AIDS Retrovirus Research Articles

RNA Packaging in HIV.

Successful replication of the AIDS retrovirus, HIV, requires that its genomic RNA be packaged in assembling virus particles with high fidelity. However, cellular mRNAs can also be packaged under some conditions. Viral RNA (vRNA) contains a 'packaging signal' (ψ) and is packaged as a dimer, with two vRNA monomers joined by a limited number of base pairs. It has two conformers, only one of which is capable of dimerization and packaging. Recent years have seen important progress on the 3D structure of dimeric ψ. Gag, the protein that assembles into the virus particle, interacts specifically with ψ, but this is obscured under physiological conditions by its high nonspecific affinity for any RNA. New results suggest that vRNA is selected for packaging because ψ nucleates assembly more efficiently than other RNAs.

AIDS: “It’s the bacteria, stupid!”

Acid-fast tuberculous mycobacterial infections are common in AIDS and are regarded as secondary "opportunistic infections." According to the National Institute of Allergy and Infectious Diseases, TB is the major attributable cause of death in AIDS patients. Could such bacteria play a primary or causative role in AIDS? Certainly, In screening tests for HIV, there is frequent, up to 70%, cross-reactivity, between the gag and pol proteins of HIV and patients with mycobacterial infections such as tuberculosis. By 1972, five years before gays started dying in the U.S., Rolland wrote Genital Tuberculosis, a Forgotten Disease? And ironically, in 1979, on the eve of AIDS recognition, Gondzik and Jasiewicz showed that even in the laboratory, genitally infected tubercular male guinea pigs could infect healthy females through their semen by an HIV-compatible ratio of 1 in 6 or 17%, prompting him to warn his patients that not only was tuberculosis a sexually transmitted disease, but also the necessity of the application of suitable contraceptives, such as condoms, to avoid it. Gondzik's solution and date of publication are chilling; his findings significant. Since 1982 Cantwell et al found acid-fast bacteria closely related to tuberculosis (TB) and atypical tuberculosis in AIDS tissue. On the other hand molecular biologist and virologist Duesberg, who originally defined retroviral ultrastructure, has made it clear that HIV is not the cause of AIDS and that the so-called AIDS retrovirus has never been isolated in its pure state. Dr. Etienne de Harven, first to examine retroviruses under the electron, agrees. In 1993 HIV co-discoverer Luc Montagnier reported on cell-wall-deficient (CWD) bacteria which he called "mycoplasma" in AIDS tissue. He suspected these as a necessary "co-factor" for AIDS. Remarkably, Montagnier remained silent on Cantwell's reports of acid-fast bacteria which could simulate "mycoplasma" in AIDS tissue. Mattman makes clear that the differentiation between mycoplasma and CWD bacteria is difficult at best and cites Pachas's 1985 study wherein one mycoplasma was actually mistaken for a CWD form of a bacterium closely related to the mycobacteria. It is important to realize that the statement "HIV is the sole cause of AIDS" is just a hypothesis. There are unanswered questions and controversy concerning the role of HIV "as the sole cause of AIDS." And until they are resolved, a cure is not possible. This paper explores the possible role of acid-fast tuberculous mycobacteria as "primary agents" in AIDS.

Distribution and three-dimensional structure of AIDS virus envelope spikes

Envelope glycoprotein (Env) spikes on AIDS retroviruses initiate infection of host cells and are therefore targets for vaccine development. Though crystal structures for partial Env subunits are known, the structure and distribution of native Env spikes on virions is obscure. We applied cryoelectron microscopy tomography to define ultrastructural details of spikes. Virions of wild-type human immunodeficiency virus 1 (HIV-1) and a mutant simian immunodeficiency virus (SIV) had approximately 14 and approximately 73 spikes per particle, respectively, with some clustering of HIV-1 spikes. Three-dimensional averaging showed that the surface glycoprotein (gp120) 'head' of each subunit of the trimeric SIV spike contains a primary mass, with two secondary lobes. The transmembrane glycoprotein 'stalk' of each trimer is composed of three independent legs that project obliquely from the trimer head, tripod-like. Reconciling available atomic structures with the three-dimensional whole spike density map yields insights into the orientation of Env spike structural elements and possible structural bases of their functions.

