms of other APOBEC3 proteins are poorly defined. We have now demonstrated that APOBEC3C (A3C) can be efficiently packaged into HIV-1 virions that are deficient for viral genomic RNA. Inhibition of the encapsidation of 7SL RNA into HIV-1 virions blocked the packaging of A3G, but not A3C. While the NC domain is required for efficient packaging of A3G, deletion of this domain had little effect on A3C packaging into HIV-1 Gag particles. A3C interacted with HIV-1 Gag which was MA domain-dependent and RNA-dependent. Deletion of the MA domain of HIV-1 Gag inhibited A3C but not A3G packaging into HIV-1 Gag particles. Thus, A3G and A3C have evolved to use distinct mechanisms for targeting retroviruses.     

Stoichiometry of the antiviral protein APOBEC3G in HIV-1 virions

A host cytidine deaminase, APOBEC3G (A3G), inhibits replication of human immunodeficiency virus type 1 (HIV-1) by incorporating into virions in the absence of the virally encoded Vif protein (Deltavif virions), at least in part by causing G-to-A hypermutation. To gain insight into the antiretroviral function of A3G, we determined the quantities of A3G molecules that are incorporated in Deltavif virions. We combined three experimental approaches-reversed-phase high-pressure liquid chromatography (HPLC), scintillation proximity assay (SPA), and quantitative immunoblotting-to determine the molar ratio of A3G to HIV-1 capsid protein in Deltavif virions. Our studies revealed that the amount of the A3G incorporated into Deltavif virions was proportional to the level of its expression in the viral producing cells, and the ratio of the A3G to Gag in the Deltavif virions produced from activated human peripheral blood mononuclear cells (PBMC) was approximately 1:439. Based on previous estimates of the stoichiometry of HIV-1 Gag in virions (1400-5000), we conclude that approximately 7 (+/-4) molecules of A3G are incorporated into Deltavif virions produced from human PBMCs. These results indicate that virion incorporation of only a few molecules of A3G is sufficient to inhibit HIV-1 replication.     

Functional analysis of the two cytidine deaminase domains in APOBEC3G

Human APOBEC3G (hA3G), a cytidine deaminase with two cytidine deaminase domains (CDs), has been identified as an anti-HIV-1 host factor. Although the two CDs of hA3G have been extensively characterized, there is still debate on the role of the CDs in the biological function of hA3G. In this work, we constructed three hA3G mutants CD1-1, CD2-2 and CD2-1, which contain duplicate CD1 domain, duplicate CD2 domain and position switched CD domain respectively, and investigated the effect of CD domain replacement or switch upon virion encapsidation, Vif-mediated degradation, deamination and antiviral activity of hA3G. The results showed that the two CD domains were functionally equivalent in virion encapsidation and the interaction with HIV-1 Vif of hA3G, whereas CD domain switch or replacement greatly affected the sensitivity to Vif induced degradation, editing and antiviral activity of hA3G. Although the CD2 domain was shown to possess the deamination activity, CD2-2 incorporated efficiently into HIV-1 was unable to mutate viral cDNA, suggesting that CD1 also involved in the enzymatic function. Interestingly, CD2-1 retained considerable deamination activity with a different sequence preference. Taken together, our results suggest that CD domain may play a structural role in virion encapsidation and Vif-mediated degradation of hA3G, and coordination of the two CD domains is required for its editing and antiviral activity.     

Cytidine deamination-induced perpetual immunity to SAR-CoV-2 infection is a potential new therapeutic target

As the world is racing to develop perpetual immunity to the SARS-CoV-2 virus. The emergence of new viral strains, together with vaccination and reinfections, are all contributing to a long-term immunity against the deadly virus that has taken over the world since its introduction to humans in late December 2019. The discovery that more than 95 percent of people who recovered from COVID-19 had long-lasting immunity and that asymptomatic people have a different immune response to SARS-CoV-2 than symptomatic people has shifted attention to how our immune system initiates such diverse responses. These findings have provided reason to believe that SARS-CoV-2 days are numbered. Hundreds of research papers have been published on the causes of long-lasting immune responses and variations in the numbers of different immune cell types in COVID 19 survivors, but the main reason of these differences has still not been adequately identified. In this article, we focus on the activation-induced cytidine deaminase (AID), which initiates molecular processes that allow our immune system to generate antibodies against SARS-CoV-2. To establish lasting immunity to SARS-CoV-2, we suggest that AID could be the key to unlocking it.     

Comparative analysis of the antiretroviral activity of APOBEC3G and APOBEC3F from primates

APOBEC3G and APOBEC3F exhibit antiretroviral activity primarily as a consequence of their ability to deaminate cytidines in retroviral DNA. Here, we compare the properties of APOBEC3F and APOBEC3G from human, macaque, and African green monkey (AGM). While all APOBEC proteins tested exhibited anti-HIV-1 activity, human APOBEC3F was, surprisingly, 10- to 50-fold less potent than human APOBEC3G. However, similar discrepancies in antiviral potency were not found when pairs of proteins from macaque and AGM were compared. Intrinsic differences in the ability of each APOBEC protein to induce hypermutation, rather than differences in packaging efficiency, partially accounted for variable antiretroviral activity. Each of four primate lentivirus Vif proteins reduced human and AGM APOBEC3F expression and antiviral activity, but all were only partially effective and species-specific effects were relatively minor. Overall, highly efficient and species-specific neutralization of APOBEC3G, and less efficient neutralization of APOBEC3F, appears to be a general property of Vif proteins.     

