Protection against Friend retrovirus-induced leukemia by recombinant vaccinia viruses expressing the gag gene.

High sequence variability in the envelope gene of human immunodeficiency virus has provoked interest in nonenvelope antigens as potential immunogens against retrovirus infection. However, the role of core protein antigens encoded by the gag gene in protective immunity against retroviruses is unclear. By using recombinant vaccinia viruses expressing the Friend murine leukemia helper virus (F-MuLV) gag gene, we could prime CD4+ T-helper cells and protectively immunize susceptible strains of mice against Friend retrovirus infection. Recovery from leukemic splenomegaly developed more slowly after immunization with vaccinia virus-F-MuLV gag than with vaccinia virus-F-MuLV env; however, genetic nonresponders to the envelope protein could be partially protected with Gag vaccines. Class switching of F-MuLV-neutralizing antibodies from immunoglobulin M to immunoglobulin G after challenge with Friend virus complex was facilitated in mice immunized with the Gag antigen. Sequential deletion of the gag gene revealed that the major protective epitope was located on the N-terminal hydrophobic protein p15.     

Human immunodeficiency virus infection of cells arrested in the cell cycle.

Cell proliferation is necessary for proviral integration and productive infection of most retroviruses. Nevertheless, the human immunodeficiency virus (HIV) can infect non-dividing macrophages. This ability to grow in non-dividing cells is not specific to macrophages because, as we show here, CD4+ HeLa cells arrested at stage G2 of the cell cycle can be infected by HIV-1. Proliferation is necessary for these same cells to be infected by a murine retrovirus, MuLV. HIV-1 integrates into the arrested cell DNA and produces viral RNA and protein in a pattern similar to that in normal cells. In addition, our data suggest that the ability to infect non-dividing cells is due to one of the HIV-1 core virion proteins. HIV infection of non-dividing cells distinguishes lentiviruses from other retroviruses and is likely to be important in the natural history of HIV infection.     

Animal models of AIDS

Animal models of AIDS are essential for understanding the pathogenesis of retrovirus-induced immune deficiency and encephalopathy and for development and testing of new therapies and vaccines. AIDS and related disorders are etiologically linked to members of the lentivirus subfamily of retroviruses; these lymphocytopathic lentiviruses are designated human immuno-deficiency virus type 1 (HIV-1) and human immuno-deficiency virus type 2 (HIV-2). The only animals susceptible to experimental HIV-1 infection are the chimpanzee, gibbon ape, and rabbit but AIDS-like disease has not yet been reported in these species. Macaques can be persistently infected with some strains of HIV-2 but no AIDS-like disease has resulted. It is not yet clear how suitable HIV-infected SCID-hu mice will be as a model for AIDS. Several subfamilies of naturally occurring cytopathic retroviruses cause immune suppression, including fatal immunodeficiency syndromes in chickens, mice, cats, and monkeys. Domestic cats suffer immunosuppression from both an onco-virus, feline leukemia virus, and a member of the lentivirus subfamily, feline immunodeficiency virus (FIV). Asian macaques are susceptible to fatal simian AIDS from a type D retrovirus, indigenous in macaques, and from a lentivirus, simian immunodeficiency virus (SIV), which is indigenous to healthy African monkeys. SIV is the animal lentivirus most closely related to HIV. Of these animal models, the lentivirus infections of cats (FIV) and macaques (SIV) appear to bear the closest similarity in their pathogenesis to HIV infection and AIDS. This review will summarize these various animal model systems for AIDS and illustrate their usefulness for antiviral therapy and vaccinology.     

Biochemical and virological analysis of the 18-residue C-terminal tail of HIV-1 integrase

Background

Mouse mammary tumor virus (MMTV) encodes the Rem protein, an HIV Rev-like protein that enhances nuclear export of unspliced viral RNA in rodent cells. We have shown that Rem is expressed from a doubly spliced RNA, typical of complex retroviruses. Several recent reports indicate that MMTV can infect human cells, suggesting that MMTV might interact with human retroviruses, such as human immunodeficiency virus (HIV), human T-cell leukemia virus (HTLV), and human endogenous retrovirus type K (HERV-K). In this report, we test whether the export/regulatory proteins of human complex retroviruses will increase expression from vectors containing the Rem-responsive element (RmRE).

Results

MMTV Rem, HIV Rev, and HTLV Rex proteins, but not HERV-K Rec, enhanced expression from an MMTV-based reporter plasmid in human T cells, and this activity was dependent on the RmRE. No RmRE-dependent reporter gene expression was detectable using Rev, Rex, or Rec in HC11 mouse mammary cells. Cell fractionation and RNA quantitation experiments suggested that the regulatory proteins did not affect RNA stability or nuclear export in the MMTV reporter system. Rem had no demonstrable activity on export elements from HIV, HTLV, or HERV-K. Similar to the Rem-specific activity in rodent cells, the RmRE-dependent functions of Rem, Rev, or Rex in human cells were inhibited by a dominant-negative truncated nucleoporin that acts in the Crm1 pathway of RNA and protein export.

