Roger J. Pomerantz

The cytidine deaminase CEM15 induces hypermutation in newly synthesized HIV-1 DNA

High mutation frequency during reverse transcription has a principal role in the genetic variation of primate lentiviral populations. It is the main driving force for the generation of drug resistance and the escape from immune surveillance. G to A hypermutation is one of the characteristics of primate lentiviruses, as well as other retroviruses, during replication in vivo and in cell culture. The molecular mechanisms of this process, however, remain to be clarified. Here, we demonstrate that CEM15 (also known as apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G; APOBEC3G), an endogenous inhibitor of human immunodeficiency virus type 1 (HIV-1) replication, is a cytidine deaminase and is able to induce G to A hypermutation in newly synthesized viral DNA. This effect can be counteracted by the HIV-1 virion infectivity factor (Vif). It seems that this viral DNA mutator is a viral defence mechanism in host cells that may induce either lethal hypermutation or instability of the incoming nascent viral reverse transcripts, which could account for the Vif-defective phenotype. Importantly, the accumulation of CEM15-mediated non-lethal hypermutation in the replicating viral genome could potently contribute to the genetic variation of primate lentiviral populations.

The challenge of finding a cure for HIV infection

Although combination therapy for HIV infection represents a triumph for modern medicine, chronic suppressive therapy is required to contain persistent infection in reservoirs such as latently infected CD4+ lymphocytes and cells of the macrophage-monocyte lineage. Despite its success, chronic suppressive therapy is limited by its cost, the requirement of lifelong adherence, and the unknown effects of long-term treatment. This review discusses our current understanding of suppressive antiretroviral therapy, the latent viral reservoir, and the needs for and challenges of attacking this reservoir to achieve a cure.

Human immunodeficiency virus type 1 in the semen of men receiving highly active antiretroviral therapy

BACKGROUND Highly active antiretroviral therapy can effectively decrease the levels of human immunodeficiency virus type 1 (HIV-1) virions in peripheral plasma and seminal fluid of infected men. Whether the genital tract of HIV-1-infected men who are receiving highly active antiretroviral therapy and who have no detectable virus in the peripheral plasma harbors replication-competent virus is not known. METHODS We collected peripheral-blood and semen samples from seven men with HIV-1 infections who were receiving highly active antiretroviral therapy and who had no detectable viral RNA (fewer than 50 copies per milliliter) in plasma and analyzed the samples for cell-associated proviral DNA using a quantitative polymerase-chain-reaction assay. Replication-competent viruses were evaluated by cell-coculture assays. Proviral DNA and replication-competent virus obtained from peripheral-blood and seminal cells were also analyzed by sequencing relevant viral genes. RESULTS Despite the long-term suppression of HIV-1 RNA in the plasma of the seven men, proviral DNA was detected in seminal cells in four. Replication-competent viruses were recovered from peripheral-blood cells in three men and from the seminal cells in two of these three men. The viruses recovered from the seminal cells had no genotypic mutations suggestive of resistance to antiretroviral drugs and were macrophage-tropic, a feature that is characteristic of HIV-1 strains that are capable of being sexually transmitted. CONCLUSIONS In HIV-1-infected men who are receiving highly active antiretroviral therapy and who have no detectable levels of viral RNA in plasma the virus may be present in seminal cells and therefore may be capable of being transmitted sexually.

Pathogenesis of Infection with Human Immunodeficiency Virus

The acquired immunodeficiency syndrome (AIDS) was first described in 1981, but it has since reached epidemic proportions, with over 38,000 cases reported in the United States alone and a three-year...

Cellular reservoirs of Hiv-1 in the central nervous system of infected individuals: identification by the combination of in situ polymerase chain reaction and immunohistochemistry

Objectives The majority of HIV-1-infected individuals manifest a plethora of central nervous system (CNS) diseases unrelated to opportunistic infections, including AIDS dementia complex, encephalitis, and various other disorders of the CNS. The present study sought to evaluate the cellular reservoirs and expression patterns of HIV-1 in brain tissue to gain further understanding of HIV-1 neuropathogenesis. Design CNS tissue, obtained post-mortem from 22 patients with AIDS and four HIV-1-seronegative controls, was analyzed. Methods CNS samples were evaluated using a combination of in situ DNA polymerase chain reaction (PCR), reverse transcriptase (RT)-initiated in situ PCR, and immunohistochemistry. By utilizing this triple-staining methodology, HIV-1 proviral DNA and HIV-1-specific mRNA can be identified at the single cell level. Results HIV-1 was detected in all 22 AIDS brain specimens and in none of the four brains from HIV-1-seronegative individuals. The most commonly infected cells in AIDS brains were microglia cells and macrophages, but variable levels of HIV-1 infection were demonstrated in many of the major histological cell types within the CNS, including neurons, microvascular endothelial cells (MVEC) and astrocytes. The presence of HIV-1-infected cells was not uniform with infected cells unevenly distributed throughout the brain parenchyma. The degree of HIV-1 mRNA expression varied from 39–65% of the cells in the CNS harboring HIV-1 provirus. Choroid plexus and MVEC exhibited relatively high levels of productive infection. Conclusions These findings demonstrate that several cell types in the CNS, in addition to microglia or macrophages, may become infected with HIV-1 in vivo with variable levels of HIV-1 mRNA expression. The diverse cellular reservoirs for HIV-1 in the CNS may be critically linked to the molecular mechanisms involved in HIV-1 neuropathogenesis. In addition, in vivo infection of MVEC, and possibly cells in the choroid plexus, may directly contribute to penetration of the blood-brain barrier by HIV-1.

