Anna Bergamaschi

Human TRIM Gene Expression in Response to Interferons

Background Tripartite motif (TRIM) proteins constitute a family of proteins that share a conserved tripartite architecture. The recent discovery of the anti-HIV activity of TRIM5α in primate cells has stimulated much interest in the potential role of TRIM proteins in antiviral activities and innate immunity. Principal Findings To test if TRIM genes are up-regulated during antiviral immune responses, we performed a systematic analysis of TRIM gene expression in human primary lymphocytes and monocyte-derived macrophages in response to interferons (IFNs, type I and II) or following FcγR-mediated activation of macrophages. We found that 27 of the 72 human TRIM genes are sensitive to IFN. Our analysis identifies 9 additional TRIM genes that are up-regulated by IFNs, among which only 3 have previously been found to display an antiviral activity. Also, we found 2 TRIM proteins, TRIM9 and 54, to be specifically up-regulated in FcγR-activated macrophages. Conclusions Our results present the first comprehensive TRIM gene expression analysis in primary human immune cells, and suggest the involvement of additional TRIM proteins in regulating host antiviral activities.

Restriction of HIV-1 replication in macrophages and CD4+ T cells from HIV controllers

How HIV controllers (HICs) maintain undetectable viremia without therapy is unknown. The strong CD8(+) T-cell HIV suppressive capacity found in many, but not all, HICs may contribute to long-lasting viral control. However, other earlier defense mechanisms may be involved. Here, we examined intrinsic HIC cell resistance to HIV-1 infection. After in vitro challenge, monocyte-derived macrophages and anti-CD3-activated CD4(+) T cells from HICs showed low HIV-1 susceptibility. CD4 T-cell resistance was independent of HIV-1 coreceptors and affected also SIVmac infection. CD4(+) T cells from HICs expressed ex vivo higher levels of p21(Waf1/Cip1), which has been involved in the control of HIV-1 replication, than cells from control subjects. However, HIV restriction in anti-CD3-activated CD4(+) T cells and macrophages was not associated with p21 expression. Restriction inhibited accumulation of reverse transcripts, leading to reduction of HIV-1 integrated proviruses. The block could be overcome by high viral inocula, suggesting the action of a saturable mechanism. Importantly, cell-associated HIV-1 DNA load was extremely low in HICs and correlated with CD4(+) T-cell permissiveness to infection. These results point to a contribution of intrinsic cell resistance to the control of infection and the containment of viral reservoir in HICs.

The Human Immunodeficiency Virus Type 2 Vpx Protein Usurps the CUL4A-DDB1DCAF1 Ubiquitin Ligase To Overcome a Postentry Block in Macrophage Infection

ABSTRACT The human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) genomes encode several auxiliary proteins that have increasingly shown their importance in the virus-host relationship. One of these proteins, Vpx, is unique to the HIV-2/SIVsm lineage and is critical for viral replication in macrophages. The functional basis for this requirement, as well as the Vpx mode of action, has remained unexplained, and it is all the more enigmatic that HIV type 1 (HIV-1), which has no Vpx counterpart, can infect macrophages. Here, we underscore DCAF1 as a critical host effector of Vpx in its ability to mediate infection and long-term replication of HIV-2 in human macrophages. Vpx assembles with the CUL4A-DDB1 ubiquitin ligase through DCAF1 recruitment. Precluding Vpx present in the incoming virions from recruiting DCAF1 in target macrophages leads to a postentry block characterized by defective accumulation of HIV-2 reverse transcripts. In addition, Vpx from SIVsm functionally complements Vpx-defective HIV-2 in a DCAF1-binding-dependent manner. Altogether, our data point to a mechanism in which Vpx diverts the Cul4A-DDB1DCAF1 ligase to inactivate an evolutionarily conserved factor, which restricts macrophage infection by HIV-2 and closely related simian viruses.

Host hindrance to HIV-1 replication in monocytes and macrophages

Monocytes and macrophages are targets of HIV-1 infection and play critical roles in multiple aspects of viral pathogenesis. HIV-1 can replicate in blood monocytes, although only a minor proportion of circulating monocytes harbor viral DNA. Resident macrophages in tissues can be infected and function as viral reservoirs. However, their susceptibility to infection, and their capacity to actively replicate the virus, varies greatly depending on the tissue localization and cytokine environment. The susceptibility of monocytes to HIV-1 infection in vitro depends on their differentiation status. Monocytes are refractory to infection and become permissive upon differentiation into macrophages. In addition, the capacity of monocyte-derived macrophages to sustain viral replication varies between individuals. Host determinants regulate HIV-1 replication in monocytes and macrophages, limiting several steps of the viral life-cycle, from viral entry to virus release. Some host factors responsible for HIV-1 restriction are shared with T lymphocytes, but several anti-viral mechanisms are specific to either monocytes or macrophages. Whilst a number of these mechanisms have been identified in monocytes or in monocyte-derived macrophages in vitro, some of them have also been implicated in the regulation of HIV-1 infection in vivo, in particular in the brain and the lung where macrophages are the main cell type infected by HIV-1. This review focuses on cellular factors that have been reported to interfere with HIV-1 infection in monocytes and macrophages, and examines the evidences supporting their role in vivo, highlighting unique aspects of HIV-1 restriction in these two cell types.