Conclusion

These data argue that many retroviral regulatory proteins recognize similar complex RNA structures, which may depend on the presence of cell-type specific proteins. Retroviral protein activity on the RmRE appears to affect a post-export function of the reporter RNA. Our results provide additional evidence that MMTV is a complex retrovirus with the potential for viral interactions in human cells.     

Inversion of a chicken ets-1 proto-oncogene segment in avian leukemia virus E26.

The segment of the avian leukemia virus E26 genome near the termination of the p135gag-myb-ets open reading frame contains an inversion of the chicken ets-1 sequence. The inversion contains at least 41 bp and may be as large as 46 bp. This results in the replacement of 13 amino acids of chicken ets-1, with 16 amino acids derived from reverse complement of the normal ets-1 coding strand or read-through into E26 env sequences. At least 13 of these codons are specified by the inverted ets sequences. This represents the first reported occurrence of inverted oncogene sequences in a natural retrovirus. The inverted ets sequences are immediately followed by sequences homologous to the Rous sarcoma virus Prague B env gene. Since the E26 env sequence is more closely related to subgroup B avian retroviruses than to avian retroviruses from subgroups A, C, D, or E, the progenitor of E26 was a virus belonging to avian retrovirus subgroup B.     

Isolation of a type D retrovirus from B-cell lymphomas of a patient with AIDS.

An atypical syncytial variant of a high-grade Burkitt's-type B-cell lymphoma from a patient with AIDS who was seropositive for human immunodeficiency virus type 1 was studied. A productive type D retrovirus infection was identified in early-passage cell lines derived from two lymphomas from this patient. Nucleotide and amino acid sequence analysis as well as immunologic reactivity indicated that the isolated virus was highly related to Mason-Pfizer monkey virus (MPMV). MPMV is an immunosuppressive type D retrovirus that causes an AIDS-like syndrome in rhesus macaques. Amplification of DNA from the patient's diagnostic bone marrow biopsy specimen by polymerase chain reaction generated the appropriate MPMV-specific fragments and indicated that the patient was infected with the MPMV-like retrovirus. In addition, the patient's serum contained antibodies which recognized type D viral env proteins (gp70 and gp20) and gag proteins (p27 and p14). Although there have been reports of human cell lines infected with type D retroviruses and of type D-reactive human sera, this is the first evidence of a type D retrovirus infection in a human confirmed by virus isolation, serum reactivity, and viral DNA identification in tumor tissue.     

Primate and feline lentivirus vector RNA packaging and propagation by heterologous lentivirus virions

Development of safe and effective gene transfer systems is critical to the success of gene therapy protocols for human diseases. Currently, several primate lentivirus-based gene transfer systems, such as those based on human and simian immunodeficiency viruses (HIV/SIV), are being tested; however, their use in humans raises safety concerns, such as the generation of replication-competent viruses through recombination with related endogenous retroviruses or retrovirus-like elements. Due to the greater phylogenetic distance from primate lentiviruses, feline immunodeficiency virus (FIV) is becoming the lentivirus of choice for human gene transfer systems. However, the safety of FIV-based vector systems has not been tested experimentally. Since lentiviruses such as HIV-1 and SIV have been shown to cross-package their RNA genomes, we tested the ability of FIV RNA to get cross-packaged into primate lentivirus particles such as HIV-1 and SIV, as well as a nonlentiviral retrovirus such as Mason-Pfizer monkey virus (MPMV), and vice versa. Our results reveal that FIV RNA can be cross-packaged by primate lentivirus particles such as HIV-1 and SIV and vice versa; however, a nonlentivirus particle such as MPMV is unable to package FIV RNA. Interestingly, FIV particles can package MPMV RNA but cannot propagate the vector RNA further for other steps of the retrovirus life cycle. These findings reveal that diverse retroviruses are functionally more similar than originally thought and suggest that upon coinfection of the same host, cross- or copackaging may allow distinct retroviruses to generate chimeric variants with unknown pathogenic potential.     

Natural history of the retrovirus associated with a human leukemia

A human retrovirus etiologically related to a unique leukemia, adult T-cell leukemia, has been discovered. This retrovirus is endemic in certain areas and ethnic groups, such as the Japanese in Japan and Blacks in Africa. The virus is transmitted from mother to child and husband to wife and by blood transfusion. In addition, a virus like the human retrovirus has been found in various species of monkeys in Asia and Africa. This review describes and discusses recent results on the natural history of these primate lymphotropic retroviruses.     

The PPPY motif of human T-cell leukemia virus type 1 Gag protein is required early in the budding process

Domains required late in the virus budding process (L domains) have been identified in the Gag proteins of a number of retroviruses. Here we show that the human T-cell leukemia virus type 1 candidate L domain motif PPPY is indeed required for virus production. Strikingly, however, mutation of this motif arrested virus particles at an earlier stage in the budding process than was seen for mutation of the L domain motifs thus far described for retroviruses. In view of the exchangeability of such domains, we propose that the retrovirus budding process may involve a continuum from bud formation to membrane fission.     

