Steady increase in cellular HIV-1 load during the asymptomatic phase of untreated infection despite stable plasma viremia.

Objective: To compare the dynamics of HIV-1 molecular markers in peripheral blood mononuclear cells (PBMCs) and in plasma during the asymptomatic phase of untreated HIV-1 infection.

Design And Methods: Using seminested real-time PCR assays, we measured the levels of HIV-1 proviral (pr) DNA, unspliced (us) RNA, and multiply spliced RNA in the PBMCs of 10 untreated HIV-1-infected men at multiple time points during the asymptomatic phase of infection and compared the longitudinal trends of these markers with those of viral RNA in plasma.

Results: Whereas plasma RNA levels did not significantly change in any of the individuals, levels of usRNA significantly increased with time in six out of 10 persons, and levels of prDNA in four.

Anticipating and blocking HIV-1 escape by second generation antiviral shRNAs.

Background: RNA interference (RNAi) is an evolutionary conserved gene silencing mechanism that mediates the sequence-specific breakdown of target mRNAs. RNAi can be used to inhibit HIV-1 replication by targeting the viral RNA genome. However, the error-prone replication machinery of HIV-1 can generate RNAi-resistant variants with specific mutations in the target sequence.

Titers of lentiviral vectors encoding shRNAs and miRNAs are reduced by different mechanisms that require distinct repair strategies.

RNAi-based gene therapy is a powerful approach to treat viral infections because of its high efficiency and sequence specificity. The HIV-1-based lentiviral vector system is suitable for the delivery of RNAi inducers to HIV-1 susceptible cells due to its ability to transduce nondividing cells, including hematopoietic stem cells, and its ability for stable transgene delivery into the host cell genome. However, the presence of anti-HIV short hairpin RNA (shRNA) and microRNA (miRNA) cassettes can negatively affect the lentiviral vector titers.

HIV-1 evolution: frustrating therapies, but disclosing molecular mechanisms.

Replication of HIV-1 under selective pressure frequently results in the evolution of virus variants that replicate more efficiently under the applied conditions. For example, in patients on antiretroviral therapy, such evolution can result in variants that are resistant to the HIV-1 inhibitors, thus frustrating the therapy. On the other hand, virus evolution can help us to understand the molecular mechanisms that underlie HIV-1 replication.

Burden of disease due to human coronavirus NL63 infections and periodicity of infection.

Background: The disease burden caused by recently identified respiratory viruses like HCoV-NL63 is unknown.

Objectives: We determined the burden of disease due to HCoV-NL63 infections using the population-based PRI.DE cohort of children under the age of 3 with lower respiratory tract infections (LRTIs).

Lentiviral vector engineering for anti-HIV RNAi gene therapy.

RNA interference or RNAi-based gene therapy for the treatment of HIV-1 infection has recently emerged as a highly effective antiviral approach. The lentiviral vector system is a good candidate for the expression of antiviral short hairpin RNAs (shRNA) in HIV-susceptible cells. However, this strategy can give rise to vector problems because the anti-HIV shRNAs can also target the HIV-based lentiviral vector system.

Optimization of shRNA inhibitors by variation of the terminal loop sequence.

Gene silencing by RNA interference (RNAi) can be achieved by intracellular expression of a short hairpin RNA (shRNA) that is processed into the effective small interfering RNA (siRNA) inhibitor by the RNAi machinery. Previous studies indicate that shRNA molecules do not always reflect the activity of corresponding synthetic siRNAs that attack the same target sequence. One obvious difference between these two effector molecules is the hairpin loop of the shRNA.

Cellular levels of HIV unspliced RNA from patients on combination antiretroviral therapy with undetectable plasma viremia predict the therapy outcome.

Background: Combination antiretroviral therapy (cART), the standard of care for HIV-1 infection, is considered to be successful when plasma viremia remains below the detection limit of commercial assays. Yet, cART fails in a substantial proportion of patients after the apparent success. No laboratory markers are known that are predictive of cART outcome in initial responders during the period of undetectable plasma viremia.

Of Mice and Men: On the Origin of XMRV.

The novel human retrovirus xenotropic murine leukemia virus-related virus (XMRV) is arguably the most controversial virus of this moment. After its original discovery in prostate cancer tissue from North American patients, it was subsequently detected in individuals with chronic fatigue syndrome from the same continent. However, most other research groups, mainly from Europe, reported negative results.

Lentiviral delivery of RNAi effectors against HIV-1.

RNA interference (RNAi) holds great promise as gene therapy approach against viral pathogens, including HIV-1. A specific anti-HIV-1 response can be induced via transfection of synthetic small interfering RNAs (siRNAs) or via intracellular transgene expression of short hairpin RNAs (shRNAs) or microRNAs (miRNAs). Both targeting of the viral mRNAs or the mRNAs for cellular co-factors that are required for viral replication have been shown successful in suppressing HIV-1 replication.

Towards a genetic AIDS vaccine.

We discuss a recent Nature Medicine publication by Philip Johnson and co-workers (Vector-mediated gene transfer engenders long-lived neutralizing activity and protection against SIV infection in monkeys. Nat. Med.

HIV vaccine: it may take two to tango, but no party time yet.

