HIV-1 but not SIVmac239 induces higher interferon-α antiviral state in chronic infected northern pig-tailed macaques (Macaca leonina)

Studies have shown that interferon (IFN)-α has an inhibitory effect on human immunodeficiency virus type 1 (HIV-1) replication in the acute infection stage, but its role in chronic infection is still unclear. We previously established a nonpathogenic HIV-1 and pathogenic simian immunodeficiency virus (SIV) model in northern pig-tailed macaques (NPMs, Macaca leonina). In the current study, we detected viral RNA and DNA in various tissues (axillary lymph nodes (LNs), inguinal LNs, and spleen) in HIV-1_NL4-3- and SIV_mac239-infected NPM during the chronic stage of infection. Results indicated that the levels of viral DNA and RNA were higher in the tested tissues (LNs and spleen) of the SIV_mac239-infected NPMs than in the HIV-1_NL4-3 infected NPMs. Furthermore, IFN-α expression was higher in the HIV-infected tissues than in the SIV-infected controls. The HIV restriction factors induced by IFN-α (i.e., tetherin and MX2), as well as inflammatory factors IFN-γ, tumor necrosis factor-α (TNF-α), and interleukin 6 (IL-6), were analyzed using real-time polymerase chain reaction (PCR) and immunofluorescence staining assays. Results showed that their expression levels were much higher in the HIV-infected tissues than in the SIV-infected controls. These findings were confirmed by in vitro experiments on healthy NPM peripheral blood mononuclear cells infected with HIV-1_NL4-3, which showed lower viral replication, higher IFN-α expression, and an antiviral status. This study demonstrated that HIV-1 infection, but not SIV_mac239 infection, in NPMs caused higher expression of IFN-α and induced a higher antiviral status. This may be one of the reasons why HIV-1 cannot replicate at a high level or develop into AIDS in NPMs.     

HIV-1 entry into quiescent primary lymphocytes: molecular analysis reveals a labile, latent viral structure

Productive infection of human T lymphocytes by HIV-1 is dependent upon proliferation of the infected cell. Nonproliferating quiescent T cells can be infected by HIV-1 and harbor the virus in an inactive state until subsequent mitogenic stimulation. We use a modification of the polymerase chain reaction method, which is both sensitive and quantitative, to demonstrate that HIV-1 DNA synthesis is initiated in infected quiescent T cells at levels comparable with those of activated T cells. However, unlike that of activated T cells, the viral genome is not completely reverse transcribed in quiescent cells. Although this viral DNA structure can persist in quiescent cells as a latent form, it is labile. We discuss the lability of this HIV-1 DNA structure in relation to a "self-restricting persistent infection" by HIV-1 and propose that this may explain the low percentage of infected cells in the circulation of AIDS patients.     

HIV viral sequences in seronegative people at risk detected by in situ hybridisation and polymerase chain reaction.

A study was conducted to assess the occurrence of latent infection with the human immunodeficiency virus (HIV) among seronegative people at high risk of infection. The presence of HIV genomes was analysed by molecular techniques in two seronegative children born to mothers infected with HIV and in three regular sexual partners of seropositive drug addicts. The adults were selected from a seronegative cohort at high risk of infection because of their sexual contacts and the children selected because of impaired growth. HIV retroviral sequences were detected in four of the five subjects directly at the cellular level by in situ hybridisation in peripheral blood mononuclear cells. HIV genomic sequences were confirmed by in vitro amplification of viral DNA with the polymerase chain reaction technique. The existence of a latent viral infection state in these seronegative subjects indicates the unreliability of standard serological analysis in people who have been in regular contact with infected patients.     

Temporal fluctuations in HIV quasispecies in vivo are not reflected by sequential HIV isolations

A genetic study has been made of the HIV tat gene from sequential HIV-1 isolates and the corresponding infected peripheral blood mononuclear cells. DNA was amplified by polymerase chain reaction (PCR) and cloned into a eukaryotic expression vector. Twenty clones were sequenced from each sample. Comparing the sequential HIV isolates, abrupt differences were seen between the major forms of each isolate. These progressive changes were not reflected at all among the in vitro samples. The fluctuation in the quasispecies in vivo may suggest a much more dynamic role for latently infected mononuclear cells. High frequencies of functionally defective tat genes were identified. Given such complexity and the evident differences between quasispecies in vivo and in vitro, the task of defining HIV infection in molecular terms will be difficult.     

