Class Of Retroviruses Research Articles

Determinants of Retroviral Integration and Implications for Gene Therapeutic MLV-Based Vectors and for a Cure for HIV-1 Infection.

To complete their replication cycle, retroviruses need to integrate a DNA copy of their RNA genome into a host chromosome. Integration site selection is not random and is driven by multiple viral and cellular host factors specific to different classes of retroviruses. Today, overwhelming evidence from cell culture, animal experiments and clinical data suggests that integration sites are important for retroviral replication, oncogenesis and/or latency. In this review, we will summarize the increasing knowledge of the mechanisms underlying the integration site selection of the gammaretrovirus MLV and the lentivirus HIV-1. We will discuss how host factors of the integration site selection of retroviruses may steer the development of safer viral vectors for gene therapy. Next, we will discuss how altering the integration site preference of HIV-1 using small molecules could lead to a cure for HIV-1 infection.

Foamy Viruses, Bet, and APOBEC3 Restriction.

Non-human primates (NHP) are an important source of viruses that can spillover to humans and, after adaptation, spread through the host population. Whereas HIV-1 and HTLV-1 emerged as retroviral pathogens in humans, a unique class of retroviruses called foamy viruses (FV) with zoonotic potential are occasionally detected in bushmeat hunters or zookeepers. Various FVs are endemic in numerous mammalian natural hosts, such as primates, felines, bovines, and equines, and other animals, but not in humans. They are apathogenic, and significant differences exist between the viral life cycles of FV and other retroviruses. Importantly, FVs replicate in the presence of many well-defined retroviral restriction factors such as TRIM5α, BST2 (Tetherin), MX2, and APOBEC3 (A3). While the interaction of A3s with HIV-1 is well studied, the escape mechanisms of FVs from restriction by A3 is much less explored. Here we review the current knowledge of FV biology, host restriction factors, and FV–host interactions with an emphasis on the consequences of FV regulatory protein Bet binding to A3s and outline crucial open questions for future studies.

JUMP AROUND, JUMP AROUND: TRANSPOSABLE ELEMENT ACTIVATION IN NEURODEGENERATIVE TAUOPATHY

Transposable elements, or “jumping genes,” constitute ~45% of the human genome. We have identified transposable element activation as a key mediator of neurodegeneration in tauopathies, a group of disorders that are pathologically defined by deposits of tau protein in the brain. Cellular defenses that limit transposable element mobilization include 1) formation of silencing heterochromatin and 2) generation of piwi-interacting RNAs (piRNAs) that clear transposable element transcripts. Using genetic approaches in Drosophila models of tauopathy, we find evidence for a causal relationship between tau-induced heterochromatin decondensation and piRNA depletion, transposable element mobilization, and neurodegeneration. 3TC, an FDA-approved inhibitor of reverse transcriptase, suppresses transposable element mobilization and neuronal death in tau transgenic Drosophila. We detect a significant increase in transcripts of the human endogenous retrovirus class of transposable elements in postmortem human Alzheimer’s disease brains. Our data identify transposable element activation as a conserved, pharmacologically targetable driver of neurodegeneration in tauopathy.

Pathogenic tau-induced piRNA depletion promotes neuronal death through transposable element dysregulation in neurodegenerative tauopathies.

Transposable elements, known colloquially as “jumping genes,” constitute approximately 45% of the human genome. Cells utilize epigenetic defenses to limit transposable element jumping, including formation of silencing heterochromatin and generation of piwi-interacting RNAs (piRNAs), small RNAs that facilitate clearance of transposable element transcripts. Here we identify transposable element dysregulation as a key mediator of neuronal death in tauopathies, a group of neurodegenerative disorders that are pathologically characterized by deposits of tau protein in the brain. Mechanistically, we find that heterochromatin decondensation and reduction of piwi/piRNAs drive transposable element dysregulation in tauopathy. We further report a significant increase in transcripts of the endogenous retrovirus class of transposable elements in human Alzheimer’s disease and progressive supranuclear palsy, suggesting that transposable element dysregulation is conserved in human tauopathy. Taken together, our data identify heterochromatin decondensation, piwi/piRNA depletion and consequent transposable element dysregulation as a novel, pharmacologically targetable, mechanistic driver of neurodegeneration in tauopathy.

