HIV induces both a down-regulation of IRAK-4 that impairs TLR signalling and an up-regulation of the antibiotic peptide dermcidin in monocytic cells.

HIV-infected individuals have an increased risk of invasive bacterial infections, even at early clinical stages with relatively normal CD4(+) T-cell counts. The pathogenic mechanisms behind this are not fully understood. However, an increasing number of studies indicate that HIV may impair the innate immunity to bacteria by infecting key cells of the monocyte/macrophage lineage. In this study, the effects of HIV infection on the protein profile of undifferentiated monocyte-like THP-1 cells were examined by a mass spectrometric approach based on stable isotope labelling with amino acid in cell culture (SILAC). We identified 651 proteins, of which nine proteins were down-regulated and 17 proteins were up-regulated in HIV-infected THP-1 cells as compared to uninfected controls. Most remarkably, the IL-1 receptor associated kinase 4 (IRAK-4), which is essential for virtually all TLR signalling, was suppressed, whereas the precursor for the antibiotic peptide Dermcidin was up-regulated in HIV-infected cells. Upon stimulation of either TLR2 or TLR4, the HIV-infected THP-1 cells displayed reduced TNF-alpha secretion. The HIV-induced down-regulation of IRAK-4 was reconfirmed in monocyte-derived macrophage cell cultures. These data suggests that HIV may impair the TLR signalling cascade for pathogen recognition in cells of the monocyte/macrophage lineage and thus, may reduce the ability of the innate immune system to sense invading pathogens and initiate appropriate responses.

Influence of genotype and the organ of origin on the subtype of T-cell in Moloney lymphomas induced by transfer of preleukemic cells from athymic and thymus-bearing mice.

Thymus, spleen, and bone marrow of 1-month-old neonatally Moloney murine leukemia virus-inoculated mice have been transferred to 400-R-irradiated syngeneic recipients of the opposite sex. The donor or recipient origin of T-cell lymphomas arising in the host animal was identified by the sex chromosome marker. Spleen and bone marrow of athymic BALB-nu/nu mice contain cells with the potential to develop into T-cell lymphomas upon transfer to thymus-bearing BALB/c recipients. Such lymphomas arise from at least two subsets of T-cells, one terminal deoxynucleotidyl transferase (TdT) positive and the other 20 alpha-hydroxysteroid dehydrogenase positive. The enzyme-negative precursor T-cells from the BALB-nu/nu spleen and bone marrow can thus mature to enzyme-positive cells and give rise to lymphoma in the thymus-bearing recipient. Preleukemic spleen and bone marrow, but not thymus, from CBA and BALB/c mice regularly contained cells with the potential to develop lymphoma. The subset of T-cell involved was influenced by the genotype since lymphomas arising after the transfer of CBA and BALB/c spleens were TdT positive and 20 alpha-hydroxysteroid dehydrogenase positive, respectively. In thymus-bearing mice, but not in nude mice, the transfer of preleukemic spleen cells gave lymphomas earlier than did transfer of bone marrow cells. This suggests that the more mature lymphoid cell population in the spleen of thymus-bearing mice may allow leukemic transformation to occur more rapidly than do the less mature cells in the bone marrow. In one-third of the cases, the virus produced by the preleukemic cells transferred induced new lymphomas involving recipient host cells. These de novo-induced lymphomas were all TdT positive. We suggest that leukemic transformation of TdT-positive cells may occur through a different mechanism than does transformation of cells bearing the 20 alpha-hydroxysteroid dehydrogenase marker.

Tropism for primary monocytes and for monocytoid cell lines are separate features of HIV-1 variants.

We have analyzed the phenotype of five human monocytoid cell lines (U-937, THP-1, RC.2A, Mono Mac 6, and DD) and their capacity to support replication of HIV-1 strains with known tropism for T lymphocytes and mononuclear phagocytes. HIV-1 Ba-L, a prototype monocytotropic HIV-1 strain, was unable to establish continuous replication in any of the cell lines used. In contrast, the lymphotropic strain IIIB could replicate in all CD4-positive cell lines. We conclude that the capacity of HIV-1 isolates to replicate in established human monocytoid malignant cells does not correlate with the tropism of the virus for primary mononuclear phagocytes. Since monocytoid cell lines do not distinguish HIV-1 variants tropic for mononuclear phagocytes, we suggest the use of primary cells for studies of HIV-1 tropism.