Inflammatory Reactions in HIV-1–Infected Persons after Initiation of Highly Active Antiretroviral Therapy

Highly active antiretroviral therapy (HAART)combination antiretroviral therapy that has potent in vivo effects on HIV-1 replicationhas led to significant decreases in AIDS-associated morbidity and mortality (1). Although patients receiving HAART have reduced plasma HIV-1 viral load and increased CD4+ T-lymphocyte counts, they still develop AIDS-defining events, particularly in the first 2 months of treatment (2, 3). A delay in restoration of immune function may account for the development of opportunistic infections. It is possible, however, that HAART may actually promote the clinical expression and development of such infections, as well as AIDS-related malignant conditions and other noninfectious diseases. Several authors have recently reported cases that may represent progression of previously quiescent disorders to symptomatic diseases after initiation of HAART. We searched MEDLINE for studies of such cases, which some authors have referred to as immune restoration disorders (4-8). Bibliographies of relevant studies were also reviewed. In this paper, we summarize clinical presentations (Table 1) and potential mechanisms of these conditions and describe therapeutic methods (Table 2). Table 1. Clinical Presentation of Specific Opportunistic Infections in HIV-1Infected Patients with and without Highly Active Antiretroviral Therapy Table 2. Successful Treatments Used for Opportunistic Infections in HIV-1Infected Patients after Highly Active Antiretroviral Therapy For the cases reported in this review, HAART refers to the initiation of a regimen that involves nucleoside reverse transcriptase inhibitors with one or more protease inhibitors or the addition of a protease inhibitor to a regimen of nucleoside reverse transcriptase inhibitors. Similarly, the usual response to HAART in the following cases involves marked reductions in plasma HIV-1 RNA levels and increases in CD4+ T-lymphocyte counts. It should be noted, however, that degrees of response to HAART varied. Mycobacterium avium Complex Several case reports have discussed the development of Mycobacterium avium complex and other nontuberculous mycobacterial infections in patients receiving HAART (6, 9-16). Many affected patients presented with focal or diffuse lymphadenitis within 2 months of initiating HAART. Mycobacteremia was rarely demonstrated, but the lymph nodes (some of which were suppurative) were usually culture positive for M. avium complex. In a retrospective chart review, Phillips and colleagues (17) found that patients who developed M. avium complex lymphadenitis within 12 weeks of starting HAART had higher CD4+ T-lymphocyte counts, were more likely to develop a localized draining sinus, and were less likely to have weight loss and disseminated M. avium complex disease than patients who had been receiving HAART for more than 12 weeks. Of interest, a case of recurrent leprosy, in the tuberculoid form secondary to increased antiM. leprae immunity, has been reported in an HIV-1infected person after initiation of HAART (18). Uncommon presentations of M. avium complex, such as granulomatous masses, osteomyelitis, bursitis, Addison disease, and skin nodules, were also seen after HAART (6, 11). Of note, similar focal M. avium complex disease was noted as early as 1988 in patients receiving zidovudine monotherapy (19, 20). In the above cases, antimycobacterial therapy, corticosteroids, or local surgical drainage alone was successful, usually in conjunction with HAART. Mycobacterium tuberculosis Inflammatory reactions in HIV-1infected patients receiving antituberculous therapy and HAART have been well documented (21-27). Affected patients had pansensitive pulmonary or extrapulmonary tuberculosis and developed inflammatory reactions several weeks after initiation of HAART despite initial response to antituberculous therapy. Examples of such reactions included fever, worsening pulmonary infiltrates, lymphadenopathy, and in some cases intracranial tuberculomas. Cultures of sputum and lymph nodes usually did not reveal acid-fast bacilli. All patients improved when anti-inflammatory agents were added to HAART. A case of regional lymphadenitis secondary to CalmetteGurin bacillus has also been reported in an HIV-1infected child after initiation of antiretroviral therapy (16). Narita and coworkers (24) noted that HIV-1infected patients treated with antituberculous therapy and HAART had a higher incidence of paradoxical reactions (new fever; worsening or emergence of lymphadenopathy, pulmonary infiltrates, or pleural effusion; or worsening of other tuberculous lesions) than HIV-1infected patients not treated with HAART. Patients in the former group developed paradoxical reactions a mean of 11 to 15 days after initiation of HAART and had a larger decrease in plasma HIV-1 viral load than those in the latter group. Cytomegalovirus Both ocular and extraocular cytomegalovirus infections have been reported after