Dilated Cardiomyopathy in Retrovirally Infected Mice: A Novel Model for Silent Viral DCM?

Dilated cardiomyopathy (DCM) is a clinically relevant disease that can occur independently or secondary to other diseases such as HIV infection and AIDS. To study this latter process, we used a model in which mice are infected with the LP-BM5 murine AIDS (MAIDS) retrovirus. Cardiac function of control and infected mice was determined through the in vivo analysis of left ventricular pressure-volume loops. Furthermore, the role of myocarditis was investigated through immunohistochemistry for T-cell, B-cell, and macrophage cardiac infiltrates and Northern blot analysis for tumor necrosis factor (TNF)-alpha and inducible nitric oxide synthase (iNOS). End-systolic and end-diastolic volumes were significantly increased and ventricular stiffness was significantly decreased in infected mice, consistent with DCM; however, no staining for inflammatory cellular infiltrates or TNF-alpha and iNOS was seen. These data support the conclusion that the LP-BM5 HIV model virus causes DCM in the absence of chronic cardiac inflammation. These findings support MAIDS retroviral infection as a new model of idiopathic DCM in which myo-carditis does not occur.

Improved Mg2+-based reverse transcriptase assay for detection of primate retroviruses.

The reverse transcriptase (RT) assay is a simple, relatively inexpensive, widely used assay that can detect all retroviruses (known and novel retroviruses as well as infectious and defective retroviruses) on the basis of the divalent cation requirement of their RT enzyme, i.e., Mg2+ or Mn2+. Descriptions of various RT assays have been published; however, they cannot be directly applied to the analysis of biological products or clinical samples without further standardization to determine the lower limit of virus detection (sensitivity), assay variability (reproducibility), or ability to detect different retroviruses (specificity). We describe the detection of type E and type D primate retroviruses, which may be pathogenic for humans, by a new 32P-based, Mg2+-containing RT assay. The results show that the sensitivity of detection is <3.2 50% tissue culture infective doses (TCID50s) for human immunodeficiency virus type 1 (HIV-1) and <1 TCID50 for simian immunodeficiency virus isolated from a rhesus macaque (SIVmac). Analysis of recombinant HIV-1 RT enzyme indicated that 10(-5) U, which is equivalent to 4.25 x 10(4) virions, could be detected. Additionally, genetically distinct type D retroviruses such as simian AIDS retrovirus and squirrel monkey retrovirus were also detected in the assay with similar sensitivities. Thus, the improved RT assay can be used to detect genetically divergent Mg2+-dependent retroviruses of human and simian origin that can infect human cells and that therefore pose a potential health risk to humans.

Attitudes toward early detection of infection by the AIDS retrovirus among persons at high and low risk

Several months prior to the availability of a now-common blood test, homosexual volunteers, in a longitudinal study of their health, and typical college students were questioned about their attitudes toward early detection of acquired immunodeficiency syndrome (AIDS) and toward receiving information about desirable or fear-worthy hypothetical events. Generally, the homosexual volunteers were more interested in receiving information than were the students. Most homosexual volunteers desired human immunodeficiency virus (HIV) antibody test results, which could indicate infection, and indicated that receiving them would affect their behavior. However, all the subjects showed a preference for information on most questions, especially when the information could be used in guiding behavior change and was not virtually certain to be unfavorable. We are unaware of controlled evidence assessing whether or how knowledge of infection influences the imminent health risk of people who are asymptomatic, but prior to learning their HIV status, the homosexual volunteers who at the time had antibody in their sera were less concerned in knowing the test results of hypothetical future sexual partners than those without antibody.

Antiviral activity of a synthetic double-stranded polyribonucleotide interferon inducer in a murine AIDS retrovirus model. Role of augmentation of natural killer cell activity and synergy with oral AZT.