Evolutionary Paradigms from Ancient and Ongoing Conflicts between the Lentiviral Vif Protein and Mammalian APOBEC3 Enzymes.

Lentiviruses are a unique class of retroviruses that infect a subset of mammalian species, including humans. Human immunodeficiency virus type 1 and 2 (HIV-1 and HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), maedi-visna virus (MVV), and caprine arthritis encephalitis virus (CAEV) infections result in immunodeficiency syndromes, neurological diseases, and other conditions. Disease is chronic, lifelong, and often fatal. Pathology is due in part to a capacity to immortalize by integrating into a host’s genomic DNA and a remarkable genetic plasticity that enables escape from potent adaptive immune responses without compromising vital viral functions. Lentiviruses encode three universal retroviral proteins (Gag, Pol, and Env), one lentivirus-specific protein called Vif (virus infectivity factor), and a handful of other less conserved accessory factors. Vif is required to protect lentiviruses from restriction by several members of the APOBEC3 family of DNA cytosine deaminases by forming a cellular polyubiquitin ligase complex to degrade these enzymes (Fig 1A). Viruses lacking Vif are inactivated because cytoplasmic APOBEC3 enzymes remain abundant, package into assembling virus particles, and prevent the production of a viable DNA copy of the viruses’ RNA genome by catalyzing the deamination of viral cDNA cytosines to uracils. Viral DNA genomes that make it through this gauntlet are rendered nonfunctional by the resulting guanine to adenine hypermutations (cDNA uracils template the insertion of genomic strand adenines that become immortalized as G-to-A mutations). In this Pearl, we review recent progress in understanding the Vif-APOBEC3 interaction and discuss a model that provides a molecular explanation for past zoonotic transmission events as well as present-day, likely ongoing optimizations that enable lentiviruses to thrive. Fig 1 Lentiviral Vif and APOBEC3 diversity and functionality in mammals. CBF-β is a specific co-factor for SIV and HIV Vif function Although HIV-1 was identified in 1983, Vif function evaded discovery for nearly 20 years, until APOBEC3G (A3G) emerged as a dominant cellular factor that strongly inhibited vif-deficient virus replication [1]. This advance led rapidly to elucidation of the proteasome-dependent APOBEC3 degradation mechanism, including the identification of cellular factors that Vif recruits to form the degradation complex (CUL5, ELOB, ELOC, and RBX2) (Fig 1A) [2, 3]. However, HIV-1 Vif still resisted biochemical and structural studies for nearly another decade, suggesting that the complex may be incomplete. This puzzle was solved in 2011, when the transcription cofactor CBF-β proved to be an essential part of the HIV-1 Vif ubiquitin ligase complex [4, 5]. CBF-β enabled the biochemical reconstitution of the Vif ubiquitin ligase complex [4] and, shortly thereafter, structural determination by X-ray crystallography [6]. Vif and CBF-β heterodimerize and form a scaffold for further assembly of an active ligase complex (Fig 1A and 1B). Surprisingly, CBF-β is only essential for HIV and SIV Vif function (Fig 1C) [7]. The APOBEC3 degradation activity of non-primate lentiviral Vif proteins is unaffected by cellular CBF-β [4, 5, 8–11]. These differing genetic and biochemical requirements were initially puzzling given that all known Vif proteins function to counteract host APOBEC3 enzymes (e.g., [12]). However, the HIV/SIV Vif requirement for CBF-βmay be reconciled by postulating that this adaptation occurred when an ancestral lentivirus transmitted from a non-primate mammal into a primate. Such an event likely took place millions of years ago, because an endogenous lentivirus with a putative Vif protein exists in the present-day genomes of at least two evolutionarily diverged lemur genera [13, 14]. Because exogenously spreading lentiviruses evolve too quickly to date based on cumulative genetic diversity, endogenization events such as this effectively fossilize a virus and provide rare opportunities for estimating the age of a particular viral species and making comparisons with present day descendants such as SIV and HIV.

