Tapasin modification on the intracellular epitope HBcAg18-27 enhances HBV-specific CTL immune response and inhibits hepatitis B virus replication in vivo.

HBV-specific cytotoxic T-lymphocyte (CTL) activity has a very important role in hepatitis B virus clearance. Present studies suggest that Tapasin, a endoplasmic reticulum (ER) chaperone, stabilizes the peptide-receptive MHC I conformation, allowing peptide exchange and increasing more peptides to be translocated into the ER. We have previously testified that cytoplasmic transduction peptide (CTP)-HBcAg(18-27)-Tapasin fusion protein could enter cytoplasm of dendritic cells, and enhance T cells' response to generate specific CTLs efficiently in vitro. In the present study, we evaluated specific immune responses of CTP-HBcAg(18-27)-Tapasin fusion protein in HLA-A2 transgenic mice (H-2K(b)) and anti-viral ability in HBV transgenic mice, and explored the mechanisms probably involved in. The studies showed that CTP-HBcAg(18-27)-Tapasin not only increased production of cytokine IFN-γ and interleukin-2 (IL-2), compared with CTP-HBcAg(18-27), HBcAg(18-27)-Tapasin, and PBS, but also significantly induced the higher percentages of IFN-γ+CD8(+) T cells and specific CTL responses in HLA-A2 transgenic mice. Moreover, enhancement of specific CTL activity induced by the fusion protein reduced HBV DNA and hepatitis B surface antigen (HBsAg) levels and decreased the expression of HBsAg and hepatitis B core antigen (HBcAg) in liver tissue of HBV transgenic mice. In addition, CTP-HBcAg(18-27)-Tapasin could upregulate the expression of JAK2, Tyk2, STAT1, and STAT4 in T lymphocytes in HLA-A2 transgenic mice splenocytes. However, there was no significant difference on the expressions of JAK1, JAK3, and STAT6 between each group. In conclusion, CTP-HBcAg(18-27)-Tapasin fusion protein could enhance not only the percentages of CTLs but also induce robust specific CTL activity and inhibits hepatitis B virus replication in vivo, which was associated with activation of the JAK/STAT signaling pathway.

Intracellular pathogen detection by RIG-I-like receptors.

The RIG-I-like receptors (RLRs) RIG-I, MDA5, and LGP2 trigger innate immune responses against viral infections that serve to limit virus replication and to stimulate adaptive immunity. RLRs are cytosolic sensors for virus-derived RNA and thus responsible for intracellular immune surveillance against infection. RLR signaling requires the adapter protein MAVS to induce type I interferon, interferon-stimulated genes, and proinflammatory cytokines. This review focuses on the molecular and cell biological requirements for RLR signal transduction.

IFN-α and lipopolysaccharide upregulate APOBEC3 mRNA through different signaling pathways.

APOBEC3 (A3) proteins are virus-restriction factors that provide intrinsic immunity against infections by viruses like HIV-1 and mouse mammary tumor virus. A3 proteins are inducible by inflammatory stimuli, such as LPS and IFN-α, via mechanisms that are not fully defined. Using genetic and pharmacological studies on C57BL/6 mice and cells, we show that IFN-α and LPS induce A3 via different pathways, independently of each other. IFN-α positively regulates mouse APOBEC3 (mA3) mRNA expression through IFN-αR/PKC/STAT1 and negatively regulates mA3 mRNA expression via IFN-αR/MAPKs-signaling pathways. Interestingly, LPS shows some variation in its regulatory behavior. Although LPS-mediated positive regulation of mA3 mRNA occurs through TLR4/TRIF/IRF3/PKC, it negatively modulates mA3 mRNA via TLR4/MyD88/MAPK-signaling pathways. Additional studies on human peripheral blood mononuclear cells reveal that PKC differentially regulates IFN-α and LPS induction of human A3A, A3F, and A3G mRNA expression. In summary, we identified important signaling targets downstream of IFN-αR and TLR4 that mediate A3 mRNA induction by both LPS and IFN-α. Our results provide new insights into the signaling targets that could be manipulated to enhance the intracellular store of A3 and potentially enhance A3 antiviral function in the host.

Critical role of IRF-8 in negative regulation of TLR3 expression by Src homology 2 domain-containing protein tyrosine phosphatase-2 activity in human myeloid dendritic cells.