Antiviral activity of a synthetic double-stranded polyribonucleotide interferon inducer in a murine AIDS retrovirus model. Role of augmentation of natural killer cell activity and synergy with oral AZT.

Dissemination of enteric Mycobacterium avium infections in mice rendered immunodeficient by thymectomy and CD4 depletion or by prior infection with murine AIDS retroviruses.

This study shows that infection of mice with the murine AIDS virus LP-BM5 or Du5H profoundly depressed the capacity of splenic T cells from these animals to respond to the T-cell mitogen phytohemagglutinin or concanavalin A or to alloantigens. Similar effects were also observed if mice were thymectomized and then infused with monoclonal anti-CD4 antibody (TxCD4- mice). When such mice were infected intravenously with Mycobacterium avium, growth of the infection was markedly exacerbated in the TxCD4- mice or in mice given murine AIDS virus 2 months earlier. In view of these data, we then investigated whether such treatments might cause dissemination of M. avium following enteric implantation of bacteria into the mouse cecum; this route was chosen in an attempt to model events in AIDS patients, in which the gut appears to be one of the major portals of M. avium infection. In this model, the entry and hematogenous dissemination of four clinical isolates of M. avium were monitored against time and found to be accelerated and enhanced in T-cell-deficient mice. In view of this finding, these novel approaches for enteric infection that use immunodeficient mice are presented as potential new models for the evaluation of immunotherapy and chemotherapy in a setting that bears some similarity to events believed to occur in AIDS patients.

Human immunodeficiency virus (HIV) type 1 can superinfect HIV-2-infected cells: pseudotype virions produced with expanded cellular host range.

In studies on viral interference, cloned T-cell lines chronically infected with human immunodeficiency virus (HIV) type 1 or HIV-2 were inoculated with several strains of these two AIDS retrovirus subtypes. HIV-2UC1-infected cells, which still express the CD4 receptor, could be superinfected with a variety of HIV-1 and HIV-2 strains. This event was accompanied by cytopathic effects in the cells and production of pseudotype virions with an expanded cellular host range. HIV-1- or HIV-2-infected clonal cell lines, which did not express CD4, could not be superinfected by any HIV strains but were coinfected after transfection of molecular clones into the persistently infected cells. These observations indicate that viral interference with HIV occurs at the cell surface and involves a down-modulation of the CD4 molecule. If the CD4 protein is expressed, superinfection can take place, and phenotypically mixed virus particles are produced. Since HIV-1 and HIV-2 dually infected individuals have been detected, these in vitro observations may have relevance to the in vivo state.

Protease Inhibitors as Potential Therapeutic Agents for AIDS

A decade since the epidemic of the acquired immunodeficiency syndrome (AIDS) was first recognized, a wealth of information has accumulated on the molecular biology of the causative agents, the human immunodeficiency viruses (HIV). Of particular interest is knowledge of the viral enzymes involved in the formation of new virus particles. Such enzymes constitute attractive targets for efforts aimed at selecting agents that interfere with virus multiplication and subsequent spread and pathogenesis. Already, several agents that inhibit the viral reverse transcriptase (e.g., nucleoside analogs such as Zidovudine) have proved to have a beneficial effect on the course off the disease, but their prolonged use has been associated with significant toxicity and the emergence of resistant mutants. A second enzyme that has recently attracted attention is the virus-coded protease. This enzyme is involved in the cleavage of viral precursor polyproteins into the final products that constitute the mature virus particle. Protease inhibitors interfere with the process of virus maturation which is required for the formation of infective virus particles. Several custom-made inhibitors with a high selective action against HIV protease have been produced recently. They are nonhydrolyzable peptide analogs that mimic the cleavage sequences of the natural substrate of the enzyme during the transition state of the cleavage reaction. It is hoped that a similar selectivity in vivo may make protease inhibitors a promising new category of AIDS therapeutics.

