Human Retroviral Infections Part 1

Retroviruses have been recognized as important animal pathogens for more than 70 years. However, retroviruses were identified as etiologic agents in human disease only within the past 2 decades.1,2 Three retroviruses have been recognized to cause infections in humans and to produce well-defined clinical disease: human T cell lymphotropic virus type I (HTLV-I), HIV type 1 (HIV-1), and HIV type 2 (HIV-2). One retrovirus (human T cell lymphotropic virus type II [HTLV-II]) has been associated with certain rare hematologic malignancies, but evidence confirming an etiologic role of HTLV-II in these disorders is lacking. In addition, human foamy virus (HFV), a member of the genus Spumavirus, is likely acquired as a zoonotic infection after severe primate bites, but it has not been associated with human disease.3

Definition and Classification

Retroviruses are named for the action of a unique viral-encoded enzyme called reverse transcriptase. The normal flow of genetic information is from DNA to RNA to protein. In a reversal of that process, reverse transcriptase makes a double-stranded DNA copy (complementary DNA, or cDNA) of the single-stranded viral RNA genome—hence, the prefix retro. Retro-viruses are characterized morphologically by their appearance on electron microscopy and by their genomic, antigenic, and pathophysiologic characteristics.4 The family Retroviridae comprises a large group of ubiquitous viruses that infect all classes of vertebrates. On the basis of similarities in amino acid sequences in the reverse transcriptase proteins of retroviruses, seven retro-virus genera have been classified. Humans can be or have been infected with several retroviruses from five of the seven retro-virus genera [see Table I]. The alpharetroviruses, betaretrovirus-es, and gammaretroviruses are considered simple retroviruses; the deltaretroviruses, epsilonretroviruses, lentiviruses, and spuma-viruses are considered complex. The simple retroviruses encode only structural and envelope proteins, whereas the complex retroviruses encode these proteins and also small regulatory proteins with a range of functions.4 With regard to clinical infections in humans, the most important retroviruses (HTLV and HIV) and the least important retroviruses (human retrovirus type 5 [HRV-5]5 and HFV) are acquired by exogenous infection (i.e., by sexual transmission or by transmission from mother to infant through breast-feeding or by parenteral exposure).

A defining feature of retroviruses is the integration of the reverse-transcribed viral cDNA into the genome of the host cell. The integrated viral cDNA is termed the provirus; it serves as the template for viral replication. A persistent infection may result from this process of integration, or the infected cell may be transformed and a malignancy induced. Although many retroviruses can cause cytopathology in the host cell, most replicate without killing the infected cell. For the endogenous human retroviruses (human endogenous retrovirus type K [HERV-K], HERV-W, and several others) [see Table I], multiple copies of endogenous provi-ral DNA sequences are integrated into chromosomal DNA and are transmitted in the germline. Interestingly, endogenous provi-ral sequences represent 0.1% or more of human DNA sequences and were acquired sometime in our evolutionary past.

Retroviral Biology

Structure and replication

All human retroviruses are similar in structure, genomic organization, and mode of replication.4 The virus particles are approximately 100 nm in diameter and have a lipid envelope composed of components from the host cell-derived plasma membrane.6 The envelope renders the virus particles very susceptible to inactivation by environmental drying and by the action of detergents and various chemical disinfectants. The lipid envelope surrounds a dense nucleocapsid core that contains two copies of the unique single-stranded RNA genome. The RNA genomes range from 8 kb in length for HTLV to approximately 10 kb in length for HIV. In the virion, the RNA genomes are associated with the virus-encoded reverse transcriptase, integrase, vpr protein in HIV-1, vpx protein in HIV-2, and a primer transfer RNA (tRNA) that is derived from the host cell.7 The HIV virion must also incorporate a cellular protein, cyclophilin A, which binds to viral capsid matrix protein p17; this protein is necessary for successful viral disassembly.8 The tightly packaged HIV nucleocap-sid protein protects the enclosed RNA genome from degradation by ribonuclease (RNase) in plasma and other body fluids. As a durable marker of HIV-1 replication, the measurement of viral particle-associated RNA plays a central role in monitoring HIV disease progression and response to therapy.

The retroviral RNA genome and the cDNA provirus contain both noncoding and coding sequences [see Figure I]. The non-coding sequences, which are important recognition sites for DNA or RNA synthesis, integration, and polyadenylation, are located at the 5′ and 3′ terminal ends of the genome. All retro-viruses are terminally redundant and contain identical se-quences, called long terminal repeats (LTRs). The coding sequences include the gag gene, which encodes group-specific structural antigens; the pol gene, which encodes RNA-depen-dent DNA polymerase or reverse transcriptase, integrase, and protease; and the env gene, which encodes envelope structural proteins. The gag gene encodes a precursor polypeptide that is cleaved by viral-encoded protease to form several internal structural proteins—namely, matrix protein (MA), capsid protein (CA), and nucleic acid binding protein (nucleocapsid, or NC).