Despite extensive studies that unraveled ligands and signal transduction pathways triggered by TLRs, little is known about the regulation of TLR gene expression. TLR3 plays a crucial role in the recognition of viral pathogens and induction of immune responses by myeloid DCs. IFN regulatory factor (IRF)-8, a member of the IRF family, is a transcriptional regulator that plays essential roles in the development and function of myeloid lineage, affecting different subsets of myeloid DCs. In this study, we show that IRF-8 negatively controls TLR3 gene expression by suppressing IRF-1- and/or polyinosinic-polycytidylic acid-stimulated TLR3 expression in primary human monocyte-derived DCs (MDDCs). MDDCs expressed TLR3 increasingly during their differentiation from monocytes to DCs with a peak at day 5, when TLR3 expression was further enhanced upon stimulation with polyinosinic-polycytidylic acid and then was promptly downregulated. We found that both IRF-1 and IRF-8 bind the human TLR3 promoter during MDDC differentiation in vitro and in vivo but with different kinetic and functional effects. We demonstrate that IRF-8-induced repression of TLR3 is specifically mediated by ligand-activated Src homology 2 domain-containing protein tyrosine phosphatase association. Indeed, Src homology 2 domain-containing protein tyrosine phosphatase-dephosphorylated IRF-8 bound to the human TLR3 promoter competing with IRF-1 and quashing its activity by recruitment of histone deacetylase 3. Our findings identify IRF-8 as a key player in the control of intracellular viral dsRNA-induced responses and highlight a new mechanism for negative regulation of TLR3 expression that can be exploited to block excessive TLR activation.

Cutting edge: mucosal application of a lyophilized viral vector vaccine confers systemic and protective immunity toward intracellular pathogens.

A major problem of current vaccines is storage stability, often requiring strict maintenance of cold chains. In the course of the eradication of smallpox, a freeze-dried vaccinia virus (Dryvax), which proved to be very stable, was used to overcome this limitation. However, Dryvax needs to be reconstituted before usage and is administered using a bifurcated needle, procedures that pose a number of additional health risks. We report in this study that a stable, lyophilized, modified vaccinia virus Ankara (MVA) vaccine can be directly applied to the nostrils of mice without previous reconstitution. This direct mucosal application induced systemic Ab and T cell responses comparable to those achieved by i.m. administration. Importantly, mucosal application of lyophilized MVA induced long-lasting protective immunity against lethal bacterial and viral challenges. These data clearly demonstrate the potency of a simple needle-free vaccination, combining the advantages of mucosal application with the stability and efficiency of lyophilized MVA.

Critical negative regulation of type 1 T cell immunity and immunopathology by signaling adaptor DAP12 during intracellular infection.

Transmembrane signaling adaptor DAP12 has increasingly been recognized for its important role in innate responses. However, its role in the regulation of antimicrobial T cell responses has remained unknown. In our current study, we have examined host defense, T cell responses, and tissue immunopathology in models of intracellular infection established in wild-type and DAP12-deficient mice. During mycobacterial infection, lack of DAP12 leads to pronounced proinflammatory and Th1 cytokine responses, overactivation of Ag-specific CD4 and CD8 T cells of type 1 phenotype, and heightened immunopathology both in the lung and lymphoid organs. DAP12-deficient airway APC display enhanced NF-kappaB activation and cytokine responses upon TLR stimulation or mycobacterial infection in vitro. Of importance, adoptive transfer of Ag-loaded DAP12-deficient APC alone could lead to overactivation of transferred transgenic or endogenous wild-type T cells in vivo. We have further found that the immune regulatory role by DAP12 is not restricted only to intracellular bacterial infection, since lack of this molecule also leads to uncontrolled type 1 T cell activation and severe immunopathology and tissue injury during intracellular viral infection. Our study thus identifies DAP12 as an important novel immune regulatory molecule that acts, via APC, to control the level of antimicrobial type 1 T cell activation and immunopathology.

Adenovirus type 5 exerts multiple effects on the expression and activity of cytosolic phospholipase A2, cyclooxygenase-2, and prostaglandin synthesis.

