Stephen D. Barr

The interferon response inhibits HIV particle production by induction of TRIM22.

Treatment of human cells with Type 1 interferons restricts HIV replication. Here we report that the tripartite motif protein TRIM22 is a key mediator. We used transcriptional profiling to identify cellular genes that were induced by interferon treatment and identified TRIM22 as one of the most strongly up-regulated genes. We confirmed, as in previous studies, that TRIM22 over-expression inhibited HIV replication. To assess the role of TRIM22 expressed under natural inducing conditions, we compared the effects of interferon in cells depleted for TRIM22 using RNAi and found that HIV particle release was significantly increased in the knockdown, implying that TRIM22 acts as a natural antiviral effector. Further studies showed that TRIM22 inhibited budding of virus-like particles containing Gag only, indicating that Gag was the target of TRIM22. TRIM22 did not block the release of MLV or EIAV Gag particles. Inhibition was associated with diffuse cytoplasmic staining of HIV Gag rather than accumulation at the plasma membrane, suggesting TRIM22 disrupts proper trafficking. Mutational analyses of TRIM22 showed that the catalytic amino acids Cys15 and Cys18 of the RING domain are required for TRIM22 antiviral activity. These data disclose a pathway by which Type 1 interferons obstruct HIV replication.

Integration Targeting by Avian Sarcoma-Leukosis Virus and Human Immunodeficiency Virus in the Chicken Genome

ABSTRACT We have analyzed the placement of sites of integration of avian sarcoma-leukosis virus (ASLV) and human immunodeficiency virus (HIV) DNA in the draft chicken genome sequence, with the goals of assessing species-specific effects on integration and allowing comparison to the distribution of chicken endogenous retroviruses (ERVs). We infected chicken embryo fibroblasts (CEF) with ASLV or HIV and sequenced 863 junctions between host and viral DNA. The relationship with cellular gene activity was analyzed by transcriptional profiling of uninfected or ASLV-infected CEF cells. ASLV weakly favored integration in active transcription units (TUs), and HIV strongly favored active TUs, trends seen previously for integration in human cells. The ERVs, in contrast, accumulated mostly outside TUs, including ERVs related to ASLV. The minority of ERVs present within TUs were mainly in the antisense orientation; consequently, the viral splicing and polyadenylation signals would not disrupt cellular mRNA synthesis. In contrast, de novo ASLV integration sites within TUs showed no orientation bias. Comparing the distribution of de novo ASLV integration sites to ERVs indicated that purifying selection against gene disruption, and not initial integration targeting, probably determined the ERV distribution. Further analysis indicated that ERVs in humans, mice, and rats showed similar distributions, suggesting purifying selection dictated their distributions as well.

Human HERC5 restricts an early stage of HIV-1 assembly by a mechanism correlating with the ISGylation of Gag

BackgroundThe identification and characterization of several interferon (IFN)-induced cellular HIV-1 restriction factors, defined as host cellular proteins or factors that restrict or inhibit the HIV-1 life cycle, have provided insight into the IFN response towards HIV-1 infection and identified new therapeutic targets for HIV-1 infection. To further characterize the mechanism underlying restriction of the late stages of HIV-1 replication, we assessed the ability of IFNbeta-induced genes to restrict HIV-1 Gag particle production and have identified a potentially novel host factor called HECT domain and RCC1-like domain-containing protein 5 (HERC5) that blocks a unique late stage of the HIV-1 life cycle.ResultsHERC5 inhibited the replication of HIV-1 over multiple rounds of infection and was found to target a late stage of HIV-1 particle production. The E3 ligase activity of HERC5 was required for blocking HIV-1 Gag particle production and correlated with the post-translational modification of Gag with ISG15. HERC5 interacted with HIV-1 Gag and did not alter trafficking of HIV-1 Gag to the plasma membrane. Electron microscopy revealed that the assembly of HIV-1 Gag particles was arrested at the plasma membrane, at an early stage of assembly. The mechanism of HERC5-induced restriction of HIV-1 particle production is distinct from the mechanism underlying HIV-1 restriction by the expression of ISG15 alone, which acts at a later step in particle release. Moreover, HERC5 restricted murine leukemia virus (MLV) Gag particle production, showing that HERC5 is effective in restricting Gag particle production of an evolutionarily divergent retrovirus.ConclusionsHERC5 represents a potential new host factor that blocks an early stage of retroviral Gag particle assembly. With no apparent HIV-1 protein that directly counteracts it, HERC5 may represent a new candidate for HIV/AIDS therapy.