Macrophage tropism: fact or fiction?

Primary HIV-1 isolates can be distinguished by phenotypic qualities such as the ability to productively infect cells of established CD4-positive lines and to induce syncytia in MT-2 cells. Such viral phenotypes have also been reported to confer host cell specificity. It is perceived that primary isolates with the syncytium-inducing phenotype (SI or rapid/high) are T cell tropic and are therefore unable to infect primary cells of the monocyte/macrophage lineage. However, we have consistently found that these isolates are as capable of establishing infection in monocyte-derived macrophages (MDM) as the monocytotropic, non-syncytium-inducing variants (NSI or slow/low). It is known that differentiation, activation, and proliferation of human monocytes are affected by both isolation methods and culture conditions. Therefore, to test whether our inability to discriminate macrophage tropic HIV-1 isolates could be explained by differences in culturing techniques, we isolated monocytes by elutriation or short-term adherence and allowed the cells to mature and differentiate in either the presence or absence of autologous lymphocytes. After removal of nonadherent cells, MDM were infected with a panel of SI and NSI primary HIV-1 isolates. MDM were susceptible to infection by the SI as well as the NSI isolates, regardless of whether or not the cells were allowed to mature in the presence of autologous lymphocytes. However, MDM matured in the presence of autologous lymphocytes replicated HIV-1 isolates (both NSI and SI) to a higher titre than MDM matured in the absence of lymphocytes. In light of these findings and recently published reports on HIV-1 phenotype and chemokine receptor usage we believe that the term macrophage-tropic strains of HIV-1 is no longer appropriate.

Replicative capacity, cytopathic effect and cell tropism of HIV.

Naturally occurring HIV variants show distinct biologic features that correspond to the severity of HIV infection. Virus from asymptomatic HIV carriers or individuals with mild disease replicates slowly and inefficiently in the patients' peripheral blood mononuclear cell cultures. Attempts to passage these viruses in CD4-positive cell lines usually fail or result in transient replication only. In contrast, viruses from patients with severe immunodeficiency replicate rapidly and efficiently in peripheral blood mononuclear cell cultures as well as cell lines: hence the designation slow/low and rapid/high, respectively. These two groups of viruses can also be distinguished by the type of cytopathogenicity exerted in peripheral blood mononuclear cells. Rapid/high viruses are characterized by extensive syncytia formation, whereas syncytia are rarely seen with slow/low viruses. Instead cultures infected with slow/low viruses show signs of cell death or no cytopathic changes at all. It has also been observed that shift from the slow/low type of virus to rapid/high may occur in the same individual over time. Whether this change signals the emergence of HIV variants with increased virulence or reflects the damage to the immune system that can no longer control virus replication remains to be seen. Rapid/high and slow/low viruses can also be distinguished by cell tropism when tested in a model system on indicator cell lines of T-lymphoid or monocytoid origin. Infection by rapid/high viruses activates chloramphenicol acetyl transferase in both T-lymphoid and monocytoid indicator cells, whereas slow/low viruses activate chloramphenicol acetyl transferase only in monocytoid cell lines. A difference between slow/low and rapid/high viruses cannot be demonstrated in fresh normal macrophage cultures, since most isolates can be successfully passaged in macrophages. Whether the viruses that infect macrophages are truly macrophage-tropic or dual-tropic, and infect macrophages and lymphocytes with equal efficiency, remains to be studied.

Activation of CD8 T cells normalizes and correlates with the level of infectious provirus in tonsils during highly active antiretroviral therapy in early HIV-1 infection.

