Immunodeficiency virus rev trans-activator modulates the expression of the viral regulatory genes

The pathogenic human retrovirus human immunodeficiency virus type 1 (HIV-1) encodes two trans-acting nuclear proteins, tat and rev, whose functional expression is essential for viral replication in vitro. The tat protein greatly enhances the expression of both structural and regulatory genes of HIV-1 (linked to the viral long-terminal-repeat promoter element), whereas the rev gene product (previously termed art or trs) has only been shown to be required for the synthesis of structural proteins. Here, we demonstrate that rev also moderates the expression of regulatory genes of HIV-1. It decreases the expression of messenger RNAs that encode the full-length form of the viral tat gene product or the rev protein itself, and induces the synthesis of a previously unreported, truncated tat protein. These actions of rev are mediated by a dramatic shift in the ratio of spliced to unspliced cytoplasmic HIV-1 mRNA. Therefore rev not only activates the synthesis of the viral structural proteins, but also modulates the level and quality of HIV-1 regulatory gene expression.     

Inhibition of HIV replication by pokeweed antiviral protein targeted to CD4+ cells by monoclonal antibodies

Functional impairment and selective depletion of CD4+ T cells, the hallmark of AIDS, are at least partly caused by human immunodeficiency virus (HIV-1) type 1 binding to the CD4 molecule and infecting CD4+ cells. It may, therefore, be of therapeutic value to target an antiviral agent to CD4+ cells to prevent infection and to inhibit HIV-1 production in patients' CD4+ cells which contain proviral DNA. We report here that HIV-1 replication in normal primary CD4+ T cells can be inhibited by pokeweed antiviral protein, a plant protein of relative molecular mass 30,000, which inhibits replication of certain plant RNA viruses, and of herpes simplex virus, poliovirus and influenza virus. Targeting pokeweed antiviral protein to CD4+ T cells by conjugating it to monoclonal antibodies reactive with CD5, CD7 or CD4 expressed on CD4+ cells, increased its anti-HIV potency up to 1,000-fold. HIV-1 replication is inhibited at picomolar concentrations of conjugates of pokeweed antiviral protein and monoclonal antibodies, which do not inhibit proliferation of normal CD4+ T cells or CD4-dependent responses. These conjugates inhibit HIV-1 protein synthesis and also strongly inhibit HIV-1 production in activated CD4+ T cells from infected patients.     

Vpx relieves inhibition of HIV-1 infection of macrophages mediated by the SAMHD1 protein

Macrophages and dendritic cells have key roles in viral infections, providing virus reservoirs that frequently resist antiviral therapies and linking innate virus detection to antiviral adaptive immune responses. Human immunodeficiency virus 1 (HIV-1) fails to transduce dendritic cells and has a reduced ability to transduce macrophages, due to an as yet uncharacterized mechanism that inhibits infection by interfering with efficient synthesis of viral complementary DNA. In contrast, HIV-2 and related simian immunodeficiency viruses (SIVsm/mac) transduce myeloid cells efficiently owing to their virion-associated Vpx accessory proteins, which counteract the restrictive mechanism. Here we show that the inhibition of HIV-1 infection in macrophages involves the cellular SAM domain HD domain-containing protein 1 (SAMHD1). Vpx relieves the inhibition of lentivirus infection in macrophages by loading SAMHD1 onto the CRL4(DCAF1) E3 ubiquitin ligase, leading to highly efficient proteasome-dependent degradation of the protein. Mutations in SAMHD1 cause Aicardi-Goutières syndrome, a disease that produces a phenotype that mimics the effects of a congenital viral infection. Failure to dispose of endogenous nucleic acid debris in Aicardi-Goutières syndrome results in inappropriate triggering of innate immune responses via cytosolic nucleic acids sensors. Thus, our findings show that macrophages are defended from HIV-1 infection by a mechanism that prevents an unwanted interferon response triggered by self nucleic acids, and uncover an intricate relationship between innate immune mechanisms that control response to self and to retroviral pathogens.     

Identification of a protein encoded by the vpu gene of HIV-1

Human immunodeficiency virus 1 (HIV-1) is the aetiological agent of AIDS. The virus establishes lytic, latent and non-cytopathic productive infection in cells in culture. The complexity of virus-host cell interaction is reflected in the complex organization of the viral genome. In addition to the genes that encode the virion capsid and envelope proteins and the enzymes required for proviral synthesis and integration common to all retroviruses, HIV-1 is known to encode at least four additional proteins that regulate virus replication, the tat, art, sor and 3' orf proteins, as well as a protein of unknown function from the open reading frame called R. Close examination of the nucleic acid sequences of the genomes of multiple HIV isolates raised the possibility that the virus encodes a previously undetected additional protein. Here we report that HIV-1 encodes a ninth protein and that antibodies to this protein are detected in the sera of people infected with HIV-1. This protein distinguishes HIV-1 isolates from the other human and simian immunodeficiency viruses (HIV-2 and SIV) that do not have the capacity to encode a similar protein.     