Viruses from Within

Early human embryos contain virus-like structures in the cytoplasm that appear to originate with an endogenous retrovirus, according to a new study [1]. The viral proteins turn-on an antiviral response and also may regulate the expression of many cellular RNAs. Only recently have technologies emerged to examine endogenous retroviruses closely during early development— and it’s clear that they are up to something. These retoviruses are highly expressed after fertilization, and some of their transcripts exhibit peculiar behavior—such as fusing with cellular RNAs to form RNA chimeras. Some endogenous retroviruses have been implicated in the initiation or maintenance of pluripotency. In the new study, Edward Grow et al. examined data from a previous single-cell analysis of RNA profiles in early human embryos. Similar to previous findings, the researchers found that distinct classes of retroviruses were transcribed at distinct stages. The researchers then focused on the most recently acquired retrovirus—HERVK (HML_2). Nearly 1/3 of its proviruses in the human genome are unique to humans. Unlike most other endogenous retroviruses, HERVK has retained the ability to encode intact retroviral proteins. It is normally transcriptionally silent, but can be activated in germ cell tumors, melanoma, and other pathological conditions. Grow et al. show that HERVK becomes active in eight-cellstage human embryos, and forms virus-like particles. The retrovirus remains active in epiblast cells, and shuts down in cultured epiblast cells as they begin to lose pluripotency. In cell culture, HERVK expression bumped up the cell-surface expression of IFITM1, a protein that combats viral infection, and thwarted infection by a flu virus. The researchers also provided hints about how HERVK is turned on—its expression was induced in human cells by the combination of high levels of the pluripotency factor OCT-4 (POU5F1) and low methylation levels (conditions also present in the early embryo). By turning on an antiviral response, HERVK may serve to protect the embryo from destruction by microbes (like the flu) and from endogenous retroviruses attempting to re-enter a cell. HERVK seems to also have a broader regulatory role: one HERVK protein, Rec, bound numerous cellular RNAs and appeared to regulate their expression. Also, HERVK could possibly re-integrate into the genome, which is a question for future studies.

Identification of Tf1 integration events in S. pombe under nonselective conditions

Integration of retroviral elements into the host genome is a phenomena observed among many classes of retroviruses. Much information concerning the integration of retroviral elements has been documented based on in vitro analysis or expression of selectable markers. To identify possible Tf1 integration events within silent regions of the Schizosaccharomyces pombe genome, we focused on performing an in vivo genome-wide analysis of Tf1 integration events from the nonselective phase of the retrotransposition assay. We analyzed 1000 individual colonies streaked from four independent Tf1 transposed patches under nonselection conditions. Our analysis detected a population of G418(S)/neo(+) Tf1 integration events that would have been overlooked during the selective phase of the assay. Further RNA analysis from the G418(S)/neo(+) clones revealed 50% of clones expressing the neo selectable marker. Our data reveals Tf1's ability to insert within silent regions of S. pombe's genome.

The tale of xenotropic murine leukemia virus-related virus.

In 2006, a new retrovirus was isolated from prostate cancer patient tissue. Named xenotropic murine leukemia virus-related virus (XMRV), this was potentially the third class of retrovirus to be pathogenic in humans. XMRV made a more dramatic impact on the wider scientific community, and indeed the media, in 2009 when it was reported to be present in a remarkably high proportion of patients with chronic fatigue syndrome as well as a significant, albeit smaller, proportion of healthy controls. The apparent strong link to disease and the fear of a previously unknown retrovirus circulating in the general population lead to a surge in XMRV research. Subsequent studies failed to find an association of XMRV with disease and, in most cases, failed to find the virus in human samples. In 2011, the case against XMRV and human disease strengthened, ending with several decisive publications revealing the origin of the virus and demonstrating contamination of samples. In this review, we outline the passage of research on XMRV and its potential association with disease from its isolation to the present day, where we find ourselves at the end of a turbulent story.