In this study, we examine how infection of murine and human fibroblasts by adenovirus (Ad) serotype 5 (Ad5) affects the expression and activity of cytosolic phospholipase A2 (cPLA2), cyclooxygenase-2 (COX-2), and production of PGs. Our experiments showed that infection with Ad5 is accompanied by the rapid activation of cPLA2 and the cPLA2-dependent release of [3H]arachidonic acid ([3H]AA). Increased expression of COX-2 was also observed after Ad infection, as was production of PGE2 and PGI2. Later, however, as the infection progressed, release of [3H]AA and production of PGs stopped. Late-stage Ad5-infected cells also did not release [3H]AA or PGs following treatment with a panel of biologically diverse agents. Experiments with UV-inactivated virus confirmed that Ad infection is accompanied by the activation of a host-dependent response that is later inhibited by the virus. Investigations of the mechanism of suppression of the PG pathway by Ad5 did not reveal major effects on the expression or activity of cPLA2 or COX-2. We did note a change in the intracellular position of cPLA2 and found that cPLA2 did not translocate normally in infected cells, raising the possibility that Ad5 interferes with the PG pathway by interfering with the intracellular movement of cPLA2. Taken together, these data reveal dynamic interactions between Ad5 and the lipid mediator pathways of the host and highlight a novel mechanism by which Ad5 evades the host immune response. In addition, our results offer insight into the inflammatory response induced by many Ad vectors lacking early region gene products.

The CD16+ monocyte subset is more permissive to infection and preferentially harbors HIV-1 in vivo.

HIV-1 persists in peripheral blood monocytes in individuals receiving highly active antiretroviral therapy (HAART) with viral suppression, despite these cells being poorly susceptible to infection in vitro. Because very few monocytes harbor HIV-1 in vivo, we considered whether a subset of monocytes might be more permissive to infection. We show that a minor CD16+ monocyte subset preferentially harbors HIV-1 in infected individuals on HAART when compared with the majority of monocytes (CD14highCD16-). We confirmed this by in vitro experiments showing that CD16+ monocytes were more susceptible to CCR5-using strains of HIV-1, a finding that is associated with higher CCR5 expression on these cells. CD16+ monocytes were also more permissive to infection with a vesicular stomatitis virus G protein-pseudotyped reporter strain of HIV-1 than the majority of monocytes, suggesting that they are better able to support HIV-1 replication after entry. Consistent with this observation, high molecular mass complexes of apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G (APOBEC3G) were observed in CD16+ monocytes that were similar to those observed in highly permissive T cells. In contrast, CD14highCD16- monocytes contained low molecular mass active APOBEC3G, suggesting this is a mechanism of resistance to HIV-1 infection in these cells. Collectively, these data show that CD16+ monocytes are preferentially susceptible to HIV-1 entry, more permissive for replication, and constitute a continuing source of viral persistence during HAART.

Principles of intracellular viral recognition.

In mammalian cells, the products of microbial infection are recognized by pathogen recognition receptor (PRR) proteins. Virus recognition is mediated in part by PRRs that comprise a subset of Toll-like receptors or a family of RNA helicases, the latter of which contain caspase activation and recruitment domains, both of which induce interferons alpha and beta and antiviral immune defenses. Recent studies show that PRR engagement of specific pathogen-associated molecular patterns (PAMPs) within viral products, including viral proteins and nucleic acid, is facilitated by the discrete subcellular distribution of PRRs to sites that intersect with processes of virus entry and replication. PAMP structure and the subcellular context of PRR distribution form a basis of self versus nonself discrimination during the antiviral response. Understanding the virus/host interface of PRR function and PAMP recognition will advance therapeutic strategies for immune response regulation.

Screening and identification of severe acute respiratory syndrome-associated coronavirus-specific CTL epitopes.

Severe acute respiratory syndrome (SARS) is a highly contagious and life-threatening disease that emerged in China in November 2002. A novel SARS-associated coronavirus was identified as its principal etiologic agent; however, the immunopathogenesis of SARS and the role of special CTLs in virus clearance are still largely uncharacterized. In this study, potential HLA-A*0201-restricted spike (S) and nucleocapsid protein-derived peptides were selected from an online database and screened for potential CTL epitopes by in vitro refolding and T2 cell-stabilization assays. The antigenicity of nine peptides which could refold with HLA-A*0201 molecules was assessed with an IFN-gamma ELISPOT assay to determine the capacity to stimulate CTLs from PBMCs of HLA-A2(+) SARS-recovered donors. A novel HLA-A*0201-restricted decameric epitope P15 (S411-420, KLPDDFMGCV) derived from the S protein was identified and found to localize within the angiotensin-converting enzyme 2 receptor-binding region of the S1 domain. P15 could significantly enhance the expression of HLA-A*0201 molecules on the T2 cell surface, stimulate IFN-gamma-producing CTLs from the PBMCs of former SARS patients, and induce specific CTLs from P15-immunized HLA-A2.1 transgenic mice in vivo. Furthermore, significant P15-specific CTLs were induced from HLA-A2.1-transgenic mice immunized by a DNA vaccine encoding the S protein; suggesting that P15 was a naturally processed epitope. Thus, P15 may be a novel SARS-associated coronavirus-specific CTL epitope and a potential target for characterization of virus control mechanisms and evaluation of candidate SARS vaccines.

