Strand Transfer and Elongation of HIV-1 Reverse Transcription Is Facilitated by Cell Factors In Vitro

Recent work suggests a role for multiple host factors in facilitating HIV-1 reverse transcription. Previously, we identified a cellular activity which increases the efficiency of HIV-1 reverse transcription in vitro. Here, we describe aspects of the activity which shed light on its function. The cellular factor did not affect synthesis of strong-stop DNA but did improve downstream DNA synthesis. The stimulatory activity was isolated by gel filtration in a single fraction of the exclusion volume. Velocity-gradient purified HIV-1, which was free of detectable RNase activity, showed poor reverse transcription efficiency but was strongly stimulated by partially purified cell proteins. Hence, the cell factor(s) did not inactivate an RNase activity that might degrade the viral genomic RNA and block completion of reverse transcription. Instead, the cell factor(s) enhanced first strand transfer and synthesis of late reverse transcription suggesting it stabilized the reverse transcription complex. The factor did not affect lysis of HIV-1 by Triton X-100 in the endogenous reverse transcription (ERT) system, and ERT reactions with HIV-1 containing capsid mutations, which varied the biochemical stability of viral core structures and impeded reverse transcription in cells, showed no difference in the ability to be stimulated by the cell factor(s) suggesting a lack of involvement of the capsid in the in vitro assay. In addition, reverse transcription products were found to be resistant to exogenous DNase I activity when the active fraction was present in the ERT assay. These results indicate that the cell factor(s) may improve reverse transcription by facilitating DNA strand transfer and DNA synthesis. It also had a protective function for the reverse transcription products, but it is unclear if this is related to improved DNA synthesis

Cloning and Characterization of the Antiviral Activity of Feline Tetherin/BST-2

Human Tetherin/BST-2 has recently been identified as a cellular antiviral factor that blocks the release of various enveloped viruses. In this study, we cloned a cDNA fragment encoding a feline homolog of Tetherin/BST-2 and characterized the protein product. The degree of amino acid sequence identity between human Tetherin/BST-2 and the feline homolog was 44.4%. Similar to human Tetherin/BST-2, the expression of feline Tetherin/BST-2 mRNA was inducible by type I interferon (IFN). Exogenous expression of feline Tetherin/BST-2 efficiently inhibited the release of feline endogenous retrovirus RD-114. The extracellular domain of feline Tetherin/BST-2 has two putative N-linked glycosylation sites, N79 and N119. Complete loss of N-linked glycosylation by introduction of mutations into both sites resulted in almost complete abolition of its antiviral activity. In addition, feline Tetherin/BST-2 was insensitive to antagonism by HIV-1 Vpu, although the antiviral activity of human Tetherin/BST-2 was antagonized by HIV-1 Vpu. Our data suggest that feline Tetherin/BST-2 functions as a part of IFN-induced innate immunity against virus infection and that the induction of feline Tetherin/BST-2 in vivo may be effective as a novel antiviral strategy for viral infection

HIV research in Australia: linking basic research findings with clinical and public health outcomes

Despite a population of only 20 million and sustained low prevalence of HIV infection in Australia, Australian researchers have provided many substantial original findings to the fields of HIV pathogenesis, treatment and prevention. More recently, Australian clinicians and scientists have turned their attention to assisting other countries in developing effective responses, particularly within the Asia-Pacific region. It is therefore fitting that the 4th International AIDS Society (IAS) Conference on HIV Pathogenesis, Treatment and Prevention will be held in Sydney in July 2007. The meeting is expected to attract over 5000 participants and will have a dynamic and innovative programme within the three major themes of HIV basic science, clinical research and biomedical prevention

Potent Inhibition of HIV-1 Replication by a Tat Mutant

Herein we describe a mutant of the two-exon HIV-1 Tat protein, termed Nullbasic, that potently inhibits multiple steps of the HIV-1 replication cycle. Nullbasic was created by replacing the entire arginine-rich basic domain of wild type Tat with glycine/alanine residues. Like similarly mutated one-exon Tat mutants, Nullbasic exhibited transdominant negative effects on Tat-dependent transactivation. However, unlike previously reported mutants, we discovered that Nullbasic also strongly suppressed the expression of unspliced and singly-spliced viral mRNA, an activity likely caused by redistribution and thus functional inhibition of HIV-1 Rev. Furthermore, HIV-1 virion particles produced by cells expressing Nullbasic had severely reduced infectivity, a defect attributable to a reduced ability of the virions to undergo reverse transcription. Combination of these inhibitory effects on transactivation, Rev-dependent mRNA transport and reverse transcription meant that permissive cells constitutively expressing Nullbasic were highly resistant to a spreading infection by HIV-1. Nullbasic and its activities thus provide potential insights into the development of potent antiviral therapeutics that target multiple stages of HIV-1 infection