Reciprocal cross-packaging of primate lentiviral (HIV-1 and SIV) RNAs by heterologous non-lentiviral MPMV proteins

Retroviral RNA packaging signal (ψ) allows the preferential packaging of genomic RNA into virus particles through its interaction with the nucleocapsid protein. The specificity of this interaction came into question when it was shown that primate retroviruses, such as HIV-1, could cross-package RNA from its simian cousin, SIV, and vice versa and that feline retrovirus, FIV could cross-package RNA from a distantly related primate retrovirus, MPMV. To study the generality of this phenomenon further, we determined whether there is a greater packaging restriction between the lentiviral class of retroviruses (HIV-1 and SIV) and a non-lentivirus, MPMV. Our results revealed that primate lentiviral RNAs can be cross-packaged by primate non-lentiviral particles reciprocally, but the cross-packaged RNAs could not be propagated by the heterologous particles. Packaging of RNA in the context of both retroviral vectors as well as non-retroviral RNA containing SIV, HIV, and MPMV packaging determinants by each others proteins further confirmed the specificity of cross-packaging conferred by the packaging sequences. These results reveal the promiscuous nature of retroviral packaging determinants and raise caution against their wide spread presence on retroviral vectors to be used for human gene therapy.

Molecular approach to human leukemia: Isolation and characterization of the first human retrovirus HTLV-1 and its impact on tumorigenesis in Adult T-cell Leukemia

Molecular biology of mouse and chicken retroviruses had identified oncogenes and provided a revolutionary concept in understanding of cancers. A human retrovirus was established during 1980–1982 in linkage with a unique human leukemia, concurrently in Japan and USA. This review covers our efforts on the discovery of new retrovirus, Human T-cell Leukemia Virus Type 1 (HTLV-1), first introducing to a new class of retroviruses with a unique regulatory factors, Tax and Rex. Then it is followed by analyses of molecular interaction of the vial Tax with cellular machineries involved in the pathogenesis of Adult T-cell Leukemia (ATL). And then a probable mechanism of pathogenesis of ATL is proposed including recent findings on HBZ after our efforts.

Characterization of the replication-competent porcine endogenous retrovirus class B molecular clone originated from Korean domestic pig

Xenotransplantation from pigs offers an opportunity to resolve the shortage of human organs. The porcine endogenous retrovirus (PERV) cannot be eliminated because of its presence in the germline DNA. Three subgroups of the replication-competent PERV (PERV-A, PERV-B, and PERV-C) have been identified in pigs. We constructed a molecular clone of PERV-B from a Korean domestic pig BAC clone containing PERV genomes, and its replication competency was characterized in human cells. The pol region of PERV-B was detected in the genomic DNA of 293T cells transfected with PERV-B (465D1) and in the genomic DNA of 293T cells infected with PERV-B (465D1) viruses. The 293T cells transfected with PERV-B (465D1) were maintained for 140 days. PERV-B (465D1) showed the stationary replication competence with no toxicity in the cell growth.

Selective Up-Regulation of Intact, but Not Defective env RNAs of Endogenous Modified Polytropic Retrovirus by the Sgp3 Locus of Lupus-Prone Mice

Endogenous retroviruses are implicated in the pathogenesis of systemic lupus erythematosus (SLE). Because four different classes of endogenous retroviruses, i.e., ecotropic, xenotropic, polytropic, or modified polytropic (mPT), are expressed in mice, we investigated the possibility that a particular class of endogenous retroviruses is associated with the development of murine SLE. We observed >15-fold increased expression of mPT env (envelope) RNA in livers of all four lupus-prone mice, as compared with those of nine nonautoimmune strains of mice. This was not the case for the three other classes of retroviruses. Furthermore, we found that in addition to intact mPT transcripts, many strains of mice expressed two defective mPT env transcripts which carry a deletion in the env sequence of the 3' portion of the gp70 surface protein and the 5' portion of the p15E transmembrane protein, respectively. Remarkably, in contrast to nonautoimmune strains of mice, all four lupus-prone mice expressed abundant levels of intact mPT env transcripts, but only low or nondetectable levels of the mutant env transcripts. The Sgp3 (serum gp70 production 3) locus derived from lupus-prone mice was responsible for the selective up-regulation of the intact mPT env RNA. Finally, we observed that single-stranded RNA-specific TLR7 played a critical role in the production of anti-gp70 autoantibodies. These data suggest that lupus-prone mice may possess a unique genetic mechanism responsible for the expression of mPT retroviruses, which could act as a triggering factor through activating TLR7 for the development of autoimmune responses in mice predisposed to SLE.