The CDK Inhibitor p21Cip1/WAF1 Is Induced by FcγR Activation and Restricts the Replication of Human Immunodeficiency Virus Type 1 and Related Primate Lentiviruses in Human Macrophages

ABSTRACT Macrophages are major targets of human immunodeficiency virus type 1 (HIV-1). We have previously shown that aggregation of activating immunoglobulin G Fc receptors (FcγR) by immune complexes inhibits reverse transcript accumulation and integration of HIV-1 and related lentiviruses in monocyte-derived macrophages. Here, we show that FcγR-mediated restriction of HIV-1 is not due to enhanced degradation of incoming viral proteins or cDNA and is associated to the induction of the cyclin-dependent kinase inhibitor p21Cip1/WAF1 (p21). Small interfering RNA-mediated p21 knockdown rescued viral replication in FcγR-activated macrophages and enhanced HIV-1 infection in unstimulated macrophages by increasing reverse transcript and integrated DNA levels. p21 induction by other stimuli, such as phorbol myristate acetate and the histone deacetylase inhibitor MS-275, was also associated with preintegrative blocks of HIV-1 replication in macrophages. Binding of p21 to reverse transcription/preintegration complex-associated HIV-1 proteins was not detected in yeast two-hybrid, pulldown, or coimmunoprecipitation assays, suggesting that p21 may affect viral replication independently of a specific interaction with an HIV-1 component. Consistently, p21 silencing rescued viral replication from the FcγR-mediated restriction also in simian immunodeficiency virus SIVmac- and HIV-2-infected macrophages. Our results point to a role of p21 as an inhibitory factor of lentiviral infection in macrophages and to its implication in FcγR-mediated restriction.

The AP-1 Binding Sites Located in the pol Gene Intragenic Regulatory Region of HIV-1 Are Important for Viral Replication

Our laboratory has previously identified an important intragenic region in the human immunodeficiency virus type 1 (HIV-1) genome, whose complete functional unit is composed of the 5103 fragment, the DNaseI-hypersensitive site HS7 and the 5105 fragment. These fragments (5103 and 5105) both exhibit a phorbol 12-myristate 13-acetate (PMA)-inducible enhancer activity on the herpes simplex virus thymidine kinase promoter. Here, we characterized the three previously identified AP-1 binding sites of fragment 5103 by showing the PMA-inducible in vitro binding and in vivo recruitment of c-Fos, JunB and JunD to this fragment located at the end of the pol gene. Functional analyses demonstrated that the intragenic AP-1 binding sites are fully responsible for the PMA-dependent enhancer activity of fragment 5103. Moreover, infection of T-lymphoid Jurkat and promonocytic U937 cells with wild-type and mutant viruses demonstrated that mutations of the intragenic AP-1 sites individually or in combination altered HIV-1 replication. Importantly, mutations of the three intragenic AP-1 sites led to a decreased in vivo recruitment of RNA polymerase II to the viral promoter, strongly supporting that the deleterious effect of these mutations on viral replication occurs, at least partly, at the transcriptional level. Single-round infections of monocyte-derived macrophages confirmed the importance of intragenic AP-1 sites for HIV-1 infectivity.

The HIV-2 Vpx protein usurps the Cul4A-DDB1-DCAF1 ubiquitin ligase to overcome a post-entry block in macrophage infection

HIV and SIV genomes encode several auxiliary proteins which have increasingly shown their importance in the virus-host relationship. One of these proteins, Vpx, is unique to the HIV-2/SIVsm lineage and is critical for viral replication in macrophages. The functional basis for this requirement as well as the Vpx mode of action have remained a mystery for quite some time. We have previously shown that HIV-1 Vpr induces cell cycle arrest by recruiting the CUL4A-DDB1DCAF1 ubiquitin ligase. This presumably leads to the degradation of a host factor required for cell cycle progression into mitosis. Vpr and Vpx proteins are evolutionary related and show significant sequence similarity even though they are not functionally redundant. This prompted us to address whether Vpx, like Vpr, recruits the CUL4A-DDB1DCAF1 ubiquitin ligase and whether Vpx uses this functional property to enable efficient macrophage infection by HIV-2. Confirming this hypothesis, our results show that DCAF1 is a critical host effector of Vpx in its ability to mediate infection and long-term replication of HIV-2 in human macrophages. WT Vpx associates with the DDB1 component of the CUL4A ubiquitin ligase through DCAF1 binding. In contrast the Q76R Vpx mutant is unable to bind DCAF1 and recruit DDB1. When placed in the HIV-2 provirus, Q76R Vpx severely diminished viral replication in macrophages. Vpx is incorporated into newly formed virions, suggesting an early function in the next infection cycle. Accordingly, precluding Vpx present in the incoming virions from recruiting DCAF1 in target macrophages leads to a postentry block characterized by defective accumulation of HIV-2 reverse transcripts. In addition Vpx from SIVsm functionally complements Vpx-defective HIV-2 in a DCAF1-binding dependent manner. Altogether, our data point to a mechanism in which Vpx diverts the DCAF1 ubiquitin ligase to inactivate an evolutionary conserved factor (RF: restriction factor), which restricts infection of macrophages by HIV-2 and closely related simian viruses (Figure 1). This function of Vpx challenges the previous idea that Vpx complements the lack of cellular factors necessary for viral replication in macrophages. Vpx therefore acts similarly to other HIV auxiliary proteins (Vif, Vpu, Nef) known to inactivate cellular factors in order to create an advantageous environment for the virus.

The CDK inhibitor p21Cip1/WAF1 is induced by FcγR activation and restricts HIV-1 replication in human macrophages

Background Macrophages are major targets of human immunodeficiency virus type 1 (HIV-1) infection. We have previously shown that the engagement of the activating Fc receptors of the IgG (FcγR) by immune complexes (IC) suppresses the replication of HIV-1 and related lentiviruses in monocyte-derived macrophages, inhibiting both the accumulation of reverse transcripts and the proviral integration [1].