Ubiquitination of APOBEC3 proteins by the Vif-Cullin5-ElonginB-ElonginC complex

APOBEC3 proteins are antiviral host factors for a wide variety of retroviruses. HIV-1 Vif overcomes the antiviral activity of APOBEC3G by ubiquitinating the protein. In this study, we examined the ability of Vif to antagonize other family members of APOBEC3 proteins, together with its mechanism. Using HIV infectivity, virion incorporation, immunoprecipitation, and in vitro ubiquitin conjugation assays, we show that the ability of Vif to inhibit antiviral activity of APOBEC3 proteins positively correlates with its ability to bind and ubiquitinate these proteins by a Vif-Cullin5-ElonginB-ElonginC (Vif-BC-Cul5) complex. These results suggest that Vif exhibits its anti-APOBEC3 activity by the ubiquitin ligase activity of the Vif-BC-Cul5 complex.     

Molecular and functional interactions of cat APOBEC3 and feline foamy and immunodeficiency virus proteins: different ways to counteract host-encoded restriction

Defined host-encoded feline APOBEC3 (feA3) cytidine deaminases efficiently restrict the replication and spread of exogenous retroviruses like Feline Immunodeficiency Virus (FIV) and Feline Foamy Virus (FFV) which developed different feA3 counter-acting strategies. Here we characterize the molecular interaction of FFV proteins with the diverse feA3 proteins. The FFV accessory protein Bet is the virus-encoded defense factor which is shown here to bind all feA3 proteins independent of whether they restrict FFV, a feature shared with FIV Vif that induces degradation of all feA3s including those that do not inactivate FIV. In contrast, only some feA3 proteins bind to FFV Gag, a pattern that in part reflects the restriction pattern detected. Additionally, one-domain feA3 proteins can homo- and hetero-dimerize in vitro, but a trans-dominant phenotype of any of the low-activity feA3 forms on FFV restriction by one of the highly-active feA3Z2 proteins was not detectable.     

Allergy and infection: understanding their relationship

Sensitization to indoor allergens has long been recognized as a risk factor for asthma and its associated phenotypes. Immunoglobulin E antibody quantification may be used to confirm that a particular sensitization has a role in the development of respiratory symptoms. However, in order to use quantitative information effectively, it is essential to understand the factors that contribute to the deterioration of symptoms and cause exacerbations. One such risk factor is high allergen exposure. Worsening asthma control is also associated with virus infections, in particular rhinovirus infection. Each of these factors in isolation is unlikely to have a major effect on symptoms. However, the combination of respiratory virus infection, allergen sensitization and concurrent high exposure to allergens markedly increases the risk for hospitalization. Further investigation of this synergism could help to understand the mechanisms of asthma exacerbations and lead to the development of strategies for prevention.     

Stress proteins may provide a link between the immune response to infection and autoimmunity

Stress proteins are frequently the target of humoral and cell-mediatedimmune responses to infection. These proteins belong to highlyconserved gene families and there is substantial sequence homologybetween antigens produced by pathogenic organisms and the correspondingproteins from mammalian cells. Human T cells from sites of infectiousand autoimmune lesions proliferate in response to stress proteins,and mapping of antigenic determinants on a mycobacterial stressprotein shows that both species specific and highly conserved,‘self-like’, regions of the molecule can take partin immune recognition. It is proposed that the lymphocyte populationinduced in response to stress proteins of pathogens during infectionincludes cells capable of autolmmune recognition of the correspondingself protein. Local accumulation of self stress proteins—inresponse to viral infection, for example—may subsequentlyprovide a stimulus for proliferation of such autoreactive lymphocytes,thereby triggering a cycle of events which may contribute tothe pathological damage associated with autoimmune disease.     

Quantitative PCR as a method for monitoring retroviral infection on the gene level

For monitoring retroviral infection on the gene level, we propose the use of quantitative PCR with two internal standards: one for a fragment of the viral genome and the other for the host cell marker gene. The standards (one for HIV and the other for a human DNA marker gene HLA-DQ) were constructed by PCR-mediated joining of DNA fragments and were found to be effective in quantitative PCR despite rather different structures of amplified fragments in target and standard DNAs. The number of HIV DNA copies was found to be 2–500 per 1000 lymphocytes in blood from HIV-infected patients and up to 5000+ per 1000 cells in chronically infected cell lines. The degree of infection thus measured was found to change over the course of treatment.     

