Direct and quantitative single-cell analysis of human immunodeficiency virus type 1 reactivation from latency

The ability of human immunodeficiency virus type 1 (HIV-1) to establish latent infections in cells has received renewed attention owing to the failure of highly active antiretroviral therapy to eradicate HIV-1 in vivo. Despite much study, the molecular bases of HIV-1 latency and reactivation are incompletely understood. Research on HIV-1 latency would benefit from a model system that is amenable to rapid and efficient analysis and through which compounds capable of regulating HIV-1 reactivation may be conveniently screened. We describe a novel reporter system that has several advantages over existing in vitro systems, which require elaborate, expensive, and time-consuming techniques to measure virus production. Two HIV-1 molecular clones (NL4-3 and 89.6) were engineered to express enhanced green fluorescent protein (EGFP) under the control of the viral long terminal repeat without removing any viral sequences. By using these replication-competent viruses, latently infected T-cell (Jurkat) and monocyte/macrophage (THP-1) lines in which EGFP fluorescence and virus expression are tightly coupled were generated. Following reactivation with agents such as tumor necrosis factor alpha, virus expression and EGFP fluorescence peaked after 4 days and over the next 3 weeks each declined in a synchronized manner, recapitulating the establishment of latency. Using fluorescence microscopy, flow cytometry, or plate-based fluorometry, this system allows immediate, direct, and quantitative real-time analysis of these processes within single cells or in bulk populations of cells. Exploiting the single-cell analysis abilities of this system, we demonstrate that cellular activation and virus reactivation following stimulation with proinflammatory cytokines can be uncoupled.     

Chimeric human immunodeficiency virus type 1 virions that contain the simian immunodeficiency virus nef gene are cyclosporin A resistant

We have previously shown that human immunodeficiency virus type 1 (HIV-1) virions which have their own nef gene deleted and are trans complemented to contain HIV-2 or simian immunodeficiency virus (SIV) Nef become resistant to treatment with cyclosporin A. To expand and confirm these studies, we have tested an HIV-1 isolate in which the HIV-1 nef gene has been replaced by the nef gene from SIV in a multiround infectivity assay using more physiologically relevant cell types. Our results confirm that HIV-1 virions that contain SIV nef can replicate in a cyclophilin-independent fashion.     

Role of the human immunodeficiency virus type 1 envelope gene in viral fitness

A human host offers a variety of microenvironments to the infecting human immunodeficiency virus type 1 (HIV-1), resulting in various selective pressures, most of them directed against the envelope (env) gene. Therefore, it seems evident that the replicative capacity of the virus is largely related to viral entry. In this study we have used growth competition experiments and TaqMan real-time PCR detection to measure the fitness of subtype B HIV-1 primary isolates and autologous env-recombinant viruses in order to analyze the contribution of wild-type env sequences to overall HIV-1 fitness. A significant correlation was observed between fitness values obtained for wild-type HIV-1 isolates and those for the corresponding env-recombinant viruses (r = 0.93; P = 0.002). Our results suggest that the env gene, which is linked to a myriad of viral characteristics (e.g., entry into the host cell, transmission, coreceptor usage, and tropism), plays a major role in fitness of wild-type HIV-1. In addition, this new recombinant assay may be useful for measuring the contribution of HIV-1 env to fitness in viruses resistant to novel antiretroviral entry inhibitors.     

The human immunodeficiency virus type 1 vpr gene prevents cell proliferation during chronic infection.

Human immunodeficiency virus type 1 (HIV-1) is a retrovirus that can cause extensive cytopathicity in T cells. However, long-term productive infection of T-cell lines has been described. Here we show that although Vpr has no effect on the initial cytopathic effect of HIV-1, viruses that contain an intact vpr gene are unable to establish a chronic infection of T cells. However, virus with a mutated vpr gene can readily establish such long-term cultures. The effect of Vpr is independent of the env gene and the nef gene. Furthermore, expression of Vpr alone affects the progression of cells in the cell cycle. These results suggest that HIV-1 has evolved a viral gene to prevent chronic infection of T cells.     

Spontaneous mutations in the human immunodeficiency virus type 1 gag gene that affect viral replication in the presence of cyclosporins.

Human immunodeficiency virus type 1 mutants that are resistant to inhibition by cyclosporins arise spontaneously in vitro during propagation in a HeLa-CD4+ cell line in the presence of a nonimmunosuppressive analog of cyclosporin A. Interestingly, the phenotype of all of the mutants examined is drug resistant and drug dependent, with both cyclosporin A and its analog. Four independently isolated mutants have been analyzed genetically by construction of recombinant proviruses in the NL4-3 parental strain background and subsequent testing of the chimeric viruses in HeLa cells. The cyclosporin-resistant, cyclosporin-dependent phenotype consistently transfers with a 1.3-kb fragment of gag, within which the four mutants share one of two possible single amino acid exchanges in a proline-rich stretch in the capsid domain of Pr55gag. These mutants provide the first evidence that mutations in human immunodeficiency virus type 1 gag confer resistance to cyclosporins; however, replication is conditional on the presence of the drug. In the T-cell line CEM, replication of the recombinant mutant viruses is also cyclosporin dependent. The drug-dependent replication in HeLa cells is stringent, and in the absence of cyclosporin only revertant viruses with the parental phenotype grow out of cultures infected with cyclosporin-dependent virus. In at least one isolate examined, the revertant phenotype appears to be due to suppressor mutations near the proline-rich region.     