Structure of the DNA deaminase domain of the HIV-1 restriction factor APOBEC3G

The human APOBEC3G (apolipoprotein B messenger-RNA-editing enzyme, catalytic polypeptide-like 3G) protein is a single-strand DNA deaminase that inhibits the replication of human immunodeficiency virus-1 (HIV-1), other retroviruses and retrotransposons. APOBEC3G anti-viral activity is circumvented by most retroelements, such as through degradation by HIV-1 Vif. APOBEC3G is a member of a family of polynucleotide cytosine deaminases, several of which also target distinct physiological substrates. For instance, APOBEC1 edits APOB mRNA and AID deaminates antibody gene DNA. Although structures of other family members exist, none of these proteins has elicited polynucleotide cytosine deaminase or anti-viral activity. Here we report a solution structure of the human APOBEC3G catalytic domain. Five alpha-helices, including two that form the zinc-coordinating active site, are arranged over a hydrophobic platform consisting of five beta-strands. NMR DNA titration experiments, computational modelling, phylogenetic conservation and Escherichia coli-based activity assays combine to suggest a DNA-binding model in which a brim of positively charged residues positions the target cytosine for catalysis. The structure of the APOBEC3G catalytic domain will help us to understand functions of other family members and interactions that occur with pathogenic proteins such as HIV-1 Vif.     

Drosophila RNAi screen identifies host genes important for influenza virus replication

All viruses rely on host cell proteins and their associated mechanisms to complete the viral life cycle. Identifying the host molecules that participate in each step of virus replication could provide valuable new targets for antiviral therapy, but this goal may take several decades to achieve with conventional forward genetic screening methods and mammalian cell cultures. Here we describe a novel genome-wide RNA interference (RNAi) screen in Drosophila that can be used to identify host genes important for influenza virus replication. After modifying influenza virus to allow infection of Drosophila cells and detection of influenza virus gene expression, we tested an RNAi library against 13,071 genes (90% of the Drosophila genome), identifying over 100 for which suppression in Drosophila cells significantly inhibited or stimulated reporter gene (Renilla luciferase) expression from an influenza-virus-derived vector. The relevance of these findings to influenza virus infection of mammalian cells is illustrated for a subset of the Drosophila genes identified; that is, for three implicated Drosophila genes, the corresponding human homologues ATP6V0D1, COX6A1 and NXF1 are shown to have key functions in the replication of H5N1 and H1N1 influenza A viruses, but not vesicular stomatitis virus or vaccinia virus, in human HEK 293 cells. Thus, we have demonstrated the feasibility of using genome-wide RNAi screens in Drosophila to identify previously unrecognized host proteins that are required for influenza virus replication. This could accelerate the development of new classes of antiviral drugs for chemoprophylaxis and treatment, which are urgently needed given the obstacles to rapid development of an effective vaccine against pandemic influenza and the probable emergence of strains resistant to available drugs.     

Modeling the intracellular dynamics for Vif-APO mediated HIV-1 virus infection

The viral infectivity factor (Vif) was found to be essential for controlling HIV-1 virus infectivity. It targets cellular antiviral proteins in APOBEC family (APO) to trigger its fast degradation and inhibits APO packaging into nascent virion. In the present study, we propose a mathematical model to quantitatively study the intracellular dynamics of these typical virus-host interactions. Four sets of published experimental data were compared with simulation results to justify the model. Systematic parameter sensitivity and perturbation analysis showed that parameters related to APO are crucial to the infectivity of newly synthesized HIV-1 virus. Interestingly, we discovered that the synthesis rate of the viral structure protein Gag and the required number per nascent virion are optimized to achieve high virion production with minimal level of packaged APO, and large portion of model parameters are beneficial to virus only within a relatively small range. Furthermore, minor variations in several parameters related to viral protein Tat, the activator of viral RNA synthesis, were found to induce switch-like behaviors on both incorporated Vif and APO. These findings may provide new insights for understanding the high mutation rate of HIV-1 virus and its latency, as well as help identify key targets for therapeutic design.     