Table I Genera of the Family Retroviridae*

In addition, the 3′ end of the gag gene reading frame overlaps with the pol gene reading frame to encode for the virus-specific protease. The pol gene encodes for a precursor polypeptide that is cleaved by the virus-specific protease enzyme to form three enzymes: protease, reverse transcriptase, and integrase. The env gene encodes for a 160 kd precursor protein, which is cleaved posttranscriptionally into two noncovalently associated envelope glycoproteins during transport through the endoplasmic reticulum and the Golgi complex by host proteases, termed fu-rins. The first env protein is gp120, which is a highly charged glycoprotein that is external to the viral envelope; gp120 binds to cell-specific viral receptors (e.g., CD4+ T cell receptor in the case of HIV) and ancillary coreceptors (e.g., chemokine receptors for HIV-1). There are five domains of the env gene—V1 through V5. Some of these are highly variable (of these domains, the V3 loop is the most highly variable). These variable domains are responsible for defining multiple variants or quasi-species that contribute to viral evasion from neutralizing antibodies produced by the host in response to viral infection. The second env protein, gp41, is a hydrophobic transmembrane gly-coprotein that anchors the oligomeric surface subunit glycopro-tein to the viral envelope membrane. The virion surface is studded with approximately 72 knobs; each is composed of three heterodimers of the gp120env/gp41env complex.7,11 Fusion of the retroviral envelope with the target cell plasma membrane is facilitated by the transmembrane glycoprotein through confor-mational change mediated by a helix coiled-coil mechanism. This mechanism is present in enveloped viruses other than retroviruses (e.g., influenzavirus)12 [see Figure 2].

The complexity of the human retroviruses is best exemplified by the array of viral proteins that are responsible for regulating viral replication and the host cell response to infection.4,7 HTLV-I has a region between the env gene and the 3′ LTR that encodes for two regulatory proteins, tax and rex, which are produced from messages that are spliced differently from distinct overlapping reading frames. The tax protein induces the expression of cell transcription factors that alter host cell gene expression, and the rex protein regulates the expression of viral messenger RNA (mRNA). The HIV viruses have a larger genome than HTLV. In addition, HIV has a larger translated region (regulatory genes) between the pol and env genes, which encode portions of several regulatory proteins that depend on the overlapping open reading frame (ORF) into which the mRNA is spliced.

These retroviral regulatory proteins express the following functions13,14 [see Table 2]. The tat protein augments the expression of virus from the LTR region. The rev protein regulates RNA splicing and RNA transport, or both, in HIV and may function in a manner similar to that of the rex protein in HTLV. The nef protein downregulates CD4 protein, which is the cellular receptor for HIV; it alters host T cell activation pathways by decreasing major histocompatibility complex (MHC) class I antigen expression; and it enhances viral infectivity. The vif protein is necessary for the proper assembly of the HIV nucleopro-tein core; without the vif protein, viral cDNA is not efficiently produced. The vpr protein (in HIV-1) and the vpx protein (in HIV-2) facilitate transport of the viral cDNA into the nucleus

Figure 1 Depicted is the genomic organization of the three most important human retroviruses. The linear double-stranded proviral DNA forms of HTLV and HIV-1 show similar patterns of genomic organization. The structural genes gag, pol, and env give rise to several proteins: matrix (MA), capsid (CA), nucleic-acid binding (NB), nucleocapsid core proteins (NC), protease (PR), reverse transcriptase (RT), surface subunit glycoprotein (SU), and a smaller transmembrane protein (TM). In addition, HIV pol encodes an integrase (IN). There are additional regulatory gene products translated. HTLV-I and HTLV-II have tax and rex genes with exons on either side of the env gene. HIV-1 and HIV-2 have six accessory gene products: tat, rev, vif, nef, vpr, and either vpu (in HIV-1) or vpx (in HIV-2). Further information about these gene products is provided131 [see Table 2].

Figure 2 Illustrated is the binding of HIV-1 with a CD4+ T cell. In unbound virions, gp41 exists in a stable, nonfusogenic conformation in which the fusion peptides are buried within the envelope trimer complex (a). When gp120 binds to the CD4 receptor, a conformational change exposes the chemokine receptor attachment site on gp120 (either CXC chemokine receptor-4 [CXCR4] or CC chemokine receptor-5 [CCR5]) (b). This in turn triggers a transition of gp41 to the prehairpin intermediate (c), with exposure of the fusion peptide attached to the trimeric coiled-coil N-peptide region. The fusin peptide inserts into the target membrane (c). In this form, the C-peptide has not associated with the N-peptide because of continued association with gp120; at this stage, the intermediate gp41 polypeptide is vulnerable to C-peptide inhibition (e.g., T-20). When the C-peptide region binds to the N-peptide region coiled-coil, the complex adopts a helical conformation of the fusion-active hairpin, which brings the two membranes into apposition (d). The precise mechanism of membrane fusion is not clear, but after fusion is complete, the fusion peptide and transmembrane segment of gp41 lie in the same membrane (e). A similar mechanism presumably applies to the fusion of a cell infected with HIV-1 that expresses viral envelope on the plasma membrane surface with an uninfected CD4+ T cell, which leads to syncytium formation among infected and uninfected cells in vitro.