Functional classes of bronchial mucosa genes that are differentially expressed in asthma

Background  

Asthma pathogenesis and susceptibility involves a complex interplay between genetic and environmental factors. Their interaction modulates the airway inflammation and remodelling processes that are present even in mild asthma and governs the appearance and severity of symptoms of airway hyperresponsiveness. While asthma is felt to develop as the result of interaction among many different genes and signalling pathways, only a few genes have been linked to an increased risk of developing this condition.     

Effect of linker insertion mutations in the human immunodeficiency virus type 1 gag gene on activation of viral protease expressed in bacteria.

We have expressed the human immunodeficiency virus type 1 (HIV-1) protease (PR) in bacteria as a Gag-PR polyprotein (J. Luban and S.P. Goff, J. Virol. 65:3203-3212, 1991). The protein displays enzymatic activity, cleaving the Gag polyprotein precursor Pr55gag to the expected products. The PR enzyme is only active as a dimer, and we hypothesized that PR activation might be used as an indicator of polyprotein multimerization. We constructed 25 linker insertion mutations throughout gag and assessed the PR activity of mutant Gag-PR polyproteins by the appearance of Gag cleavage products in bacterial lysates. All mutant constructs produced stable protein in bacteria. PR activity of the majority of the Gag-PR mutants was indistinguishable from that of the wild type. Six mutants, one with an insertion in the matrix (MA), four with insertions in the capsid (CA), and one with insertions in the nucleocapsid (NC), globally disrupted polyprotein processing. When PR was provided in trans on a separate plasmid, the Gag proteins were cleaved with wild-type efficiency. These results suggest that the gag mutations identified as disruptive of polyprotein processing did not conceal the scissile bonds of the polyprotein. Rather, the mutations prevented PR activation in the context of a Gag-PR polyprotein, perhaps by preventing polyprotein dimerization.     

The effect of viral regulatory protein expression on gene delivery by human immunodeficiency virus type 1 vectors produced in stable packaging cell lines.

We describe the generation of stable human immunodeficiency virus type 1 (HIV-1)-packaging lines that constitutively express high levels of HIV-1 structural proteins in either a Rev-dependent or a Rev-independent fashion. These cell lines were used to assess gene transfer by using an HIV-1 vector expressing the hygromycin B resistance gene and to study the effects of Rev, Tat, and Nef on the vector titer. The Rev-independent cell lines were created by using gag-pol and env expression vectors that contain the Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE). Vector titers approaching 10(4) CFU/ml were routinely obtained with these cell lines, as well as with the Rev-dependent cell lines, with HeLa-CD4 cells as targets. The presence of Nef and Tat in the producer cell each increased the vector titer 5- to 10-fold. Rev, on the other hand, was absolutely essential for gene transfer, unless the MPMV CTE was present in the vector. In that case, by using the Rev-independent cell lines for packaging, Rev could be completely eliminated from the system without a reduction in vector titer.     

Cellular and viral specificities of human immunodeficiency virus type 1 vif protein

The vif gene of human immunodeficiency virus type 1 (HIV-1) greatly enhances the infectivity of HIV-1 virions that are released from cells classified as nonpermissive (e.g., lymphocytes, macrophages, and H9 leukemic T cells) but is irrelevant in permissive cells (e.g., HeLa or COS cells). Recently, it was reported that vif expression in nonpermissive cells dramatically increases infectivity not only of HIV-1 but also of other enveloped viruses, including murine leukemia viruses (MLVs). This was surprising in part because MLVs and other murine retroviruses lack vif genes yet replicate efficiently in T lymphocytes. To investigate these issues, we first developed improved methods for producing substantial quantities of HIV-1 virions with vif deletions from healthy H9 cells. These virions had approximately the same amounts of major core proteins and envelope glycoproteins as the control wild-type virions but were only approximately 1% as infectious. We then produced H9 cells that contained wild-type or vif deletion HIV-gpt proviruses, which lack a functional env gene. After superinfection with either xenotropic or amphotropic MLVs, these cells released HIV-gpt virions pseudotyped with an MLV envelope plus replication-competent MLV. Interestingly, the pseudotyped HIV-gpt (vif deletion) virions were noninfectious, whereas the MLV virions simultaneously released from the same H9 cells were fully infectious. These results strongly suggest that the Vif protein functions in a manner that is both cell specific and at least substantially specific for HIV-1 and related lentiviruses. In addition, these results confirm that vif deletion HIV-1 virions from nonpermissive cells are blocked at a postpenetration stage of the infection pathway.