Therapeutic effect of a Gag-nuclease fusion protein against retroviral infection in vivo.

Recently, remarkable progress has been made in developing effective combination drug therapies that can control but not cure retroviral replication. Even when effective, these drug regimens are toxic, they require demanding administration schedules, and resistant viruses can emerge. Thus the need for new gene-based therapies continues. In one such approach, capsid-targeted viral inactivation (CTVI), nucleases fused to viral coat proteins are expressed in infected cells and become incorporated during virion assembly. CTVI can eliminate infectious murine retrovirus titer in tissue culture. Here we describe transgenic mice expressing fusions of the Moloney murine leukemia virus (Mo-MuLV) Gag protein to staphylococcal nuclease. This work tests the protective effect and demonstrates in vivo proof-of-principle of CTVI in transgenic mice expressing endogenous proviral copies of Mo-MuLV. The antiviral protein-expressing mice are phenotypically normal, attesting to the lack of toxicity of the fusion protein. The Mo-MuLV infection was much less virulent in transgenic littermates than in nontransgenic littermates. Gag-nuclease expression reduced infectious titers in blood up to 10-fold, decreased splenomegaly and leukemic infiltration, and increased life spans up to 2.5-fold in transgenic relative to nontransgenic infected animals. These results suggest that gene therapies based on similar fusion proteins, designed to attack human immunodeficiency virus or other retroviruses, could provide substantial therapeutic benefits.

HIV-1 drug resistance profiles in children and adults with viral load of <50 copies/ml receiving combination therapy.

CONTEXT: The continued release of human immunodeficiency virus type 1 (HIV-1) into plasma at very low levels during highly active antiretroviral therapy (HAART) can be detected using specialized techniques, but the nature and significance of this low-level viremia, especially as related to acquisition of drug resistance mutations, are unclear. OBJECTIVE: To determine genetic resistance profiles of low-level plasma HIV-1 in patients with prolonged viral suppression (<50 copies/mL of plasma HIV-1 RNA) while receiving HAART. DESIGN AND SETTING: Cross-sectional study conducted at a US academic hospital from November 1999 to February 2001 using a novel method for amplification of low levels of viral genomes in plasma. PATIENTS: Eighteen HIV-1-infected patients (7 children and 11 adults), enrolled in a longitudinal study of HIV-1 reservoirs, who had suppression of viral replication while receiving protease inhibitor-containing combination therapy. Two patients (1 adult and 1 child) with less optimal suppression of viral replication were included to assess virus predominating when plasma HIV-1 RNA levels are low but detectable (<1000 copies/mL). Follow-up analyses were conducted in 3 patients. MAIN OUTCOME MEASURE: Detection of drug resistance mutations in clones amplified from low-level plasma virus. RESULTS: Viral sequences were amplified from 8 of the 18 patients with simultaneous plasma HIV-1 measurements of less than 50 copies/mL and from 2 patients with 231 and 50 copies/mL. Clones from 3 treatment-naive patients with less than 50 copies/mL of plasma HIV-1 RNA showed continued release, for as long as 42 months, of wild-type drug-sensitive virus. The 7 patients with prior nonsuppressive therapy, with viral loads below 50 copies/mL and during "blips" to 231 and 64 copies/mL, had only resistance mutations consistent with pre-HAART therapy (although reverse transcriptase inhibitor mutations may have continued to occur). New HAART-related mutations were seen in a control patient with prior viral load levels of about 400 to 1000 copies/mL. For phylogenetic analysis, sequences were available for both resting CD4(+) T cells and plasma HIV for 7 of 10 patients and showed patient-specific clustering of sequences and a close relationship between virus in the plasma and the latent reservoir. CONCLUSIONS: Based on the samples that could be amplified, low-level viremia in children and adults receiving HAART with prolonged suppression of viremia to less than 50 copies/mL of HIV-1 RNA may result primarily from archival, pre-HAART virus, reflecting earlier treatment conditions, and does not appear to require development of new, HAART-selected mutations reflecting partial resistance to therapy. Low-level viremia below 50 copies/mL may represent less of a concern regarding impending drug failure of current HAART regimens. However, the archival drug-resistant virus may be relevant regarding future treatment strategies.