Pathogenetic tryptic

The biologic properties of the human retroviruses, summarized above, orchestrate a viral pathogenesis tryptic defined by latency, transformation, and cytopathicity; this process occurs through two replication phases [see Figure 3]. In the first replication phase, the viral-encoded proteins that are packed within the virion nucleocapsid enter the cell; this eventually results in the formation of the integrated cDNA provirus. In the second replication phase, the enzymatic machinery of the host cell is utilized to replicate the viral RNA genome and to transcribe and translate viral proteins from the provirus. With the exception of retroviral latency, the pathogenesis tryptic leads to cell dysfunction (and sometimes cell death), with eventual immunosup-pression and clinical disease. Both HTLV-I and HTLV-II immortalize primary human peripheral blood T cells in vitro. Immortalization is defined as interleuken-2 (IL-2)-dependent, long-term growth in culture. Subsequently, cells become fully transformed—that is, they are subject to continuous cell proliferation in the absence of IL-2. Cellular transformation is an early step in the pathogenic process of HTLV and is distinct from oncogenesis or malignancy.4 As such, HTLV transformation of T cells results in a pool of proliferating cells that are not oncogenic themselves but that provide a population from which a malignant clone may subsequently arise.4 This accounts for the long latent period between infection and tumor development in HTLV disease. In contrast, HIV does not transform the infected cell but rather produces a cytopathic effect that results in cell dysfunction and, eventually, cell death.

The first phase of viral replication starts when the retrovirus binds to a specific cell receptor, which for HIV is the CD4 molecule on the cell surface and an associated chemokine corecep-tor.15 The HTLV receptors have not been characterized; however, cellular targets for HTLV-I include several types of T cells, the primary one being the CD4+ T cell. HTLV-II preferentially infects CD8+ T cells.16 The retroviral nucleocapsid complex enters the cell cytoplasm, and the viral genomic RNA is reverse-transcribed to a slightly longer, double-stranded cDNA molecule that actively enters the nucleus as a nucleoprotein-preinte-gration complex and integrates permanently at a single random site in the cell chromosome. The integration of linear viral cDNA occurs during division of the cell; as such, nondividing cells are generally resistant to retroviral infection. However, the one exception to this is HIV, which is able to infect resting cells (the Gj phase of the cell cycle), albeit less efficiently than activated cells in the S phase of the cell cycle. The inhibition of reverse transcription by combinations of nucleotide, nucleoside, or non-nucleoside reverse transcriptase inhibitors (NNRTIs) is a cornerstone of current HIV-1 therapeutics.

The second replication phase begins with the production of viral genomic RNA and mRNA and protein synthesis; the production of mRNA and protein synthesis occur almost exclusive ly through the enzymatic machinery of the host cell under the influence of viral gene regulatory products. Processing of virion proteins begins in the endoplasmic reticulum and Golgi complex. Virion assembly begins at the plasma membrane, and the nascent virions are released from the cell surface through budding. The budding viral envelope, which has a phospholipid composition different from that of the plasma membrane of the cell, may incorporate some cell membrane surface proteins (e.g., P2-microglobulin and MHC class I and II proteins), along with virus-specific glycoproteins.6,7 Extracellular HIV undergoes further maturation by continued proteolytic cleavage of the nucle-ocapsid polypeptide, which results in the mature infectious viri-on with a distinctive cone-shaped nucleocapsid core. The inhibition of viral protease through virus-specific protease inhibitor therapy, in combination with reverse transcriptase inhibitor therapy, is an important aim of current antiretroviral therapeutics. However, inhibition of reverse transcriptase with combinations of nucleoside reverse transcriptase inhibitors (NRTIs) and NNRTIs is equally efficacious in many cases.

Table 2 Genes and Proteins of HIV¹³

*HIV-1 only tHIV-2 only.

Pathogenesis of Retroviral Infection

The clinical consequences of retrovirus infection reflect a precarious balance between the type of invading retrovirus, its tro-pism for specific cells and tissues, and the patient’s natural and acquired resistance. The pathophysiologic model of retrovirus infection can be approached by understanding viral mechanisms of infection, latency and persistence, cell injury, and immune system evasion and modulation. Some aspects of this model are shared by other RNA and DNA viruses; other aspects are unique to the human retroviruses.