HAART. One of the earliest reports described five HIV-1infected patients with no history of ocular disease who received a first diagnosis of cytomegalovirus retinitis after CD4+ T-lymphocyte counts increased from less than 0.085 109 cells/L to more than 0.195 109 cells/L with HAART (28). Similarly, cytomegalovirus viremia, colitis, pancreatitis, and submandibular inflammation have also been reported after HAART in patients with no history of cytomegalovirus infection (29). Other authors have described the development of vitritis in patients treated with HAART who previously had active or inactive cytomegalovirus retinitis (30-35). Patients with a history of unilateral cytomegalovirus retinitis developed vitritis in the same eye after HAART. In one patient with vitritis, anterior chamber paracentesis showed no evidence of cytomegalovirus by polymerase chain reaction or antibody titer and no evidence of other infectious agents. Most cases improved without evidence of relapse, regardless of therapy (topical medications, periocular injection of steroids, or anticytomegalovirus agents). Karavellas and colleagues (36) conducted a cohort study of 30 patients with cytomegalovirus retinitis who responded to HAART. Nineteen patients (63%) developed immune recovery vitritis after a median of 43 weeks. All affected patients reported floaters, and 17 of 19 had decreased visual acuity. All patients had inactive cytomegalovirus retinitis in the affected eye when vitritis was diagnosed. In a retrospective review by Jabs and coworkers (37), this disorder was observed in only 6 of 33 patients, perhaps because of previous therapy with ganciclovir implants for cytomegalovirus retinitis. Hepatitis C Virus Several patients with previously quiescent hepatitis C virus (HCV) infection developed acute hepatitis, cirrhosis, or an HCV-associated disorder, such as cryoglobulinemia, within 1 to 9 months after initiation of HAART (5, 38-41). Hepatitis C virus was implicated because HCV IgG antibody developed or plasma HCV RNA level increased after introduction of HAART, although this was not observed in all cases. Some patients improved with interferon- therapy; however, in other patients, protease inhibitor therapy had to be discontinued. The response of plasma HCV RNA to HAART in patients co-infected with HIV-1 has been evaluated (40, 42-46). One study found that mean plasma HCV RNA level initially increased 6 weeks after addition of HAART and later decreased to below baseline, with minimal changes in serum aminotransferase levels (42). Other studies, however, noted a moderate increase in plasma HCV RNA level after HAART, and liver biopsy showed associated lobular necrosis and inflammation in some patients (40, 43, 46). Hepatitis B Virus Some studies have attributed acute hepatitis in the setting of HAART to hepatitis B virus (HBV) (47-51). Affected patients had previous HBV infection and developed clinical hepatitis 5 to 12 weeks after beginning HAART. Most of these patients demonstrated newly detectable plasma HBV RNA or increased levels of HBV RNA during acute hepatitis. In some cases, serologic tests for HBV also showed active HBV infection. Researchers discontinued HAART in one patient (51), but most other patients improved without changes in therapy. Cryptococcus and Histoplasma Species A case report of Histoplasma capsulatum infection and a retrospective review of cryptococcal infection occurring after HAART have been described. Bottaro and coworkers (52) described a 35-year-old South American HIV-1infected man with a CD4+ T-lymphocyte count of 0.005 109 cells/L who received a diagnosis of disseminated histoplasmosis on the basis of blood culture results. Successful treatment with amphotericin B was followed by itraconazole therapy. Three weeks after initiation of HAART, the patients inguinal lymph node became enlarged; excisional biopsy revealed a granulomatous reaction and H. capsulatum by culture. The CD4+ T-lymphocyte count had increased to 0.066 109 cells/L. Itraconazole therapy and HAART were continued, and the patients symptoms improved. A retrospective review of all HIV-1infected patients with culture-proven cryptococcal meningitis between 1996 and 1999 identified three patients who developed cryptococcal meningitis in temporal association with initiation of HAART (53). Two patients developed clinical meningitis within 1 to 6 weeks after introduction of HAART. Studies of cerebrospinal fluid were remarkable for leukocytosis (leukocyte count 14 109 cells/L) and the presence of Cryptococcus neoformans by culture. A third patient developed recrudescence of previously suppressed C. neoformans meningitis, also marked by cerebrospinal fluid leukocytosis (leukocyte count, 100 109 cells/L) and an elevated cerebrospinal fluid C. neoformans antigen titer within 10 days of beginning HAART. Therapy was not changed in this patient, and meningeal signs and symptoms resolved over 2 weeks. Herpes Zoster Several studies have reported development of herpes zoster after HAART (8, 54, 55).