A Re-Examination of Global Suppression of RNA Interference by HIV-1

The nature of the interaction between replicating HIV-1 and the cellular RNAi pathway has been controversial, but it is clear that it can be complex and multifaceted. It has been proposed that the interaction is bi-directional, whereby cellular silencing pathways can restrict HIV-1 replication, and in turn, HIV-1 can suppress silencing pathways. Overall suppression of RNAi has been suggested to occur via direct binding and inhibition of Dicer by the HIV-1 Tat protein or through sequestration of TRBP, a Dicer co-factor, by the structured TAR element of HIV-1 transcripts. The role of Tat as an inhibitor of Dicer has been questioned and our results support and extend the conclusion that Tat does not inhibit RNAi that is mediated by either exogenous or endogenous miRNAs. Similarly, we find no suppression of silencing pathways in cells with replicating virus, suggesting that viral products such as the TAR RNA elements also do not reduce the efficacy of cellular RNA silencing. However, knockdown of Dicer does allow increased viral replication and this occurs at a post-transcriptional level. These results support the idea that although individual miRNAs can act to restrict HIV-1 replication, the virus does not counter these effects through a global suppression of RNAi synthesis or processing

Modifications in host cell cytoskeleton structure and function mediated by intracellular HIV-1 Tat protein are greatly dependent on the second coding exon

Supplementary Data are available at NAR OnlineThe human immunodeficiency virus type 1 (HIV-1) regulator Tat is essential for viral replication because it achieves complete elongation of viral transcripts. Tat can be released to the extracellular space and taken up by adjacent cells, exerting profound cytoskeleton rearrangements that lead to apoptosis. In contrast, intracellular Tat has been described as protector from apoptosis. Tat gene is composed by two coding exons that yield a protein of 101 amino acids (aa). First exon (1–72aa) is sufficient for viral transcript elongation and second exon (73–101 aa) appears to contribute to non-transcriptional functions. We observed that Jurkat cells stably expressing intracellular Tat101 showed gene expression deregulation 4-fold higher than cells expressing Tat72. Functional experiments were performed to evaluate the effect of this deregulation. First, NF-iB-, NF-AT- and Sp1-dependent transcriptional activities were greatly enhanced in Jurkat-Tat101, whereas Tat72 induced milder but efficient activation. Second, cytoskeleton-related functions as cell morphology, proliferation, chemotaxis, polarization and actin polymerization were deeply altered in Jurkat- Tat101, but not in Jurkat-Tat72. Finally, expression of several cell surface receptors was dramatically impaired by intracellular Tat101 but not by Tat72. Consequently, these modifications were greatly dependent on Tat second exon and they could be related to the anergy observed in HIV-1-infected T cells.Plan Nacional del SIDA (MVI 1434/05–5), FIPSE 36584/ 06 and 36633/07, VIRHORST Network from Comunidad de Madrid (Spain), FIS PI040614 and PI0808752, ISCIII-RETIC RD06/0006, EUROPRISE Network of Excellence of the EU (Grant no. LSHP CT-2006- 037611), and BIO2008-04384 from the Ministerio de Ciencia e Innovacio´ n, Espan˜ a. Funding for open access charge: Instituto de Salud Carlos III, Ministry of Science and Technology, Spain.Peer reviewe

The Herpesvirus Associated Ubiquitin Specific Protease, USP7, Is a Negative Regulator of PML Proteins and PML Nuclear Bodies

The PML tumor suppressor is the founding component of the multiprotein nuclear structures known as PML nuclear bodies (PML-NBs), which control several cellular functions including apoptosis and antiviral effects. The ubiquitin specific protease USP7 (also called HAUSP) is known to associate with PML-NBs and to be a tight binding partner of two herpesvirus proteins that disrupt PML NBs. Here we investigated whether USP7 itself regulates PML-NBs. Silencing of USP7 was found to increase the number of PML-NBs, to increase the levels of PML protein and to inhibit PML polyubiquitylation in nasopharyngeal carcinoma cells. This effect of USP7 was independent of p53 as PML loss was observed in p53-null cells. PML-NBs disruption was induced by USP7 overexpression independently of its catalytic activity and was induced by either of the protein interaction domains of USP7, each of which localized to PML-NBs. USP7 also disrupted NBs formed from some single PML isoforms, most notably isoforms I and IV. CK2α and RNF4, which are known regulators of PML, were dispensable for USP7-associated PML-NB disruption. The results are consistent with a novel model of PML regulation where a deubiquitylase disrupts PML-NBs through recruitment of another cellular protein(s) to PML NBs, independently of its catalytic activity