TRIM22: A Diverse and Dynamic Antiviral Protein.

The tripartite motif (TRIM) family of proteins is an evolutionarily ancient group of proteins with homologues identified in both invertebrate and vertebrate species. Human TRIM22 is one such protein that has a dynamic evolutionary history that includes gene expansion, gene loss, and strong signatures of positive selection. To date, TRIM22 has been shown to restrict the replication of a number of viruses, including encephalomyocarditis virus (EMCV), hepatitis B virus (HBV), and human immunodeficiency virus type 1 (HIV-1). In addition, TRIM22 has also been implicated in cellular differentiation and proliferation and may play a role in certain cancers and autoimmune diseases. This comprehensive paper summarizes our current understanding of TRIM22 structure and function.

Identification of HIV integration sites in infected host genomic DNA.

The integration of the Human Immunodeficiency Virus (HIV) genetic information into the host genome is fundamental for its replication and long-term persistence in the host. Isolating and characterizing the integration sites can be useful for obtaining data such as identifying the specific genomic location of integration or understanding the forces dictating HIV integration site selection. The methods outlined in this article describe a highly efficient and precise technique for identifying HIV integration sites in the host genome on a small scale using molecular cloning techniques and standard sequencing or on a massive scale using 454 pyrosequencing.

Interferon-induced HERC5 is evolving under positive selection and inhibits HIV-1 particle production by a novel mechanism targeting Rev/RRE-dependent RNA nuclear export

BackgroundType I interferon (IFN) inhibits virus replication by activating multiple antiviral mechanisms and pathways. It has long been recognized that type I IFNs can potently block HIV-1 replication in vitro; as such, HIV-1 has been used as a system to identify and characterize IFN-induced antiviral proteins responsible for this block. IFN-induced HERC5 contains an amino-terminal Regulator of Chromosome Condensation 1 (RCC1)-like domain and a carboxyl-terminal Homologous to the E6-AP Carboxyl Terminus (HECT) domain. HERC5 is the main cellular E3 ligase that conjugates the IFN-induced protein ISG15 to proteins. This E3 ligase activity was previously shown to inhibit the replication of evolutionarily diverse viruses, including HIV-1. The contribution of the RCC1-like domain to the antiviral activity of HERC5 was previously unknown.ResultsIn this study, we showed that HERC5 inhibits HIV-1 particle production by a second distinct mechanism that targets the nuclear export of Rev/RRE-dependent RNA. Unexpectedly, the E3 ligase activity of HERC5 was not required for this inhibition. Instead, this activity required the amino-terminal RCC1-like domain of HERC5. Inhibition correlated with a reduction in intracellular RanGTP protein levels and/or the ability of RanGTP to interact with RanBP1. Inhibition also correlated with altered subcellular localization of HIV-1 Rev. In addition, we demonstrated that positive evolutionary selection is operating on HERC5. We identified a region in the RCC1-like domain that exhibits an exceptionally high probability of having evolved under positive selection and showed that this region is required for HERC5-mediated inhibition of nuclear export.ConclusionsWe have identified a second distinct mechanism by which HERC5 inhibits HIV-1 replication and demonstrate that HERC5 is evolving under strong positive selection. Together, our findings contribute to a growing body of evidence suggesting that HERC5 is a novel host restriction factor.

Inhibition or deficiency of cathepsin B leads defects in HIV-1 Gag pseudoparticle release in macrophages and HEK293T cells ☆

Human immunodeficiency virus type 1 (HIV-1) egresses from infected cells through utilizing the host membrane budding mechanisms. Assembly of HIV-1 Gag particles occurs on membranes where the Gag multimers subsequently bud off and form enveloped viral particles. In certain cell types such as macrophages, HIV-1 Gag particles have shown to be released into intracellular virus containing compartments (VCC) such as late endosomes, multivesicular bodies (MVBs) or invaginated plasma membrane pockets. Here, we showed that macrophages or HEK293T cells treated with the cathepsin B (CTSB)-specific inhibitor CA-074Me or cells deficient in CTSB failed to release HIV-1 Gag pseudoparticles into the extracellular environment. Based on immunofluorescence and electron microscopy, these cells retained the pseudoparticles in heterogeneous intracellular VCC. CA-074Me was also able to inhibit propagation of two enveloped viruses, herpes simplex virus and influenza A virus, but not non-enveloped enterovirus. These results suggest that CTSB is required for the efficient release of HIV-1 Gag pseudoparticles and targeting CTSB can be a new therapeutic strategy for inhibiting egress of HIV-1 and other enveloped viruses.