Maintenance of HIV-specific CD4+ T cell help distinguishes HIV-2 from HIV-1 infection.

Unlike HIV-1-infected people, most HIV-2-infected subjects maintain a healthy CD4+ T cell count and a strong HIV-specific CD4+ T cell response. To define the cellular immunological correlates of good prognosis in HIV-2 infection, we conducted a cross-sectional study of HIV Gag-specific T cell function in HIV-1- and HIV-2-infected Gambians. Using cytokine flow cytometry and lymphoproliferation assays, we show that HIV-specific CD4+ T cells from HIV-2-infected individuals maintained proliferative capacity, were not terminally differentiated (CD57-), and more frequently produced IFN-gamma or IL-2 than CD4+ T cells from HIV-1-infected donors. Polyfunctional (IFN-gamma+/IL-2+) HIV-specific CD4+ T cells were found exclusively in HIV-2+ donors. The disparity in CD4+ T cell responses between asymptomatic HIV-1- and HIV-2-infected subjects was not associated with differences in the proliferative capacity of HIV-specific CD8+ T cells. This study demonstrates that HIV-2-infected donors have a well-preserved and functionally heterogeneous HIV-specific memory CD4+ T cell response that is associated with delayed disease progression in the majority of infected people.

NS1 interaction with CKII alpha: novel protein complex mediating parvovirus-induced cytotoxicity.

During a productive infection, the prototype strain of the parvovirus minute virus of mice (MVMp) induces dramatic morphological alterations in permissive A9 fibroblasts, culminating in cell lysis at the end of infection. These cytopathic effects (CPE) result from rearrangements and destruction of the cytoskeletal micro- and intermediate filaments, while other structures such as the nuclear lamina and particularly the microtubule network remain protected throughout the infection (J. P. F. Nüesch et al., Virology 331:159-174, 2005). In order to unravel the mechanism(s) by which parvoviruses trigger CPE, we searched for NS1 interaction partners by differential affinity chromatography, using distinct NS1 mutants debilitated specifically for this function. Thereby, we isolated an NS1 partner polypeptide, whose interaction with NS1 correlated with the competence of the viral product for CPE induction, and further identified it by tandem mass spectrometry and Western blotting analyses to consist of the catalytic subunit of casein kinase II, CKIIalpha. This interaction of NS1 with CKIIalpha suggested interference by the viral protein with intracellular signaling. Using permanent cell lines expressing dominant-negative CKIIalpha mutants, we were able to show that this kinase activity was indeed specifically involved in parvoviral CPE and progeny particle release. Furthermore, the NS1/CKIIalpha complex proved to be able to specifically phosphorylate viral capsids, indicating a mediator function of NS1 for CKII activity and specificity, at least in vitro. Altogether our data suggest that parvovirus-induced CPE is mediated by NS1 interference with intracellular CKII signaling.

Different domains of CD81 mediate distinct stages of hepatitis C virus pseudoparticle entry.

The CD81 tetraspanin was first identified as a hepatitis C virus (HCV) receptor by its ability to bind the soluble ectodomain of envelope glycoprotein E2 (sE2). More recently, it has been suggested that CD81 is necessary but not sufficient for HCV entry into target cells. Here we present further evidence that putative human hepatocyte-specific factors act in concert with CD81 to mediate sE2 binding and HCV pseudoparticle (HCVpp) entry. Moreover, we show that CD81-mediated HCVpp entry entails E2 binding to residues in the large extracellular loop as well as molecular events mediated by the transmembrane and intracellular domains of CD81. The concept that CD81 receptor function progresses in stages is further supported by our finding that anti-CD81 monoclonal antibodies inhibit HCVpp entry by different mechanisms. The half-life of CD81-HCVpp binding was determined to be approximately 17 min, and we propose that binding is followed by CD81 oligomerization, partitioning into cholesterol-rich membrane domains, or other, lateral protein-protein interactions. This results in the formation of a receptor-virus complex that undergoes endocytosis and pH-dependent membrane fusion.