Impact of viral accessory proteins of SIVsmmPBj on early steps of infection of quiescent cells

Although lentiviruses like HIV-1 are able to infect non-dividing cells, particular resting cells such as non-stimulated primary peripheral blood mononuclear cells (PBMC) are resistant to infection. In contrast to other lentiviruses, SIVsmmPBj can replicate in non-stimulated PBMC. Moreover, SIVsmmPBj-derived, but not HIV-1-derived, replication-incompetent vectors enable gene transfer into G(0)-arrested human cell lines and primary human monocytes. Here, we demonstrate that transduction of G(0)-arrested cell lines by SIVsmmPBj-derived vectors is independent of the viral accessory proteins Vif, Vpx, Vpr, or Nef. In contrast, for the transduction of primary human monocytes, the Vpx protein proved to be essential. However, trans-complementation of HIV-1 vectors with SIVsmmPBj Vpx did not provide the property of gene transfer into monocytes. Taken together, these data indicate that Vpx is essential for the infection of primary monocytes by SIVsmmPBj. Additionally, further genome functions besides the accessory proteins are required for the particular capacity of SIVsmmPBj in transduction or infection events.     

Phosphorylation of a novel SOCS-box regulates assembly of the HIV-1 Vif-Cul5 complex that promotes APOBEC3G degradation

HIV-1 Vif (viral infectivity factor) protein overcomes the antiviral activity of the DNA deaminase APOBEC3G by targeting it for proteasomal degradation. We report here that Vif targets APOBEC3G for degradation by forming an SCF-like E3 ubiquitin ligase containing Cullin 5 and Elongins B and C (Cul5-EloB-EloC) through a novel SOCS (suppressor of cytokine signaling)-box that binds EloC. Vif binding to EloC is negatively regulated by serine phosphorylation in the BC-box motif of the SOCS-box. Vif ubiquitination is promoted by Cul5 in vitro and in vivo, and requires an intact SOCS-box. Thus, autoubiquitination of Vif occurs within the assembled Vif-Cul5 complex, analogous to F-box proteins that are autoubiquitinated within their SCF (Skp1-Cullin-F-box) complex. These findings suggest mechanisms that regulate the assembly and activity of Cul5 E3 complexes through phosphorylation or autoubiquitination of the SOCS-box protein, and identify interactions between Vif and host cell proteins that may be therapeutic targets.     

Cytosine deamination and selection of CpG suppressed clones are the two major independent biological forces that shape codon usage bias in coronaviruses

Using the complete genome sequences of 19 coronavirus genomes, we analyzed the codon usage bias, dinucleotide relative abundance and cytosine deamination in coronavirus genomes. Of the eight codons that contain CpG, six were markedly suppressed. The mean NNU/NNC ratio of the six amino acids using either NNC or NNU as codon is 3.262, suggesting cytosine deamination. Among the 16 dinucleotides, CpG was most markedly suppressed (mean relative abundance 0.509). No correlation was observed between CpG abundance and mean NNU/NNC ratio. Among the 19 coronaviruses, CoV-HKU1 showed the most extreme codon usage bias and extremely high NNU/NNC ratio of 8.835. Cytosine deamination and selection of CpG suppressed clones by the immune system are the two major independent biochemical and biological selective forces that shape codon usage bias in coronavirus genomes. The underlying mechanism for the extreme codon usage bias, cytosine deamination and G+C content in CoV-HKU1 warrants further studies.     

Establishment of a therapeutic model for retroviral infection using the genetic resistance mechanism of the host

Resistance to retroviral infection is often regulated by multiple genes that control different aspects of the host-virus interaction. Genetically distinct inbred strains of mice differ in their susceptibility to retrovirus and have allowed the identification of several host-resistant loci that regulate the host defense mechanism to retroviral Infection. Using the murine retrovirus infection system, a therapeutic model has been developed of retrovirus Infection In association with the resistant mechanism of host genes. The most effective result achieved with the model was when using bone marrow transplantation of retrovirus-reslstant cells with receptor interference function, which was genetlcally defined by the Fv-4 resistant gene. The possible appllcatlon of these findings to the gene therapy of retrovirus-induced disease of humans Is discussed.     

How the study of Helicobacter infection can contribute to the understanding of carcinoma development

The inflammatory environment dramatically impacts the formation of cancer at many levels, acting on the stem cell to foster the initiation of cancer all the way through its contribution to metastatic disease. Using Helicobacter-induced gastric cancer as an example, it can be seen that, early on, chronic inflammation exhausts tissue stem cells, forcing the remaining stem cells to work overtime and calling in replacement cells from marrow sources. Marrow-derived stromal cells orchestrate growth and remodelling through secreted factors and cell–cell communication. Once cancer is present, the inflammatory environment is responsible for the continued growth signals to the cancer stem cells and to the stromal cells which become a vital part of the cancer niche as well as the pre-metastatic niche which will effectively lure cancer cells into peripheral organs for distant growth. This understanding of the inflammatory environment and its many effects on cancer throughout its natural history provides intervention targets directed at the unique aspects of cancer behaviour.