Cells nonproductively infected with HIV-1 exhibit an aberrant pattern of viral RNA expression: A molecular model for latency

U1 and ACH-2 cells are subclones of HIV-1-infected monocyte/macrophage-like and T lymphocyte cell lines, respectively, which express the HIV-1 genome at very low levels. We have examined whether they might provide a model of HIV-1 latency. The patterns of HIV-1-specific RNA expressed in these cells consisted of singly and multiply spliced RNA species, with little or no full-length genomic RNA. Upon stimulation with agents that activate the HIV-1 long terminal repeat in these cells, a marked rise in the amount of small mRNAs, encoding the viral regulatory proteins, preceded the increase in the unspliced RNA. Thus, U1 and ACH-2 cells maintain HIV-1 in a state equivalent to the early phase of a lytic infection and, after stimulation, recapitulate the events of a single cycle infection of highly susceptible target cells.

Detection of Human Immunodeficiency Virus Type 1 Provirus in Mononuclear Cells by in Situ Polymerase Chain Reaction

BACKGROUND Studies of human immunodeficiency virus type 1 (HIV-1) infection have attempted to quantitate the viral load correlate it with the degree of immune deficiency. In one study, only about 1 in 10,000 peripheral-blood mononuclear cells (PBMC) expressed HIV-1, but in other studies, at least 1 in 100 CD4-positive cells was infected and harbored the HIV-1 provirus. METHODS We developed a new, highly sensitive in situ polymerase-chain-reaction (PCR) method that amplifies selected genetic regions within intact single cells. We used this technique to determine the proportion of PBMC carrying HIV-1 provirus in infected patients in different stages of disease. RESULTS None of the PBMC from 11 HIV-1--seronegative patients were found to be positive for HIV-1 provirus by the in situ PCR method. In 56 patients infected with HIV-1, the percentage of PBMC with HIV-1 ranged from 0.1 percent to 13.5 percent. The mean percentage of infected mononuclear cells was greater in 13 patients with persistent generalized adenopathy (mean, 6.6 percent) and 19 with the acquired immunodeficiency syndrome (Stages IV-A to IV-C) (4.6 percent) than in 19 patients with asymptomatic HIV-1 infection (0.9 percent) (P less than 0.001). However, in five patients with Kaposis sarcoma (Stage IV-D), an average of only 1.6 percent of mononuclear cells were infected. CONCLUSIONS In HIV-1 infection, the proportion of PBMC that are infected appears to be at least 10 times higher than previously described. It is likely that most infected cells contain HIV-1 provirus in a latent or defective form that was not detected in some earlier studies.

Twenty years of therapy for HIV-1 infection.

Antiretroviral therapy, where available, has transformed HIV-1 disease into a treatable and somewhat chronic infection. This article summarizes the accomplishments thus far and what lies ahead in our struggle to improve the treatment of, and possibly eliminate, HIV-1 infection.

IL-7 is a potent and proviral strain–specific inducer of latent HIV-1 cellular reservoirs of infected individuals on virally suppressive HAART

The persistence of HIV-1 in virally suppressed infected individuals on highly active antiretroviral therapy (HAART) remains a major therapeutic problem. The use of cytokines has been envisioned as an additional therapeutic strategy to stimulate latent proviruses in these individuals. Immune activation therapy using IL-2 has shown some promise. In the present study, we found that IL-7 was significantly more effective at enhancing HIV-1 proviral reactivation than either IL-2 alone or IL-2 combined with phytohemagglutinin (PHA) in CD8-depleted PBMCs. IL-7 also showed a positive trend for inducing proviral reactivation from resting CD4(+) T lymphocytes from HIV-1-infected patients on suppressive HAART. Moreover, the phylogenetic analyses of viral envelope gp120 genes from induced viruses indicated that distinct proviral quasispecies had been activated by IL-7, as compared with those activated by the PHA/IL-2 treatment. These studies thus demonstrate that different activators of proviral latency may perturb and potentially deplete only selected, specific portions of the proviral archive in virally suppressed individuals. The known immunomodulatory effects of IL-7 could be combined with its ability to stimulate HIV-1 replication from resting CD4(+) T lymphocytes, in addition to other moieties, to potentially deplete HIV-1 reservoirs and lead to the rational design of immune-antiretroviral approaches.