Modifications in the human T cell proteome induced by intracellular HIV-1 Tat protein expression.

The effects of the human immunodeficiency virus type 1 (HIV-1) Tat protein on cellular gene expression were analysed using a Jurkat cell line that was stably transfected with tat gene in a doxycycline-repressible expression system. Expressed Tat protein (aa 1-101) was proved to present basically a nuclear localisation, and to be fully functional to induce HIV LTR transactivation. Tat expression also resulted in protection from Tunicamycin-induced apoptosis as determined by DNA staining and TUNEL assays. We applied proteomics methods to investigate changes in differential protein expression in the transfected Jurkat-Tat cells. Protein identification was performed using 2-D DIGE followed by MS analysis. We identified the down-regulation of several cytoskeletal proteins such as actin, beta-tubulin, annexin II, as well as gelsolin, cofilin and the Rac/Rho-GDI complex. Down-expression of these proteins could be involved in the survival of long-term reservoirs of HIV-infected CD4+ T cells responsible for continuous viral production. In conclusion, in addition to its role in viral mRNA elongation, the proteomic approach has provided insight into the way that Tat modifies host cell gene expression.

Innate sensors of microbial infection.

The innate immune system utilizes multiple families of pattern-recognition receptors (PRRs) to protect the host from infection. Each of these families contributes certain elements to the complement of innate effector functions that is elicited during an infection. Here we review the families of PRRs and explore examples of their cooperativity.

IL-12-independent Th1 polarization in human mononuclear cells infected with varicella-zoster virus.

T helper type 1 (Th1) cells perform a critical role in fighting intracellular organisms, and interleukin-12 (IL-12) is known to promote a Thl response. This study was conducted to identify whether an IL-12-independent Th1 reaction is induced by the varicella-zoster virus (VZV) in human beings. It was found that different intracellular microorganisms could induce IFNgamma but not IL-12 production. Induction of IFNgamma production by VZV was associated with IFNalpha production and phosphorylation of both the signal transducer and activator of transcription-1 (STAT-1) and STAT-4 in lymphocytes. In contrast, Bacillus Calmette-Guerin (BCG) induced IL-12 production in association with STAT-4 but not STAT-1 activation. Anti-IFNalpha but not anti-IL-12 antibodies blocked the VZV-induced Th1 polarization. A patient with an IL-12 receptor beta1 chain deficiency showed a normal VZV- but not a normal BCG-induced Th1 reaction, further supporting the concept of an IFNalpha-mediated, IL-12-independent Th1 reaction in response to certain intracellular infections. Identification of the early Th1 polarization induced by IFNalpha versus IL-12 in response to specific viruses may enable the development of better therapeutic strategies tailored to different infections.

Protein kinase R mediates intestinal epithelial gene remodeling in response to double-stranded RNA and live rotavirus.

As sentinels of host defense, intestinal epithelial cells respond to the viral pathogen rotavirus by activating a gene expression that promotes immune cell recruitment and activation. We hypothesized that epithelial sensing of rotavirus might target dsRNA, which can be detected by TLR3 or protein kinase R (PKR). Accordingly, we observed that synthetic dsRNA, polyinosinic acid:cytidylic acid (poly(I:C)), potently induced gene remodeling in model intestinal epithelia with the specific pattern of expressed genes, including both classic proinflammatory genes (e.g., IL-8), as well as genes that are classically activated in virus-infected cells (e.g., IFN-responsive genes). Poly(I:C)-induced IL-8 was concentration dependent (2-100 mug/ml) and displayed slower kinetics compared with IL-8 induced by bacterial flagellin (ET(50) approximately 24 vs 8 h poly(I:C) vs flagellin, respectively). Although model epithelia expressed detectable TLR3 mRNA, neither TLR3-neutralizing Abs nor chloroquine, which blocks activation of intracellular TLR3, attenuated epithelial responses to poly(I:C). Conversely, poly(I:C)-induced phosphorylation of PKR and inhibitors of PKR, 2-aminopurine and adenine, ablated poly(I:C)-induced gene expression but had no effect on gene expression induced by flagellin, thus suggesting that intestinal epithelial cell detection of dsRNA relies on PKR. Consistent with poly(I:C) detection by an intracellular molecule such as PKR, we observed that both uptake of and responses to poly(I:C) were polarized to the basolateral side. Lastly, we observed that the pattern of pharmacologic inhibition of responses to poly(I:C) was identical to that seen in response to infection by live rotavirus, indicating a potentially important role for PKR in activating intestinal epithelial gene expression in rotavirus infection.