Structure of B-MLV Capsid Amino-terminal Domain Reveals Key Features of Viral Tropism, Gag Assembly and Core Formation

The Gag polyprotein is the major structural protein found in all classes of retroviruses. Interactions between Gag molecules control key events at several stages in the cycle of infection. In particular, the capsid (CA) domain of Gag mediates many of the protein–protein interactions that drive retrovirus assembly, maturation and disassembly. Moreover, in murine leukaemia virus (MLV), sequence variation in CA confers N and B tropism that determines susceptibility to the intracellular restriction factors Fv1n and Fv1b. We have determined the structure of the N-terminal domain (NtD) of CA from B-tropic MLV. A comparison of this structure with that of the NtD of CA from N-tropic MLV reveals that although the crystals belong to different space groups, CA monomers are packed with the same P6 hexagonal arrangement. Moreover, interhexamer crystal contacts between residues located at the periphery of the discs are conserved, indicating that switching of tropism does not result in large differences in the backbone conformation, nor does it alter the quaternary arrangement of the disc. We have also examined crystals of the N-tropic MLV CA containing both N- and C-terminal domains. In this case, the NtD hexamer is still present; however, the interhexamer spacing is increased and the conserved interhexamer contacts are absent. Investigation into the effects of mutation of residues that mediate interhexamer contacts reveals that amino acid substitutions at these positions cause severe defects in viral assembly, budding and Gag processing. Based on our crystal structures and mutational analysis, we propose that in MLV, interactions between the NtDs of CA are required for packing of Gag molecules in the early part of immature particle assembly. Moreover, we present a model where proteolytic cleavage at maturation results in migration of CA C-terminal domains into interstitial spaces between NtD hexamers. As a result, NtD-mediated interhexamer contacts present in the immature particle are displaced and the less densely packed lattice with increased hexamer–hexamer spacing characteristic of the viral core is produced.

Isolation and Characterization of an Infectious Replication-Competent Molecular Clone of Ecotropic Porcine Endogenous Retrovirus Class C

Xenotransplantation of pig organs is complicated by the existence of polytropic replication-competent porcine endogenous retroviruses (PERV) capable of infecting human cells. The potential for recombination between ecotropic PERV-C and human-tropic PERV-A and PERV-B adds another level of infectious risk. Proviral PERV-C were characterized in MAX-T cells derived from d/d haplotype miniature swine. Three proviruses were cloned from a genomic library. Clone PERV-C(1312) generated infectious particles after transfection into porcine ST-IOWA cells. Electron microscopy revealed the same morphologies of virions in MAX-T cells and in ST-IOWA cells infected with cell-free PERV-C(1312) particles, indicating that MAX-T cells harbor one functional PERV-C provirus.

Transfusion‐transmitted viral infections: building bridges to transfusion medicine to reduce risks and understand epidemiology and pathogenesis

