Human RNA "rumor" viruses: the search for novel human retroviruses in chronic disease

Abstract

Retroviruses are an important group of pathogens that cause a variety of diseases in humans and animals. Four human retroviruses are currently known, including human immunodeficiency virus type 1, which causes AIDS, and human T-lymphotropic virus type 1, which causes cancer and inflammatory disease. For many years, there have been sporadic reports of additional human retroviral infections, particularly in cancer and other chronic diseases. Unfortunately, many of these putative viruses remain unproven and controversial, and some retrovirologists have dismissed them as merely "human rumor viruses." Work in this field was last reviewed in depth in 1984, and since then, the molecular techniques available for identifying and characterizing retroviruses have improved enormously in sensitivity. The advent of PCR in particular has dramatically enhanced our ability to detect novel viral sequences in human tissues. However, DNA amplification techniques have also increased the potential for false-positive detection due to contamination. In addition, the presence of many families of human endogenous retroviruses (HERVs) within our DNA can obstruct attempts to identify and validate novel human retroviruses. Here, we aim to bring together the data on "novel" retroviral infections in humans by critically examining the evidence for those putative viruses that have been linked with disease and the likelihood that they represent genuine human infections. We provide a background to the field and a discussion of potential confounding factors along with some technical guidelines. In addition, some of the difficulties associated with obtaining formal proof of causation for common or ubiquitous agents such as HERVs are discussed.

FIG. 1.

Retrovirus structure and replication. (a) Genome organization. The RNA and DNA forms of a generalized retrovirus genome are shown with conserved features. R, repeated region at termini of RNA genome; U5 and U3, unique elements close to the 5′ and 3′ ends, respectively, of the RNA genome; PBS, primer binding site used for initiation of reverse transcription; Ψ, encapsidation signal; PPT, polypurine tract. All infectious retroviruses have at least one splice donor (SD) and one splice acceptor (SA) site used for expression of a spliced transcript encoding env; some retroviruses have additional splice sites. During reverse transcription, the LTR is formed, which contains gene promoter and enhancer elements. At least four genes are present in all infectious retroviruses, gag, pro, pol, and env. Retroviral proteins are synthesized as large polyprotein precursors and later cleaved into the mature viral proteins matrix (MA), capsid (CA), nucleocapsid (NC), protease (PR), reverse transcriptase (RT), and integrase (IN) and into-the-surface (SU) and transmembrane (TM) glycoproteins. Specific retroviruses encode additional proteins with specialized functions in the viral life cycle or pathogenesis. (b) Comparison of proviral structures of MLV and HTLV-1 showing arrangement of ORFs for viral genes. (Panels a and b are adapted from reference with permission from Elsevier.) (c) Structure of a generalized retrovirus particle indicating virus capsid containing two copies of the RNA genome associated with NC protein, viral enzymes, and a cellular tRNA molecule. The capsid is contained within the viral lipid envelope, which is associated with the envelope glycoproteins. (d) Replication. Retroviruses infect their target cells by adsorption to one or more specific cell surface receptors. Binding leads to conformational changes in the envelope and receptor molecules that trigger fusion of the viral and cell membranes. Depending on the specific virus, this may occur at the plasma membrane or within endosomes following endocytosis. Fusion releases the viral core into the cytoplasm (uncoating), and reverse transcription is initiated, during which the single-stranded RNA genome is converted into a double-stranded DNA form. This DNA subsequently becomes integrated into the chromosomal DNA of the cell to form the provirus. The expression of viral genes and proteins requires the host cellular machinery for transcription and translation, although some retroviruses also encode proteins that can regulate these processes. The cellular specificity of expression is dependent on enhancer elements located in the LTR. Assembly of retroviral capsids occurs either in the cytoplasm prior to budding (betaretroviruses and spumaviruses) or at the plasma membrane concomitant with budding (all other retroviral genera). Once released, the retroviral protease is activated, and the viral polyproteins become cleaved into their mature forms. This maturation step is required for infectivity.

FIG. 2.

Retrovirus-like particles described in diseased human tissues and cultured cells. (a) LM7 particles from leptomeningeal cells from MS induced by ICP0 protein of herpes simplex virus type 1. (Reprinted from reference with permission of the publisher.) (b) Particles in cultured lymphocytes from MS. (Reprinted from reference with permission from Elsevier.) (c) Particles in SS salivary gland (see arrows). (Reprinted from reference with permission of the publisher.) (d) HICRV in ICL. Bar, 0.5 μm. (Reprinted from reference with permission.) (e) Virus-like particles in human milk. (Reprinted from reference by permission from Macmillan Publishers Ltd.) (f) Particles in PBC. (Reprinted from reference with permission of the publisher. Copyright 2003 National Academy of Sciences, U.S.A.) (g) Particles in myeloproliferative disease. Bar, 100 nm. (Reprinted from reference with permission from Elsevier.) (h) HERV-K in teratocarcinoma-derived cell lines labeled with gold anti-HERV-K Gag. (Reprinted from reference with permission from Elsevier.)

FIG. 3.

Electron micrographs of cell supernatants purified by sucrose density gradient centrifugation. Culture supernatants from EBV-transformed human B lymphocytes (a, b, d, and e) and HTLV-1-infected MT-2 cells (c and f) were concentrated by ultracentrifugation and then recentrifuged through a 10 to 60% sucrose density gradient. Fractions with a density typical of retroviruses (1.15 to 1.18 g/ml) were pooled, fixed in 2.5% paraformaldehyde-0.4% glutaraldehyde, and embedded in Epon resin. Ultrathin sections were stained with 1% uranyl acetate for 1 h and 1% lead citrate for 4 min and analyzed by transmission EM. Multiple vesicular particle-like structures can be observed. The origin of these structures is unclear, although they may be derived from cellular components such as polyribosomes, exosomes, and apoptotic blebs. Alternatively, because these cells were transformed with EBV, it is also possible that these structures are viral in origin (470). Panel f shows immunogold labeling of a Unicryl-embedded section with an anti-HTLV CA antiserum. Bar, 200 nm (C. Voisset, B. Mandrand, and G. Paranhos-Baccalà, unpublished data).

FIG. 4.

Identification of novel viral sequences. A generalized scheme summarizing the approaches taken by a number of groups is shown (e.g., see references , , , , and 539). Sequence data can be generated experimentally or collected directly from expression sequence databases. Bullet points indicate alternative procedures at each stage. EST, expressed sequence tag.

FIG. 5.

Scheme for recovery of viral nucleic acid from microarray spots. Hybridized viral sequences were physically scraped from a DNA microarray spot using a tungsten wire probe mounted on a micromanipulator, while the spots were visualized under fluorescence microscopy. Subsequently, the virus was identified by nucleic acid amplification, cloning, and sequencing. (Reprinted from reference with permission.)

FIG. 6.

Activation of HERV-K superantigen. Possible mechanisms for activation of a superantigen encoded by HERV-K18 on chromosome 1 (based on data from references , , , , , and 476). IgM, immunoglobulin M.