OBJECTIVES: To study the effects of antiretroviral therapy on T cell activation in blood and tonsils from HIV-1 infected individuals in relation to CD4 cell count, plasma viremia, and infectious HIV-1 provirus. DESIGN: A 48-week study of viral load and T cell subsets in blood and tonsils from 12 HIV-1-positive individuals with a mean CD4 cell number of 400 x 10(6) cells/l treated with a combination of zidovudine, lamivudine, and indinavir. METHODS: Tonsil biopsies and blood samples were collected at regular intervals. Lymphocytes were phenotyped and quantified by three-color flow cytometry; infectious provirus was quantified by a limiting dilution assay. HIV-1-negative individuals were included as controls. RESULTS: The fraction of tonsillar CD8 T cells expressing CD69, CD38, or HLA-DR in the patients with suppressed virus replication declined to levels comparable with that in controls by 48 weeks and showed a strong positive correlation with tonsillar infectious provirus and plasma viremia. The level of CD4 T cell activation was within normal range in tonsils throughout the study. The fraction of HLA-DR+ cells within CD4 and CD8 T cells in blood declined rapidly in parallel with plasma viremia but remained slightly higher compared with that in uninfected individuals. CONCLUSION: Antiretroviral therapy normalizes tonsillar CD8 T cell activation in HIV-1-positive individuals in parallel with suppression of viral replication, indicating reduced CD8 cell turnover. Normal tonsillar CD4 T cell activation suggests limited CD4 cell turnover in early HIV infection. Activated CD8 T cells in lymphoid tissue is superior to that in blood as an immunological marker for the virological response to antiretroviral therapy.

Reduced CD4 cell counts in blood do not reflect CD4 cell depletion in tonsillar tissue in asymptomatic HIV-1 infection.

OBJECTIVE: To investigate whether the loss of CD4 cells seen in peripheral circulation of HIV-1-positive individuals reflects a similar depletion of CD4 cells from lymphoid tissue. DESIGN: CD4 and CD8 cells in tonsillar mononuclear cell suspensions were quantified relative to tonsillar B cells, as these were thought to remain numerically unchanged in the course of HIV infection. Results were related to the CD4 cell counts in blood and to the clinical status of the patients. METHODS: Blood samples and tonsillar tissue were obtained from 13 HIV-1-seropositive individuals and six seronegative controls. B cells and T-cell subsets in mononuclear cells were quantified using a three-colour flow cytometry protocol. Histological sections were morphologically classified and B-cell areas were quantified by morphometry. RESULTS: The B-cell fraction was confirmed to be relatively unchanged in asymptomatic HIV-1-seropositive individuals compared with controls. The tonsillar CD4 : B-cell ratios in asymptomatic individuals was similar to those seen in controls, whereas the CD4 : B-cell ratios in symptomatic HIV-1-infected individuals were greatly reduced. The tonsillar CD4 : CD8 cell ratios in HIV-1-infected individuals were much lower than those seen in controls, in the asymptomatic group due to a considerable expansion of the tonsillar CD8 cell subset, and in the symptomatic group also due to a loss of CD4 cells. CONCLUSIONS: We found no evidence of CD4 cell depletion in tonsillar tissue in asymptomatic HIV-1-infected individuals despite low CD4 cell counts in blood. Loss of CD4 cells from this lymphoid tissue seems to occur as a late-stage phenomenon correlated with the onset of clinical symptoms.

Neopterin concentrations in cerebrospinal fluid and serum of individuals infected with HIV-1.

Neopterin, a biochemical marker for the activation of cell-mediated immune reactions, was determined in serum and cerebrospinal fluid (CSF) from patients infected with HIV-1. A significant correlation was found between serum and CSF neopterin concentrations, although concentrations of neopterin in serum were more closely correlated with the clinical severity of HIV-1 infection than those in CSF. However, higher CSF levels were observed in patients with neurologic/psychiatric symptoms than in unaffected patients. Also, quotients of CSF neopterin versus serum neopterin concentrations were increased, indicating intrathecal production of neopterin. Positive HIV-1 isolation from peripheral blood mononuclear cells (PBMC) was associated with higher neopterin concentrations in serum, when compared with negative HIV-1 isolation. Neopterin in CSF appears to be a suitable biochemical marker in patients with HIV-1 infection for detecting overt neurologic/psychiatric disturbances. The data suggest that in HIV-1 infected patients, cell-mediated immune reactions might be activated intrathecally and might contribute to neuropsychiatric disease.