ransfusion medicine is a rapidly evolving field that now interfaces with virtually every clinical discipline and with many of the basic medical and social sciences. Transfusion medicine has been heavily influenced by, and has had a major impact on, public health and political, regulatory, and legal systems around the globe. When I was a young resident in pathology and laboratory medicine at the University of California in San Francisco (UCSF) in the early 1980s, I recall first encountering what was then referred to simply as blood banking. I was struck and intrigued by the relatively primitive understanding of the molecular basis of blood groups and by the simple methods used for compatibility and infectious disease testing. At the time, infectious disease concerns were minor and revolved primarily around possible residual risk of viral hepatitis following the recent implementation of hepatitis B surface antigen (HBsAg) screening. I had just completed my MD and PhD degrees at the University of Southern California (USC), where my thesis focused on the role of feline endogenous retroviruses in embryogenesis and lymphoma. Despite large-scale viral discovery studies at UCSF, USC, and elsewhere, no convincing evidence for a human retrovirus existed at that time. Certainly no one suspected that we were already in the early stages of a major global panT demic due to a novel class of retrovirus (lentivirus) that spread efficiently by blood transfusions. And then came acquired immunodeficiency syndrome (AIDS), and blood banking, indeed the entire world, changed forever! Fortunately for me, I was in the right place, at the right time. Thanks to relevant training, supportive mentors, wonderful collaborators, and a home base in the epicenter of the US AIDS epidemic, I was able to participate in the early characterization of transfusion-AIDS and help develop and execute many of the studies that have advanced our understanding of infectious risks associated with blood transfusions over the past 25 years. The theme of this Cooley Lectureship is “building bridges to transfusion medicine,” referring to the success that has been achieved by application of novel approaches and findings from other fields to study transfusion complications, while at the same time deriving insights from investigations of viral infections in blood donors and recipients to develop broader understanding of the epidemiology, diagnosis, and pathogenesis of relevant infections diseases with far reaching implications beyond our field. This lecture traces the history of transfusion risk analysis, demonstrating how thoughtful and creative individuals both in academia and in industry have responded to once unimaginable challenges posed by emerging transfusion-transmitted infections which, unfortunately, we now view as routine.

Effect of HIV-1 Vpr on cell cycle regulators.

Cell cycle is one of the most complex processes in the life of a dividing cell. It involves numerous regulatory proteins, which direct the cell through a specific sequence of events for the production of two daughter cells. Cyclin-dependent kinases (cdks), which complex with the cyclin proteins, are the main players in the cell cycle. They can regulate the progression of the cells through different stages regulated by several proteins including p53, p21(WAF1), p19, p16, and cdc25. Downstream targets of cyclin-cdk complexes include pRB and E2F. A cell cycle can be altered to the advantage of many viral agents, most notably polyomaviruses, papillomaviruses, adenoviruses, and retroviruses. In addition, viral protein R (Vpr) is a protein encoded by the human immunodeficiency virus type 1 (HIV-1). HIV-1, the causative agent of acquired immunodeficiency syndrome (AIDS), is a member of the lentivirus class of retroviruses. This accessory protein plays an important role in the regulation of the cell cycle by causing G(2) arrest and affecting cell cycle regulators. Vpr prevents infected cells from proliferating, and collaborates with the matrix protein (MA) to enable HIV-1 to enter the nucleus of nondividing cells. Studies from different labs including ours showed that Vpr affects the functions of cell cycle proteins, including p53 and p21(WAF1). Thus, the replication of HIV-1, and ultimately its pathogenesis, are intrinsically tied to cell-cycle control.

Lentiviral and MLV based retroviral vectors for ex vivo and in vivo gene transfer

Retroviral vectors have become an important tool for gene transfer in vitro and in vivo. Classical Moloney murine leukemia virus (MLV) based retroviral vectors have been used for over 20 years to transfer genes into dividing cells. Cell lines for production of retroviral vectors have become commonly available and modifications in retroviral vector design and use of envelope proteins have made the production of high titer, helper-free, infectious virus stocks relatively easy. More recently, lentiviral vectors, another class of retroviruses, have been modified for in vitro and in vivo gene transfer. The ability of lentiviral vectors to transduce non-dividing cells has made them especially attractive for in vivo gene transfer into differentiated, non-dividing tissues. Several improvements in helper plasmids and vectors have made lentivirus a safe vector system for ex vivo and in vivo gene transfer. This review will briefly summarize the background of these vector systems and provide some common protocols available for the preparation of MLV based retroviral vectors and HIV-1 based lentiviral vectors.