In Silico Analysis of Functional Single Nucleotide Polymorphisms in the Human TRIM22 Gene

Tripartite motif protein 22 (TRIM22) is an evolutionarily ancient protein that plays an integral role in the host innate immune response to viruses. The antiviral TRIM22 protein has been shown to inhibit the replication of a number of viruses, including HIV-1, hepatitis B, and influenza A. TRIM22 expression has also been associated with multiple sclerosis, cancer, and autoimmune disease. In this study, multiple in silico computational methods were used to identify non-synonymous or amino acid-changing SNPs (nsSNP) that are deleterious to TRIM22 structure and/or function. A sequence homology-based approach was adopted for screening nsSNPs in TRIM22, including six different in silico prediction algorithms and evolutionary conservation data from the ConSurf web server. In total, 14 high-risk nsSNPs were identified in TRIM22, most of which are located in a protein interaction module called the B30.2 domain. Additionally, 9 of the top high-risk nsSNPs altered the putative structure of TRIM22s B30.2 domain, particularly in the surface-exposed v2 and v3 regions. These same regions are critical for retroviral restriction by the closely-related TRIM5α protein. A number of putative structural and functional residues, including several sites that undergo post-translational modification, were also identified in TRIM22. This study is the first extensive in silico analysis of the highly polymorphic TRIM22 gene and will be a valuable resource for future targeted mechanistic and population-based studies.

Characterization of the 55-Residue Protein Encoded by the 9S E1A mRNA of Species C Adenovirus

ABSTRACT Early region 1A (E1A) of human adenovirus (HAdV) has been the focus of over 30 years of investigation and is required for the oncogenic capacity of HAdV in rodents. Alternative splicing of the E1A transcript generates mRNAs encoding multiple E1A proteins. The 55-residue (55R) E1A protein, which is encoded by the 9S mRNA, is particularly interesting due to the unique properties it displays relative to all other E1A isoforms. 55R E1A does not contain any of the conserved regions (CRs) present in the other E1A isoforms. The C-terminal region of the 55R E1A protein contains a unique sequence compared to all other E1A isoforms, which results from a frameshift generated by alternative splicing. The 55R E1A protein is thought to be produced preferentially at the late stages of infection. Here we report the first study to directly investigate the function of the species C HAdV 55R E1A protein during infection. Polyclonal rabbit antibodies (Abs) have been generated that are capable of immunoprecipitating HAdV-2 55R E1A. These Abs can also detect HAdV-2 55R E1A by immunoblotting and indirect immunofluorescence assay. These studies indicate that 55R E1A is expressed late and is localized to the cytoplasm and to the nucleus. 55R E1A was able to activate the expression of viral genes during infection and could also promote productive replication of species C HAdV. 55R E1A was also found to interact with the S8 component of the proteasome, and knockdown of S8 was detrimental to viral replication dependent on 55R E1A.

Lentivector Integration Sites in Ependymal Cells From a Model of Metachromatic Leukodystrophy: Non-B DNA as a New Factor Influencing Integration

The blood–brain barrier controls the passage of molecules from the blood into the central nervous system (CNS) and is a major challenge for treatment of neurological diseases. Metachromatic leukodystrophy is a neurodegenerative lysosomal storage disease caused by loss of arylsulfatase A (ARSA) activity. Gene therapy via intraventricular injection of a lentiviral vector is a potential approach to rapidly and permanently deliver therapeutic levels of ARSA to the CNS. We present the distribution of integration sites of a lentiviral vector encoding human ARSA (LV-ARSA) in murine brain choroid plexus and ependymal cells, administered via a single intracranial injection into the CNS. LV-ARSA did not exhibit a strong preference for integration in or near actively transcribed genes, but exhibited a strong preference for integration in or near satellite DNA. We identified several genomic hotspots for LV-ARSA integration and identified a consensus target site sequence characterized by two G-quadruplex-forming motifs flanking the integration site. In addition, our analysis identified several other non-B DNA motifs as new factors that potentially influence lentivirus integration, including human immunodeficiency virus type-1 in human cells. Together, our data demonstrate a clinically favorable integration site profile in the murine brain and identify non-B DNA as a potential new host factor that influences lentiviral integration in murine and human cells.