9-(2-phosphonylmethoxyethyl)adenine (PMEA) effectively inhibits retrovirus replication in vitro and simian immunodeficiency virus infection in rhesus monkeys

9-(2-Phosphonylmethoxyethyl)adenine (PMEA) is a potent and selective inhibitor of the in vitro replication of a number of retroviruses, including HIV-1 and HIV-2, simian immunodeficiency virus (SIV), simian AIDS-related virus (SRV), feline immunodeficiency virus (FIV) and Moloney murine sarcoma virus (MSV). PMEA causes a dose-dependent suppression of the induction of anti-SIVmacgp120 antibodies in SIV mac-infected rhesus monkeys. Complete suppression of anti-SIVmacgp120 antibodies was achieved in SIV-infected animals treated with PMEA at 2 x 10 or 2 x 5 mg/kg per day for 29 days. No toxic side-effects were noted during this treatment period. Antibodies against SIVmac gp120 appeared 1-2 weeks after PMEA treatment was stopped, but the antibody titre reached in these animals was significantly lower than in the SIVmac-infected animals who had not been treated with PMEA. Our data strongly suggest that PMEA should be pursued for its potential in the treatment of AIDS and other retrovirus infections.

Differences in transcriptional enhancers of HIV-1 and HIV-2. Response to T cell activation signals.

T cell activation results in high levels of HIV replication and is thought to be one mechanism leading to the conversion from latent to active viral infection. In HIV-1, the sequences that respond to these signaling events are found in the long terminal repeat (LTR) and comprise the transcriptional enhancer, which contains two conserved binding sites for the nuclear factor kappa B (NF kappa B). The corresponding region in the second AIDS retrovirus, HIV-2, contains a conserved and a divergent NF kappa B binding site. We demonstrate that the HIV-1 LTR responds better than the HIV-2 LTR to T cell activation signals. These qualitative differences in the response to T cell activation are reproduced not only when HIV-1 or HIV-2 enhancers are placed upstream of a heterologous promoter but also when these enhancers are switched between their respective LTR. In electrophoretic mobility shift assays, NF kappa B binds to both conserved sites in the HIV-1 transcriptional enhancer and only to the single conserved site in the HIV-2 transcriptional enhancer. Instead of NF kappa B, the activator protein 3 binds to the divergent site in HIV-2. In conclusion, HIV-1 and HIV-2 are differentially regulated by T cell activation signals, and this difference may account for the longer period of viral latency observed with HIV-2 than with HIV-1 infection.

Synthetic peptides of envelope proteins of two different strains of simian aids retrovirus (SRV-1 and SRV-2) represent unique antigenic determinants for serum neutralizing antibodies

There are at least three distinct serotypes of simian type D retrovirus (SRV) which exhibit extensive serological cross-reactivity, but no cross-reactivity exists at the level of serum neutralizing antibodies. Amino acid sequence analysis and hydrophobicity plots of SRV-1 and SRV-2 envelope proteins were compared in order to identify unique potential antigenic determinants to which respective neutralizing antibodies may be directed. Peptides representing residues 147–162 of SRV-1 and 96–102 of SRV-2 were synthesized and assessed for their immunoreactivity. Free peptide inhibition of strain-specific serum (rhesus) neutralizing antibodies to SRV-1 and SRV-2 was demonstrated using the SRV-1 147–162 peptide and the SRV-2 peptide, 96–102, respectively. Inhibition of serum neutralizing activity by these peptides was also strain-specific, showing no cross-inhibition. SRV-1 147–162 conjugated to a protein carrier and cross-linked to Sepharose beads specifically adsorbed neutralizing antibodies from SRV-1 immune rhesus sera. The antibodies eluted from the immunoadsorbent possessed SRV-1 neutralizing activity, but showed no effect on the infectivity of SRV-2. Peptide SRV-1 147–162 also exhibited the capacity to bind specifically with a mouse monoclonal antibody which neutralizes the infectivity of SRV-1. Mice immunized with a recombinant SRV-1 envelope protein or with whole, inactivated SRV-1 produced antibodies which bound the SRV-1 147–162 conjugate, but not the protein carrier itself. Mouse antibodies to the SRV-1 147–162 conjugate exhibited specific binding with both natlve SRV-1 and with recombinant SRV-1 envelope protein. These findings provide strong evidence that SRV-1 147–162 and SRV-2 96–102 constitute at least two unique antigenic determinants, or parts thereof, which participate in the strain-specific neutralizing antibody response. Moreover, the findings indicate that the SRV-1 neutralizing antibodies produced by monkeys and at least a certain population of neutralizing antibodies produced by mice recognize the same epitope of SRV-1.