Intraepithelial cell neutralization of HIV-1 replication by IgA.

HIV is transmitted sexually through mucosal surfaces where IgA Abs are the first line of immune defense. In this study, we used paired IgA and IgG mAbs against HIV gp160 to study intraepithelial cell neutralization and inhibition of HIV replication. African green monkey kidney cells, Vero C1008, polarizable epithelial cells transfected to express the polymeric Ig receptor (pIgR), were transfected with HIV proviral DNA, and intracellular neutralization mediated by the mAbs was assessed. D47A and D19A IgA, which neutralized HIV in a conventional assay, potently inhibited intracellular HIV replication as assessed by infecting HeLa-CD4-long terminal repeat/beta-galactosidase cells (human cervical carcinoma cell line) and CEMx174 cells (human T cell line) with apical supernatant, basolateral medium, and cell lysate from transfected cells. D47A also inhibited the production of virus as assessed by direct assay of p24. In contrast, D47 and D19 IgG, sharing the same V regions, but which were not transcytosed by the pIgR, did not inhibit intracellular HIV replication, nor did D47A and D19A IgA in pIgR- cells, incapable of transcytosing IgA. Confocal immunofluorescence microscopy showed prominent colocalization of HIV protein and D47A, in agreement with the intracellular neutralization data. D10A, which did not neutralize HIV in the conventional assay, and irrelevant IgA did not show intracellular neutralization or colocalization. Control studies with two kinds of conditioned medium confirmed that HIV neutralization had indeed occurred inside the cells. Thus, during its transcytosis through epithelial cells, HIV-specific IgA can neutralize HIV replication.

Down-modulation of the CXCR4 co-receptor by intracellular expression of a single chain variable fragment (SFv) inhibits HIV-1 entry into primary human brain microvascular endothelial cells and post-mitotic neurons.

Our laboratories previously demonstrated that expression of a single chain variable antibody fragment (SFv), anti-CXCR4 SFv, in human lymphoid cells suppresses surface display of the chemokine co-receptor CXCR4 and inhibits infectious entry of human immunodeficiency virus type I (HIV-1). We now sought to extend these results to two types of central nervous system (CNS) cells, primary isolated human brain microvascular endothelial cells (MVECs), and post-mitotic differentiated human neurons, both of which normally express significant levels of CXCR4. The anti-CXCR4 SFv expression construct was delivered using an HIV-1-based vector, and control cells received LacZ-expressing viral particles. Upon intracellular expression of the anti-CXCR4 SFv, immunostaining revealed a marked reduction in surface display of CXCR4 on both cell types. Consequently, post-mitotic neurons expressing the anti-CXCR4 SFv were significantly protected from HIV-1 infection, as measured by HIV-1 p24 antigen production, and partial protection was observed in human brain MVECs. The ability to selectively down-modulate the surface expression of CXCR4 in CNS cells may allow for the development of clinical molecular therapy strategies against HIV-1-related neurodegenerative disorders and neuroinvasion.

Blocking HIV-1 Vif restores a natural mechanism of intracellular antiviral defense.

AIDS has become the greatest pandemic in the human history counting approximately 40 millions people worldwide. To purge HIV-1 infection, new therapeutic approaches need to be searched in alternative and/or in addition to the current pharmacological ones. Recently, several independent laboratories have unveiled a non-immune intracellular anti-HIV-1 defense strategy based on the cytidine deaminase APOBEC3G, which restricts HIV-1 production by directly mutating the proviral DNA in infected cells. To counteract this defense pathway, HIV-1 has developed an evasion strategy by acquiring the accessory protein Vif, which blocks the action of APOBEC3G by inducing its proteasome-mediated degradation.