Jurkat-tat but not other tat-expressing cell lines support replication of slow/low type HIV.

The Jurkat-tat cell line, carrying the transactivator (tat) gene of HIV-1 IIIB and thus constitutively expressing the tat protein, has the capacity to support replication of HIV isolates obtained from asymptomatic individuals, so called slow/low (s/l) type virus. A major characteristic of the s/l isolates in vitro is their inability to continuously replicate in cells of CD4+ established lines. In contrast, virus isolates designated rapid/high (r/h) obtained from patients in advanced stages of the HIV-infection do not show this restriction in replicative capacity. To analyze whether introduction of the tat protein into certain cell types or an over-expression of the tat protein would render cells permissive for s/l virus replication, the tat gene was transfected into cells of monocytoid and T cell origin. The resulting cell lines were then tested for their susceptibility to infection with s/l and r/h type HIV-1 isolates. The results conclusively show that mere constitutive expression of the tat protein in established CD4+ cell lines will not provide conditions allowing for continuous replication of s/l type virus. Thus, the Jurkat-tat cell line is a unique cell system for long-term propagation of this type of virus. In addition, it is a suitable system to study virus-host cell interactions and control of virus replication.

In vitro maturation of mononuclear phagocytes and susceptibility to HIV-1 infection.

In the present report, we have studied the in vitro transition of normal blood monocytes to macrophages by changes in cell morphology, and the expression of surface antigens with a panel of monoclonal antibodies. The maturation process was accompanied by notable changes in cell-surface markers in a time-dependent manner. The percentage of cells expressing CD11c, ICAM-1, HLA-DR, and Fc receptor class III increased while the CD4 and CD35 expression was markedly decreased. After demonstrating that in vitro monocytes mature to macrophages in a recognizable manner, we studied the susceptibility to HIV-1 infection at time points representing different stages of cell maturation. The results show that monocyte/macrophages are susceptible to HIV-1 infection at all stages of differentiation. However, the kinetics of virus replication depends on the degree of maturation at the time of infection. Two major patterns of replication were observed: Infection of monocytes resulted in efficient virus production measurable by reverse transcriptase activity in culture supernatant, whereas infection of fully differentiated macrophages yielded low but sustained virus release only demonstrable by p24 antigen assay. We were not able to detect differences in the capacity of the virus to infect and replicate in monocyte/macrophages with respect to cellular origin of the virus isolate and whether the viruses were laboratory-adapted strains or low-passaged patient isolates.

Updated European recommendations for the clinical use of HIV drug resistance testing.

In most European countries, HIV drug resistance testing has become a routine clinical tool. However, its practical implementation in a clinical context is demanding. The European HIV Drug Resistance Panel was established to make recommendations to clinicians and virologists on this topic and to propose quality control measures. The panel recommends resistance testing for the following indications: i) drug-naive patients with acute or recent infection; ii) therapy failure, including suboptimal treatment response, when treatment change is considered; iii) pregnant HIV-1-infected women and paediatric patients with detectable viral load when treatment initiation or change is considered; and iv) genotype source patient when post-exposure prophylaxis is considered. In addition, for drug-naive patients with chronic infection in whom treatment is to be started, the panel suggests that resistance testing should be strongly considered and recommends testing the earliest sample for drug resistance if suspicion of resistance is high or prevalence of resistance in this population exceeds 10%. The panel does not favour genotyping over phenotype, however it is anticipated that genotyping will be used more often because of its greater accessibility, lower cost and faster turnaround time. For the interpretation of resistance data, clinically validated systems should be used to the greatest extent possible. It is mandatory that laboratories performing HIV resistance tests take regular part in quality assurance programs. Similarly, it is necessary that HIV clinicians and virologists take part in continuous education and meet regularly to discuss problematic clinical cases. Indeed, resistance test results should be used in the context of all other clinically relevant information for predicting therapy response. The panel also encourages the timely collection of epidemiological information to estimate the impact of transmission of resistant HIV and the prevalence of HIV-1 non-B subtypes in the different European countries.

Characterization of the in vitro maturation of monocytes and the susceptibility to HIV infection.