Poly(ADP-ribose) polymerase-1 is required for efficient HIV-1 integration.

Poly(ADP-ribose) polymerase-1 (PARP-1; EC ) is an abundant nuclear enzyme, activated by DNA strand breaks to attach up to 200 ADP-ribose groups to nuclear proteins. As retroviral infection requires integrase-catalyzed DNA strand breaks, we examined infection of pseudotyped HIV type I in fibroblasts from mice with a targeted deletion of PARP-1. Viral infection is almost totally abolished in PARP-1 knockout fibroblasts. This protection from infection reflects prevention of viral integration into the host genome. These findings suggest a potential for PARP inhibitors in therapy of HIV type I infection.

Identification of a reservoir for HIV-1 in patients on highly active antiretroviral therapy.

The hypothesis that quiescent CD4+ T lymphocytes carrying proviral DNA provide a reservoir for human immunodeficiency virus-type 1 (HIV-1) in patients on highly active antiretroviral therapy (HAART) was examined. In a study of 22 patients successfully treated with HAART for up to 30 months, replication-competent virus was routinely recovered from resting CD4+ T lymphocytes. The frequency of resting CD4+ T cells harboring latent HIV-1 was low, 0.2 to 16.4 per 10(6) cells, and, in cross-sectional analysis, did not decrease with increasing time on therapy. The recovered viruses generally did not show mutations associated with resistance to the relevant antiretroviral drugs. This reservoir of nonevolving latent virus in resting CD4+ T cells should be considered in deciding whether to terminate treatment in patients who respond to HAART.

Quantification of latent tissue reservoirs and total body viral load in HIV-1 infection.

The capacity of HIV-1 to establish latent infection of CD4+ T cells may allow viral persistence despite immune responses and antiretroviral therapy. Measurements of infectious virus and viral RNA in plasma and of infectious virus, viral DNA and viral messenger RNA species in infected cells all suggest that HIV-1 replication continues throughout the course of infection. Uncertainty remains over what fraction of CD4+ T cells are infected and whether there are latent reservoirs for the virus. We show here that during the asymptomatic phase of infection there is an extremely low total body load of latently infected resting CD4+ T cells with replication-competent integrated provirus (<10(7) cells). The most prevalent form of HIV-1 DNA in resting and activated CD4+ T cells is a full-length, linear, unintegrated form that is not replication competent. The infection progresses even though at any given time in the lymphoid tissues integrated HIV-1 DNA is present in only a minute fraction of the susceptible populations, including resting and activated CD4+ T cells and macrophages.

Increased length of long terminal repeats inhibits Ty1 transposition and leads to the formation of tandem multimers.

The Ty1 retrotransposon of Saccharomyces cerevisiae is bounded by long-terminal repeats (LTRs). We have constructed a variety of Ty1 elements in which the LTR length has been increased from the normal length of 334 bp to > 2 kb. Although small insertions in the LTR have minimal effects on transposition frequency, larger insertions dramatically reduce it. Nevertheless, elements with long LTRs are incorporated into the genome at a low frequency. Most of these rare insertion events represent Ty1 tandem (head to tail) multimers.

A conditionally replicating HIV-1 vector interferes with wild-type HIV-1 replication and spread.

Defective-interfering viruses are known to modulate virus pathogenicity. We describe conditionally replicating HIV-1 (crHIV) vectors that interfere with wild-type HIV-1 (wt-HIV) replication and spread. crHIV vectors are defective-interfering HIV genomes that do not encode viral proteins and replicate only in the presence of wt-HIV helper virus. In cells that contain both wt-HIV and crHIV genomes, the latter are shown to have a selective advantage for packaging into progeny virions because they contain ribozymes that cleave wt-HIV RNA but not crHIV RNA. A crHIV vector containing a triple anti-U5 ribozyme significantly interferes with wt-HIV replication and spread. crHIV vectors are also shown to undergo the full viral replicative cycle after complementation with wt-HIV helper-virus. The application of defective interfering crHIV vectors may result in competition with wt-HIVs and decrease pathogenic viral loads in vivo.