A press conference on Thursday September 24 in Bangkok, Thailand, released data that an experimental vaccine provided mild protection against HIV-1 infection. This is the first positive signal of any degree of vaccine efficacy in humans, more than a quarter-century after scientists discovered the virus that causes AIDS. The research was conducted by a team including Thai researchers, the U.

Viral load levels measured at set-point have risen over the last decade of the HIV epidemic in the Netherlands.

Background: HIV-1 RNA plasma concentration at viral set-point is associated not only with disease outcome but also with the transmission dynamics of HIV-1. We investigated whether plasma HIV-1 RNA concentration and CD4 cell count at viral set-point have changed over time in the HIV epidemic in the Netherlands.

Methodology/principal Findings: We selected 906 therapy-naïve patients with at least one plasma HIV-1 RNA concentration measured 9 to 27 months after estimated seroconversion.

Toward a durable anti-HIV gene therapy based on RNA interference.

Basic research in the field of molecular biology led to the discovery of the mechanism of RNA interference (RNAi) in Caenorhabditis elegans in 1998. RNAi is now widely appreciated as an important gene control mechanism in mammals, and several RNAi-based gene-silencing applications have already been used in clinical trials. In this review I will discuss RNAi approaches to inhibit the pathogenic human immunodeficiency virus type 1 (HIV-1), which establishes a chronic infection that would most likely require a durable gene therapy approach.

Combinatorial RNAi against HIV-1 using extended short hairpin RNAs.

RNA interference (RNAi) is a widely used gene suppression tool that holds great promise as a novel antiviral approach. However, for error-prone viruses including human immunodeficiency virus type 1(HIV-1), a combinatorial approach against multiple conserved sequences is required to prevent the emergence of RNAi-resistant escape viruses. Previously, we constructed extended short hairpin RNAs (e-shRNAs) that encode two potent small interfering RNAs (siRNAs) (e2-shRNAs).

RNAi-mediated inhibition of HIV-1 by targeting partially complementary viral sequences.

Potent antiviral RNAi can be induced by intracellular expression of short hairpin RNAs (shRNAs) and artificial microRNAs (miRNAs). Expression of shRNA and miRNA results in target mRNA degradation (perfect base pairing) or translational repression (partial base pairing). Although efficient inhibition can be obtained, error-prone viruses such as human immunodeficiency virus type 1 (HIV-1) can escape from RNAi-mediated inhibition by mutating the target sequence.

RNA-interference-based gene therapy approaches to HIV type-1 treatment: tackling the hurdles from bench to bedside.

RNA interference (RNAi) is a cellular mechanism that can be induced by small interfering RNAs (siRNAs) to mediate sequence-specific gene silencing by cleavage of the targeted messenger RNA. RNAi can be used as an antiviral approach to silence HIV type-1 (HIV-1) through stable expression of precursors, such as short hairpin RNAs (shRNAs), which are processed into siRNAs that can elicit degradation of HIV-1 RNAs. At the beginning of 2008, the first clinical trial using a lentivirus with an RNA-based gene therapy against HIV-1 was initiated.

Detailed mechanistic insights into HIV-1 sensitivity to three generations of fusion inhibitors.

Peptides based on the second heptad repeat (HR2) of viral class I fusion proteins are effective inhibitors of virus entry. One such fusion inhibitor has been approved for treatment of human immunodeficiency virus-1 (T20, enfuvirtide). Resistance to T20 usually maps to the peptide binding site in HR1.

Stringent testing identifies highly potent and escape-proof anti-HIV short hairpin RNAs.

Background: RNA interference (RNAi) is a cellular mechanism that can be induced by small interfering RNAs to mediate sequence-specific gene silencing by cleavage of the targeted mRNA. RNAi can be used as an antiviral approach to silence the human immunodeficiency virus type 1 (HIV-1) through stable expression of short hairpin RNAs (shRNAs). Previously, we used a co-transfection assay in which shRNA constructs were transfected with an HIV-1 molecular clone to identify 20 shRNA inhibitors that target highly conserved HIV-1 sequences.

Differential RNA silencing suppression activity of NS1 proteins from different influenza A virus strains.

The NS1 gene of influenza A virus encodes a multi-functional protein that plays an important role in counteracting cellular antiviral mechanisms such as the interferon (IFN), protein kinase R and retinoic acid-inducible gene product I pathways. In addition, NS1 has recently been shown to have RNA interference (RNAi) or RNA silencing suppression (RSS) activity. This study analysed the IFN antagonistic activity of NS1 and the RSS activity for several influenza subtypes: H1N1, H3N2, H5N1 and H7N7.

Optimal translation initiation enables Vif-deficient human immunodeficiency virus type 1 to escape restriction by APOBEC3G.

APOBEC3G restricts Vif-deficient human immunodeficiency virus type 1 (HIV-1) by deaminating viral cDNA cytosines to uracils. This promutagenic activity is counteracted by HIV-1 Vif, which is a natural APOBEC3G antagonist. However, we previously reported that Vif-deficient HIV-1 could evolve resistance to APOBEC3G by a novel mechanism requiring an A200-to-C/T transition mutation and Vpr inactivation.