The in vitro maturation of peripheral blood monocytes to macrophages can be followed morphologically, and by measurement of cell surface antigens (CD4, HLA-DR, and FcR III) and lysozyme production. We used these markers to correlate monocyte maturation with susceptibility to human immunodeficiency virus (HIV) infection. Maturation of peripheral blood monocytes is associated with a decrease in membrane CD4, while HLA-DR and FcR III expression increase along with lysozyme secretion. Cells at all stages of maturation were susceptible to HIV infection, even mature macrophages without CD4 detectably by immunofluorescent staining. Maximal replication was observed in 7-day-old cells.

Intersubtype recombinant HIV type 1 involving HIV-MAL-like and subtype H-like sequence in four Norwegian cases.

Suspected epidemiological links between three cases of human immunodeficiency virus type 1 (HIV-1) infection were verified by the finding of a shared unique virus genotype. A probable male index case was not available for testing. Case 1 was a female sexual partner of the index case. Case 2 was an adult son of case 1. Case 3 was a female sexual partner of case 2. The link to the index case was substantiated by the subsequent finding of another female sexual contact of the index case, harboring the same HIV-1 genotype as the three other cases. To characterize the genotype further, the complete provirus nucleotide sequence was obtained directly from blood cell DNA of case 3. HIV cultivated from case 3 demonstrated CCR5 dependence, an extreme slow-low phenotype, and some genotypic features not present in its directly sequenced counterpart. Most of the gag, pol, and vif genes of these viruses clustered with one of the earliest African HIV-1 strains, MAL, previously classified as a recombinant between the subtypes A, D, and I. Most of the rest of the genome was related to subtype H, albeit with less than 90% identity in most regions. These viruses are the only ones shown to display extensive similarity with MAL in the gag-pol region and among the first HIV-1 recombinants described involving subtype H. We postulate that the gag-pol genes of MAL and these viruses are derived from a common ancestor that is not necessarily intersubtype recombinant in the pol region.

Correlates of apoptosis of CD4+ and CD8+ T cells in tonsillar tissue in HIV type 1 infection.

The immunopathogenesis of human immunodeficiency virus type 1 (HIV-1) infection has been associated with increased death by apoptosis of T cell subsets. In the present study, we have examined correlates of apoptosis of CD4+, CD8S+CD28+, and CD8+CD28- T cells in tonsillar lymphoid tissue in persons with HIV-1. Single-cell suspensions of tonsillar lymphocytes were analyzed by flow cytometry to determine the fraction of cells showing typical characteristics of apoptosis as well as the expression of activation markers within the live and the apoptotic cell populations. The proportion of cells carrying infectious provirus was quantified by limiting dilution analysis. Compared with uninfected controls, apoptosis of both CD4+ and CD8+ T cells was enhanced in HIV-1 infection and was higher among CD8+ than among CD4+ T cells. Apoptosis of CD28-cells was more prevalent than apoptosis of CD28+ cells for both CD4+ and CD8+ T cells. Occurrence of apoptosis of CD4+ T cells correlated with provirus levels and proportional expression of the activation marker HLA-DR. Apoptosis of CD8+CD28+ cells correlated with expression of the activation markers CD69 and HLA-DR while apoptosis within CD8+CD28- cells did not correlate with any of the studied parameters. Although apoptosis was much more prevalent among CD8+ than CD4+ T cells, CD8+ T cells still accumulated in tonsillar lymphoid tissue in persons with HIV-1. Our data may be interpreted to suggest that apoptosis of CD4+, CD8+CD28+, and CD8+CD28- cells in tonsillar tissue is regulated by different mechanisms and the results are of importance to our understanding of the immunopathogenesis of HIV-1 infection.

Characterization of HIV type 1 from Romanian children: lack of correlation between V3 loop amino acid sequence and syncytium formation in MT-2 cells.