Screening for simian foamy virus infection by using a combined antigen Western blot assay: evidence for a wide distribution among Old World primates and identification of four new divergent viruses

Simian foamy viruses (SFVs) belong to a genetically and antigenically diverse class of retroviruses that naturally infect a wide range of nonhuman primates (NHPs) and can also be transmitted to humans occupationally exposed to NHPs. Current serologic detection of SFV infection requires separate Western blot (WB) testing by using two different SFV antigens [SFV AGM (African green monkey) and SFV CPZ (chimpanzee)]. However, this method is labor intensive and validation is limited to only small numbers of NHPs. To facilitate serologic SFV testing, we developed a WB assay that combines antigens from both SFV AGM and SFV CPZ. The combined-antigen WB (CA-WB) assay was validated with 145 serum samples from 129 NHPs (32 African and Asian species) and 16 humans, all with known SFV infection status determined by PCR. Concordant CA-WB results were obtained for all 145 PCR-positive or -negative primate and human specimens, giving the assay a 100% sensitivity and specificity. In addition, no reactivity was observed in sera from persons positive for human immunodeficiency virus or human T cell lymphotropic virus (HIV/HTLV) ( n = 25) or HIV/HTLV-negative U.S. blood donors ( n = 100). Using the CA-WB assay, we screened 360 sera from 43 Old World primate species and found an SFV prevalence of about 68% in both African and Asian primates. We also isolated SFV from the blood of four seropositive primates ( Allenopithecus nigroviridis, Trachypithecus françoisi, Hylobates pileatus, and H. leucogenys) not previously known to be infected with SFV. Phylogenetic analysis of integrase sequences from these isolates confirmed that all four SFVs represent new, distinct, and highly divergent lineages. These results demonstrate the ability of the CA-WB assay to detect infection in a large number of NHP species, including previously uncharacterized infections with divergent SFVs.

Intracellular trafficking of retroviral genomes during the early phase of infection: viral exploitation of cellular pathways.

Retroviruses enter cells through specific cell-surface receptors and then embark on a journey that ultimately leads to the establishment of the integrated proviral DNA. The steps of the journey include the reverse transcription of the viral RNA into DNA, the trafficking of the viral protein-DNA complex through the cytoplasm, the entry of the complex into the nucleus, and the insertion of the linear viral DNA into the host genome. All these steps are likely to involve specific interactions of viral proteins with host machinery. Our knowledge of the details of these interactions is very limited but is rapidly expanding, and should provide a deeper understanding of the pathways and components used by the different classes of retroviruses. This knowledge in turn should enable the development of better and more efficient retroviral vectors for use in gene therapy protocols in vivo.

Retroviral vectors for liver-directed gene therapy.

Retroviruses are popular gene therapy vectors because they stably integrate the DNA copy of their genome into the host chromosome during their replication cycle. The widely used murine retroviral vector systems have two components: the transfer vector for the transgene carries all the cis-acting elements necessary for the replication and efficient integration of the viral DNA; and the packaging cell line produces all the trans-acting proteins necessary for both structural and catalytic functions of the virus. Advances in design of retroviral vectors have resulted in greater degree of biosafety, expanded host range, and increased stability of the virus particles. Retroviral vectors have been widely used in the ex vivo gene therapy protocols to correct the liver diseases in a wide variety of species. In a limited number of applications, in vivo gene therapy has been achieved after the liver cells have been stimulated to regenerate. One major limitation of murine retroviral vectors is their inability to infect nondividing cells. This problem has been overcome by deriving vectors from lentiviruses (a class of retroviruses) that have the ability to infect both dividing and nondividing cells. The lentiviral vectors are derived from human immunodeficiency virus type 1 (HIV-1). Initial studies using lentiviral vectors for gene delivery to the liver in vivo show promising results. A highly crippled version of lentivirus has been generated by using producer cells in which the trans-acting components are expressed by several different coding elements and vectors that incorporate features of self-inactivation. These improvements should ensure biosafety of lentiviruses and make them useful in efficient delivery of therapeutic genes to nondividing differentiated tissues such as the liver.