Control of Stochastic Gene Expression by Host Factors at the HIV Promoter

The HIV promoter within the viral long terminal repeat (LTR) orchestrates many aspects of the viral life cycle, from the dynamics of viral gene expression and replication to the establishment of a latent state. In particular, after viral integration into the host genome, stochastic fluctuations in viral gene expression amplified by the Tat positive feedback loop can contribute to the formation of either a productive, transactivated state or an inactive state. In a significant fraction of cells harboring an integrated copy of the HIV-1 model provirus (LTR-GFP-IRES-Tat), this bimodal gene expression profile is dynamic, as cells spontaneously and continuously flip between active (Bright) and inactive (Off) expression modes. Furthermore, these switching dynamics may contribute to the establishment and maintenance of proviral latency, because after viral integration long delays in gene expression can occur before viral transactivation. The HIV-1 promoter contains cis-acting Sp1 and NF-κB elements that regulate gene expression via the recruitment of both activating and repressing complexes. We hypothesized that interplay in the recruitment of such positive and negative factors could modulate the stability of the Bright and Off modes and thereby alter the sensitivity of viral gene expression to stochastic fluctuations in the Tat feedback loop. Using model lentivirus variants with mutations introduced in the Sp1 and NF-κB elements, we employed flow cytometry, mRNA quantification, pharmacological perturbations, and chromatin immunoprecipitation to reveal significant functional differences in contributions of each site to viral gene regulation. Specifically, the Sp1 sites apparently stabilize both the Bright and the Off states, such that their mutation promotes noisy gene expression and reduction in the regulation of histone acetylation and deacetylation. Furthermore, the NF-κB sites exhibit distinct properties, with κB site I serving a stronger activating role than κB site II. Moreover, Sp1 site III plays a particularly important role in the recruitment of both p300 and RelA to the promoter. Finally, analysis of 362 clonal cell populations infected with the viral variants revealed that mutations in any of the Sp1 sites yield a 6-fold higher frequency of clonal bifurcation compared to that of the wild-type promoter. Thus, each Sp1 and NF-κB site differentially contributes to the regulation of viral gene expression, and Sp1 sites functionally “dampen” transcriptional noise and thereby modulate the frequency and maintenance of this model of viral latency. These results may have biomedical implications for the treatment of HIV latency

Rationally Designed Interfacial Peptides Are Efficient In Vitro Inhibitors of HIV-1 Capsid Assembly with Antiviral Activity

Virus capsid assembly constitutes an attractive target for the development of antiviral therapies; a few experimental inhibitors of this process for HIV-1 and other viruses have been identified by screening compounds or by selection from chemical libraries. As a different, novel approach we have undertaken the rational design of peptides that could act as competitive assembly inhibitors by mimicking capsid structural elements involved in intersubunit interfaces. Several discrete interfaces involved in formation of the mature HIV-1 capsid through polymerization of the capsid protein CA were targeted. We had previously designed a peptide, CAC1, that represents CA helix 9 (a major part of the dimerization interface) and binds the CA C-terminal domain in solution. Here we have mapped the binding site of CAC1, and shown that it substantially overlaps with the CA dimerization interface. We have also rationally modified CAC1 to increase its solubility and CA-binding affinity, and designed four additional peptides that represent CA helical segments involved in other CA interfaces. We found that peptides CAC1, its derivative CAC1M, and H8 (representing CA helix 8) were able to efficiently inhibit the in vitro assembly of the mature HIV-1 capsid. Cocktails of several peptides, including CAC1 or CAC1M plus H8 or CAI (a previously discovered inhibitor of CA polymerization), or CAC1M+H8+CAI, also abolished capsid assembly, even when every peptide was used at lower, sub-inhibitory doses. To provide a preliminary proof that these designed capsid assembly inhibitors could eventually serve as lead compounds for development of anti-HIV-1 agents, they were transported into cultured cells using a cell-penetrating peptide, and tested for antiviral activity. Peptide cocktails that drastically inhibited capsid assembly in vitro were also able to efficiently inhibit HIV-1 infection ex vivo. This study validates a novel, entirely rational approach for the design of capsid assembly interfacial inhibitors that show antiviral activity