The biological properties and amino acid sequences of the third variable domain (V3 loop and flanking regions) of the env region of 34 HIV-1 isolates obtained from Romanian children were analyzed. Unambiguous nucleic acid sequences were obtained from 31 isolates. The derived V3 amino acid sequences were highly homologous (93-100%) and clustered with the HIV-1 subtype F Romanian consensus. Five of the 31 isolates presented a syncytium-inducing phenotype in MT-2 cells and established continuous viral replication in various CD4+ cell lines (rapid/high phenotype). The V3 sequence from one of these isolates showed a slightly lesser degree of homology with the consensus sequence. The presence of positively charged amino acids at positions 306 and 320 has been strongly associated with the ability to induce syncytia in MT-2 cells, whereas negatively or uncharged amino acids at these positions are present in non-syncytium-inducing isolates (slow/low phenotype). There was, however, no correlation between phenotype and amino acid sequence in the five syncytium-inducing isolates; negatively or uncharged amino acids were conserved at positions 306 and 320 for all 31 isolates in sequences obtained from PBMCs. A tendency toward a more positive net charge in the V3 loop of syncytium-inducing isolates was noted. These data confirm the recent observations that HIV-1 isolates from Romania not only cluster in subtype F, but also show a high degree of interpatient homogeneity in the V3 region.(ABSTRACT TRUNCATED AT 250 WORDS)

CD8+ T cells from HIV type 1-seronegative individuals suppress virus replication in acutely infected cells.

CD8+ lymphocytes (CD8 cells) have been shown to inhibit replication of the human immunodeficiency virus (HIV) in vitro when cocultured with HIV-infected CD4+ lymphocytes (CD4 cells). This suppressive effect on HIV replication in experimentally infected CD4 cells has so far been demonstrated only for CD8 cells from HIV-seropositive individuals. In the present study we have investigated if CD8 cells from HIV-negative individuals can also suppress HIV replication in experimentally infected CD4 cells. Positively selected CD4 cells were infected with phenotypically different primary isolates of HIV type 1 and 2 (HIV-1 and HIV-2). Graded numbers of CD8 cells were added to the infected cultures. The T cells were activated by antibodies directed against the CD3 molecule or the T cell receptor. Culture supernatants were harvested for HIV p24 quantitation and the CD8 suppression of HIV replication was calculated by comparing p24 levels from parallel cultures in the presence or absence of CD8 cells from different donors. We show that CD8 cells from unexposed HIV-seronegative blood donors are able to control HIV-1 and HIV-2 replication in experimentally infected autologous CD4 cells. The antiviral activity of CD8 cells from and HIV-naive individual was reproducible over time and the suppressive effect was comparable to that seen with CD8 cells from HIV-positive individuals. The infected cells were not eliminated from the cultures. The suppressive effect of CD8 cells varied depending on the dose and biological phenotype of the virus used for infection. Thus, exposure to HIV in vivo is not a prerequisite for CD8 cells to exert a suppressive effect on HIV replication in acutely infected cells.

Normalization of CD4+ cell numbers and reduced levels of memory CD8+ cells in blood and tonsillar tissue after highly active antiretroviral therapy in early HIV type-1 infection.

Antiretroviral therapy increases the number of both CD4+ and CD8+ T cells in the blood of HIV-1-positive patients with advanced disease. In the present study, we have examined the kinetics of CD4+ and CD8+ T cell restoration in blood and lymphoid tissue in asymptomatic HIV-1-positive individuals with high CD4+ cell counts during highly active antiretroviral treatment. Tonsillar biopsies and blood samples were collected at baseline and at regular intervals during the following 48 weeks and from HIV-1-negative controls. Mononuclear cells from blood and tonsils were phenotyped and quantified by three-color flow cytometry. After 48 weeks of therapy, blood CD4+ cell counts in the HIV-1-infected group were comparable to those found in uninfected controls. Naive CD4+ T cells in blood increased during the initial 2 weeks in parallel with reduced plasma viremia. Both naive and memory CD4+ T cells in blood reached normal numbers by week 48, whereas the CD4+ naive/memory cell ratio in tonsils was within normal range throughout the study. The level of memory CD8+ T cells in blood declined during the first 8 weeks in parallel with a reduction in the tonsillar memory CD8+ T cells. Naive CD8+ T cells in the blood increased after 4 weeks, while the level of naive CD8+ T cells in tonsils remained unaltered. Our data indicate that in the early stages of HIV-1 infection antiretroviral therapy normalizes CD4+ cell counts and causes a decrease in the level of memory CD8+ cells in blood and lymphoid tissue, suggesting reduced CD8+ cell turnover in response to reduced viral replication.