Biology of Retroviruses: Detection, Molecular Biology, and Treatment of Retroviral Infection

Although human retrovirology can trace its roots to the early part of this century, it has been within the last decade that the field has seen its greatest advances, including the description of the first human retroviruses, HTLV-I, and the subsequent discovery of HTLV-II and the AIDS retroviruses, HIV-1 and HIV-2. The growth this field has undergone in recent years would not have been possible had it not been for key advances in the fields of cell biology, immunology, biochemistry, and molecular biology. Nowhere has this been more evident than in the description of HIV as the etiologic agent of AIDS. In the relatively short time since the disease was initially described, the agent responsible has been isolated, much of its complex biology has been defined, and a drug that helps combat it has been licensed for use. The rapidity with which these events have transpired is largely unprecedented. There remain many unanswered questions regarding HIV and AIDS, and there still is no effective vaccine or cure. Significant questions regarding the immune response to HIV and the HIV response to immune or chemotherapeutic challenge, the establishment and maintenance of HIV latency, and the all-important mechanisms of HIV cytopathicity remain unanswered. It is clear that HIV offers no easy answers, and that significant work remains to be done before death from AIDS can be halted.

Inhibitory effects of 9‐(2‐Phosphonylmethoxyethyl)adenine and 3′‐azido‐2′,3′‐dideoxythymidine on tumor development in mice inoculated intracerebrally with moloney murine sarcoma virus

9-(2-Phosphonylmethoxyethyl)adenine (PMEA), a potent inhibitor of human immunodeficiency virus (HIV), caused a dose-dependent suppression of tumor formation, and mortality associated therewith, in 6-day-old NMRI mice inoculated intracerebrally with Moloney murine sarcoma virus (MSV). Even at a dose as low as 1 mg/kg/day, PMEA effected a significant delay in tumor formation. When evaluated in parallel with PMEA, 3'-azido-2',3'-dideoxythymidine (AZT) conferred a comparable tumor-inhibitory effect at a 5- to 10-fold higher dose than PMEA. Prolonged treatment of MSV-infected mice with PMEA resulted in long-term survivors without apparent signs of tumor development. In view of the propensity of HIV to spread to the central nervous system (CNS), the marked activity shown by PMEA against experimental retrovirus infection of the brain in mice points to its potential in the treatment of AIDS and other retrovirus infections of the CNS.

Toxicity and efficacy of 2',3'-dideoxycytidine in clinical trials of pigtailed macaques infected with simian retrovirus type 2

Four dosing regimens of 2',3'-dideoxycytidine (ddC) were administered intravenously for 10 to 28 days to 18 pigtailed macaques with simian acquired immunodeficiency syndrome. Ten macaques naturally infected with simian acquired immunodeficiency syndrome retrovirus serotype 2 (SRV-2), the etiologic agent of simian acquired immunodeficiency syndrome, received ddC by continuous intravenous infusion or by a daily bolus injection for 10 to 12 days. Another eight macaques that were negative for SRV-2 and antibody received ddC prophylaxis prior to challenge with virus and continued to receive ddC therapy for up to 28 days postchallenge. All monkeys treated with a continuous intravenous dose of ddC, which maintained plasma concentrations of ddC at levels known to inhibit SRV-2 in vitro, developed dose-related toxic effects, including leukopenia, anemia, lethargy, and decreased appetite. Monkeys treated with a daily bolus injection of ddC experienced more severe toxic effects than those on the continuous intravenous regimen, including exfoliative dermatitis and peripheral neuropathy. At the concentrations of ddC administered, no significant inhibition of SRV-2 replication was detected in naturally infected macaques. However, a prophylactic regimen of ddC did have an inhibitory effect on SRV-2. Our findings suggest that ddC may be valuable as a short-term prophylactic treatment rather than as a long-term therapy.