APOBEC3 inhibits mouse mammary tumour virus replication in vivo

Genomes of all mammals encode apobec3 genes, which are thought to have a function in intrinsic cellular immunity to several viruses including human immunodeficiency virus type 1 (HIV-1). APOBEC3 (A3) proteins are packaged into virions and inhibit retroviral replication in newly infected cells, at least in part by deaminating cytidines on the negative strand DNA intermediates. However, the role of A3 in innate resistance to mouse retroviruses is not understood. Here we show that A3 functions during retroviral infection in vivo and provides partial protection to mice against infection with mouse mammary tumour virus (MMTV). Both mouse A3 and human A3G proteins interacted with the MMTV nucleocapsid in an RNA-dependent fashion and were packaged into virions. In addition, mouse A3-containing and human A3G-containing virions showed a marked decrease in titre. Last, A3(-/-) mice were more susceptible to MMTV infection, because virus spread was more rapid and extensive than in their wild-type littermates.     

Prevention of HIV-2 and SIVsm infection by passive immunization in cynomolgus monkeys.

Infection of macaques with simian immunodeficiency virus (SIV) and human immunodeficiency virus type 2 (HIV-2) are useful models for studies of immunotherapy and vaccination against HIV as well as for testing of antiviral drugs. Vaccine research showing protective immunity in immunized monkeys has indicated that it will be possible to develop a vaccine for prevention of human HIV infection, although many hurdles remain. The design of an HIV vaccine would be helped if the basis of the protective immunity could be elucidated. Passive immune prophylaxis offers a means to determine the relative role of antibodies in protection against infection. We have studied whether a transfer of antibodies can prevent HIV-2 and SIVsm (SIV of sooty mangabey origin) infection in cynomolgus monkeys. Sera with high antibody titres were collected, heat-treated and injected into naive animals 6 h before challenge with 10-100 monkey-infectious doses of live homologous virus. All control animals treated with normal monkey serum (n = 6) or no serum (n = 39) became infected by the challenge virus, whereas five out of seven animals pretreated with antibody-containing serum at a dose of 9 ml kg-1 resisted infection. Thus passively transferred antibodies can protect against a low-dose lentivirus challenge in a nonhuman primate.     

The gene product Murr1 restricts HIV-1 replication in resting CD4+ lymphocytes

Although human immunodeficiency virus-1 (HIV-1) infects quiescent and proliferating CD4+ lymphocytes, the virus replicates poorly in resting T cells. Factors that block viral replication in these cells might help to prolong the asymptomatic phase of HIV infection; however, the molecular mechanisms that control this process are not fully understood. Here we show that Murr1, a gene product known previously for its involvement in copper regulation, inhibits HIV-1 growth in unstimulated CD4+ T cells. This inhibition was mediated in part through its ability to inhibit basal and cytokine-stimulated nuclear factor (NF)-kappaB activity. Knockdown of Murr1 increased NF-kappaB activity and decreased IkappaB-alpha concentrations by facilitating phospho-IkappaB-alpha degradation by the proteasome. Murr1 was detected in CD4+ T cells, and RNA-mediated interference of Murr1 in primary resting CD4+ lymphocytes increased HIV-1 replication. Through its effects on the proteasome, Murr1 acts as a genetic restriction factor that inhibits HIV-1 replication in lymphocytes, which could contribute to the regulation of asymptomatic HIV infection and the progression of AIDS.     

Cyclophilin A retrotransposition into TRIM5 explains owl monkey resistance to HIV-1

In Old World primates, TRIM5-alpha confers a potent block to human immunodeficiency virus type 1 (HIV-1) infection that acts after virus entry into cells. Cyclophilin A (CypA) binding to viral capsid protects HIV-1 from a similar activity in human cells. Among New World primates, only owl monkeys exhibit post-entry restriction of HIV-1 (ref. 1). Paradoxically, the barrier to HIV-1 in owl monkey cells is released by capsid mutants or drugs that disrupt capsid interaction with CypA. Here we show that knockdown of owl monkey CypA by RNA interference (RNAi) correlates with suppression of anti-HIV-1 activity. However, reintroduction of CypA protein to RNAi-treated cells did not restore antiviral activity. A search for additional RNAi targets unearthed TRIMCyp, an RNAi-responsive messenger RNA encoding a TRIM5-CypA fusion protein. TRIMCyp accounts for post-entry restriction of HIV-1 in owl monkeys and blocks HIV-1 infection when transferred to otherwise infectable human or rat cells. It seems that TRIMCyp arose after the divergence of New and Old World primates when a LINE-1 retrotransposon catalysed the insertion of a CypA complementary DNA into the TRIM5 locus. This is the first vertebrate example of a chimaeric gene generated by this mechanism of exon shuffling.