Residual human immunodeficiency virus type 1 infection in lymphoid tissue during highly active antiretroviral therapy: quantitation and virus characterization.

HIV-1 can persist in infected patients despite undetectable plasma viremia. To characterize the residual viral load, repetitive blood and tonsillar samples were collected from 11 HIV-1-positive individuals before and during 96 weeks of therapy with zidovudine, lamivudine, and indinavir. HIV-1 RNA in tonsils was quantified by RT-PCR and infectious HIV-1 provirus by the limiting dilution assay. Genotypic resistance analyses and biological characterization were performed on plasma virus, blood, and tonsillar isolates. Tonsillar infectious HIV-1 provirus and HIV-1 RNA declined by 2 and 3 log(10), respectively, but 10(3)-10(4) cells, less than 0.5% of the total body CD4(+) T cell population carrying infectious HIV-1 provirus, remained involved in active viral replication of drug-sensitive R5 viruses. Thus, the dominant HIV-1 residual infection consists of < or = 10(6) latently infected CD4(+) cells. Plasma HIV-1 RNA decline of > 1.5 log(10) during the first 2 weeks of therapy may indicate low levels of this latent reservoir. Whereas the reservoir of latently infected cells remains stable, actively replicating HIV-1 continuously declines during prolonged antiretroviral therapy. Thus, although viral eradication seems unlikely, antiretroviral therapy may induce an extended period of virologic latency in HIV-1-positive individuals.

Determinants for the syncytium-inducing phenotype of HIV-1 subtype F isolates are located in the V3 region.

The HIV-1 syncytium-inducing phenotype is determined by virus replication and the presence of cytopathic effects in MT-2 cells. There is a strong correlation between the syncytium-inducing/MT-2-tropic phenotype and positively charged amino acids at positions 306 and 320 in the V3 loop for HIV-1 subtypes A, B, D, and E. In contrast, a lack of correlation between signature amino acids and syncytium formation in MT-2 cells for subtype F viruses from Romania has been reported. Virus phenotype and V3 loop amino acid sequences from Romanian HIV-1 subtype F isolates were further investigated in the present study. While the determinants of MT-2 tropism are clearly harbored in the V3 loop of subtype F isolates from Romania, the induction of syncytium formation occurs in the presence or absence of positively charged amino acids at positions 306, 320, and/or 324. However, the net positive charge of V3 loop sequences derived from syncytium-inducing viruses was higher than that of the nonsyncytium-inducing isolate.

Radioimmunoprecipitation and Western blotting with sera of human immunodeficiency virus infected patients: a comparative study.

The sensitivity and specificity of radioimmunoprecipitation assay (RIPA) and Western blot (WB) test were compared by use of a collection of 183 sera, representing different categories of individuals, noninfected or infected with the human immunodeficiency virus (HIV). The sera were subdivided on the basis of their reactivity in at least two anti-HIV enzyme-linked immunosorbent assays (ELISA); 53 sera were negative and 61 sera were positive in both tests, whereas 69 sera showed ambiguous reactions. The reaction patterns in RIPA could be divided into 6 different groups. The same grouping could to some extent be applied to the results of WB test. RIPA provided the most efficient means for identification of the large viral envelope glycoproteins, gp120/160, whereas gp41 was detected more effectively by WB. Internal virus components reacted to a varying extent with specific antibodies in the two tests. The reaction with pol products was more pronounced with the WB tests in which extracellular material was used as antigen. In a few WB tests, however, the reaction with internal components did not reflect a prior HIV infection. No such ambiguity was observed with RIPA, reflecting the advantage of a test that uses a minimally denaturated antigen and provides appropriate conditions for identification of the large viral glycoproteins. The practical choice of confirmatory tests to be used in diagnostic laboratories requires evaluation of both the sensitivity and specificity of the tests but also their economy and convenience.