Infection of Macaque Monkeys With Simian Immunodeficiency Virus From African Green Monkeys: Virulence and Activation of Latent Infection

The virulence of three isolates of simian immunodeficiency virus from African green monkeys (SIVagm) was studied in rhesus and pigtailed macaques. None of 15 rhesus monkeys and one of four pigtailed monkeys died from infection during the time they were studied (up to 33 months). SIVagm was only isolated from rhesus monkeys for up to 2 months after inoculation. However, when these animals were secondarily infected with Simian acquired immunodeficiency syndrome retrovirus type 1 (SRV-1), SIVagm was activated and isolated. Dual infection caused increased mortality.

Mucosal Epithelial Cells and Langerhans Cells are Targets for Infection by the Immunosuppressive Type D Retrovirus Simian AIDS Retrovirus Serotype 1

Simian acquired immune deficiency syndrome (SAIDS) caused by the type D retrovirus SRV-1 results in opportunistic infections and a spectrum of oral lesions similar to those seen in humans with AIDS. To better understand the pathogenesis of these oral lesions we have retrospectively examined the oral mucosa from ten rhesus monkeys that died with SAIDS and prospectively examined the oral mucosa of ten additional animals inoculated with SRV-1 to determine at what time, and in what cells SRV-1 infection of the oral mucosa occurs. Using single and double label immunohistologic techniques, and electron microscopy we detected SRV-1 in clusters of oral epithelial cells and rare Langerhans cells as early as 1 month postinoculation.

Herpes Simplex Virus Type 2 Infection of Unstimulated Human T-Lymphocytes

Unstimulated human leukemia T-cell lines (MOLT-4, MT-4) were tested for their susceptibility to herpes simplex virus type 2 (HSV-2) infection. Permissive infection of MT-4 cells was demonstrated by growth curve and infectious center assays. In growth curve experiments new progeny virus replication was detected by 24 hrs and maximum titers of HSV-2 replication were measured by 72 hrs after infection of MT-4 cells, whereas, MOLT-4 cells did not produce detectable infectious HSV-2 in growth curve experiments. It may be that a T-cell subset is involved with infectious HSV-2 production, since 5.7% of MT-4 cells were scored as infectious centers after HSV-2 infection compared to only 0.06% of MOLT-4 cells. Furthermore, HSV-2 infected MT-4 (45% of cells) and MOLT-4 cells (30% of cells) expressed viral induced antigen(s) detected by immunofluorescence assays. These data provide the first evidence of infectious HSV-2 replication in T-cells not prestimulated in vitro with mitogens, pharmacologic agents or growth factors. The establishment of T-cell systems that permit rapid and efficient replication of HSV-2 could greatly facilitate studies on interactions between human herpesviruses and AIDS retroviruses since recent published evidence indicates possible synergistic interactions between these virus groups.

Current strategies in the treatment of HIV infection.

N 1981, a new clinical entity was described when previously healthy, homosexual men were diagnosed with Pneumocystis carinii pneumonia (PCP), a disease generally seen in immunosuppressed patients, and Kaposi’s sarcoma (KS), a rare form of cancer. This illness was characterized by a profound underlying cellular immune dysfunction and was named Acquired Immunodeficiency Syndrome (AIDS) by the Centers for Disease Control (CDC) in 1982.’ In 1983, a human T-cell lymphotrophic retrovirus was identified as the etiologic agent of AIDS.2 Known variously as human T-cell lymphotrophic virus (HTLV-III), lymphadenopathy-associated virus (LAV) and AIDS-related virus (ARV),3 the International Committee on the Taxonomy of Viruses proposed that the AIDS retrovirus be officially designated human immunodeficiency virus (HIV).4 Soon after discovery of the virus, serologic tests for detecting antibody to HIV (ELISA, Western blot) also became available.5