HIV Rev-isited

The human immunodeficiency virus type 1 (HIV-1) proteome is expressed from alternatively spliced and unspliced genomic RNAs. However, HIV-1 RNAs that are not fully spliced are perceived by the host machinery as defective and are retained in the nucleus. During late infection, HIV-1 bypasses this regulatory mechanism by expression of the Rev protein from a fully spliced mRNA. Once imported into the nucleus, Rev mediates the export of unprocessed HIV-1 RNAs to the cytoplasm, leading to the production of the viral progeny. While regarded as a canonical RNA export factor, Rev has also been linked to HIV-1 RNA translation, stabilization, splicing and packaging. However, Rev''s functions beyond RNA export have remained poorly understood. Here, we revisit this paradigmatic protein, reviewing recent data investigating its structure and function. We conclude by asking: what remains unknown about this enigmatic viral protein?     

Meaning of DNA detection during the follow-up of HIV-1 infected patients: a brief review

A growing body of evidence indicates that proviral DNA load quantitation is an important parameter in establishing the dynamics of HIV infection. Proviral DNA load can be determined during the follow-up of infected individuals to evaluate reservoir status in addition to viral replication. Hence, the study of viral reservoirs, represented by HIV-1 latently infected cells, including resting memory CD4+ T cells, monocytes and macrophages, by which HIV-1 can be reactivated, opens new perspectives in the assessment and the comprehension of HIV-1 infection. However, the identification of viral reservoirs, that can store both wild and drug resistance viruses, is one of the most important steps in developing treatment strategies because it is now clear that viral reservoirs not only prevent sterilizing immunity but also represent a major obstacle to curing the infection with the potent antiretroviral drugs currently in use. Even if only careful evaluation of virological and immunological markers is necessary to fully characterize the course of HIV-1 infection and to provide a more complete laboratory-based assessment of disease progression, the availability of a new standardized assay such as DNA proviral load will be important to assess the true extent of virological suppression in treated patients and to verify the efficacy of new immune-based therapies aimed at purging HIV-1 DNA reservoirs. Several studies demonstrate, in fact, that HIV-1 cellular DNA load may be an indicator of spread of infection whereas the plasma RNA load is indicates active infection. This article will review the importance of monitoring HIV-1 proviral load DNA during the follow-up of HIV-1 infected subjects, suggesting that additional information complementing HIV RNA load could provide crucial information to monitor viral replication and the effectiveness of HAART therapy.     

Discontinuous sequence change of human immunodeficiency virus (HIV) type 1 env sequences in plasma viral and lymphocyte-associated proviral populations in vivo: implications for models of HIV pathogenesis.

Sequence change in different hypervariable regions of the external membrane glycoprotein (gp120) of human immunodeficiency virus type 1 (HIV-1) was studied. Viral RNA associated with cell-free virus particles circulating in plasma and proviral DNA present in HIV-infected peripheral blood mononuclear cells (PBMCs) were extracted from blood samples of two currently asymptomatic hemophiliac patients over a 5-year period. HIV sequences were amplified by polymerase chain reaction to allow analysis in the V3, V4, and V5 hypervariable regions of gp120. Rapid sequence change, consisting of regular replacements by a succession of distinct viral populations, was found in both plasma virus and PBMC provirus populations. Significant differences between the frequencies of sequence variants in DNA and RNA populations within the same sample were observed, indicating that at any one time point, the predominant plasma virus variants were antigenically distinct from viruses encoded by HIV DNA sequences in PBMCs. How these findings contribute to current models of HIV pathogenesis is discussed.     

Isolation and characterization of a virus-specific ribonucleoprotein complex from reticuloendotheliosis virus-transformed chicken bone marrow cells.

Chicken bone marrow cells transformed by reticuloendotheliosis virus (REV) produce in the cytoplasm a ribonucleoprotein (RNP) complex which has a sedimentation value of approximately 80 to 100S and a density of 1.23 g/cm3. This RNP complex is not derived from the mature virion. An endogenous RNA-directed DNA polymerase activity is associated with the RNP complex. The enzyme activity was completely neutralized by anti-REV DNA polymerase antibody but not by anti-avian myeloblastosis virus DNA polymerase antibody. The DNA product from the endogenous RNA-directed DNA polymerase reaction of the RNP complex hybridized to REV RNA but not to avian leukosis virus RNA. The RNA extracted from the RNP hybridized only to REV-specific complementary DNA synthesized from an endogenous DNA polymerase reaction of purified REV. The size of the RNA in the RNP is 30 to 35S, which represents the subunit size of the genomic RNA. No 60S mature genomic RNA was found within the RNP complex. The significance of finding the endogenous DNA polymerase activity in the viral RNP in infected cells and the maturation process of 60S virion RNA of REV are discussed.