An siRNA Screen of Membrane Trafficking Genes Highlights Pathways Common to HIV-1 and M-PMV Virus Assembly and Release

The assembly and release of retroviruses from the host cells requires a coordinated series of interactions between viral structural proteins and cellular trafficking pathways. Although a number of cellular factors involved in retrovirus assembly have been identified, it is likely that retroviruses utilize additional trafficking factors to expedite their assembly and budding that have not yet been defined. We performed a screen using an siRNA library targeting host membrane trafficking genes in order to identify new host factors that contribute to retrovirus assembly or release. We utilized two retroviruses that follow very distinct assembly pathways, HIV-1 and Mason-Pfizer monkey virus (M-PMV) in order to identify host pathways that are generally applicable in retrovirus assembly versus those that are unique to HIV or M-PMV. Here we report the identification of 24 host proteins identified in the screen and subsequently validated in follow-up experiments as contributors to the assembly or release of both viruses. In addition to identifying a number of previously unsuspected individual trafficking factors, we noted multiple hits among proteins involved in modulation of the actin cytoskeleton, clathrin-mediated transport pathways, and phosphoinositide metabolism. Our study shows that distant genera of retroviruses share a number of common interaction strategies with host cell trafficking machinery, and identifies new cellular factors involved in the late stages of retroviral replication.     

Pseudotypes of vesicular stomatitis virus with the envelope properties of mammalian and primate retroviruses.

By employing improved techniques it has been possible to produce and characterize a representative spectrum of mammalian and primate retrovirus pseudotypes of vesicular stomatitis virus (VSV). Selection of appropriate cell lines for both the production and subsequent detection of the VSV pseudotypes has been the most important factor in permitting their demonstration. The host range for penetration of these retrovirus pseudotypes of VSV has been defined and found to differ from that reported for the replication of the corresponding retroviruses. Additionally, retroviruses having an identical host range for replication were distinguishable by differences in their host range for penetration, implying that restriction of replication may be occurring by different mechanisms. Studies of the plaque-forming efficiency of retrovirus pseudotypes of VSV in cell lines nonpermissive for replication of the corresponding retroviruses permitted a distinction to be made between the restriction of replication occurring as a consequence of postpenetration events and that occurring as a consequence of a block of penetration itself. The demonstration of primate retrovirus pseudotypes of VSV permits the use of VSV as a probe for the detection of this group of viruses.     

Human immunodeficiency virus pseudotypes with expanded cellular and species tropism.

One mechanism for expanding the cellular tropism of a virus is through the formation of phenotypically mixed particles or pseudotypes, a process commonly occurring during viral assembly in cells infected with two or more viruses. We report here that dual infection of cells with human immunodeficiency virus (HIV) and a murine amphotropic retrovirus leads to the production of HIV pseudotypes that have acquired the host range of the amphotropic retrovirus and are capable of infecting not only CD4- human cells but also mouse cells. The replication of the HIV pseudotypes in the various CD4- cells was determined by measuring the appearance of HIV antigens in the supernatants, by cocultivation of CD4+ CEM cells with the infected CD4- cells, and in some cases by assaying the culture supernatants directly for infectious virus. Of the cells tested, human foreskin fibroblasts were the best host cells, and by in situ cytohybridization, we were able to document that all cells in the culture were infected. In addition, the temporal appearance of HIV-specific proteins in the HIV pseudotype-infected fibroblasts was similar to that seen in CD4+ CEM cells. If the human fibroblasts were first infected with the amphotropic retrovirus, they demonstrated the property of superinfection exclusion and were resistant to subsequent infection by the HIV pseudotype. In other cell lines, including the human glioblastoma-derived cell line U373MG, HeLa cells, BALB/c mouse embryo cells, and SC-1 wild mouse cells, although the HIV pseudotype infection appeared to be less efficient, substantial amounts of HIV were nevertheless produced. These results indicate that the HIV (amphotropic retrovirus) pseudotypes may be useful for studying the molecular biology of HIV infections in a wide range of cells.     

T helper cell activation and human retroviral pathogenesis.

T helper (Th) cells are of central importance in regulating many critical immune effector mechanisms. The profile of cytokines produced by Th cells correlates with the type of effector cells induced during the immune response to foreign antigen. Th1 cells induce the cell-mediated immune response, while Th2 cells drive antibody production. Th cells are the preferential targets of human retroviruses. Infections with human T-cell leukemia virus (HTLV) or human immunodeficiency virus (HIV) result in the expansion of Th cells by the action of HTLV (adult T-cell leukemia) or the progressive loss of T cells by the action of HIV (AIDS). Both retrovirus infections impart a high-level activation state in the host immune cells as well as systemically. However, diverging responses to this activation state have contrasting effects on the Th-cell population. In HIV infection, Th-cell loss has been attributed to several mechanisms, including a selective elimination of cells by apoptosis. The induction of apoptosis in HIV infection is complex, with many different pathways able to induce cell death. In contrast, infection of Th cells with HTLV-1 affords the cell a protective advantage against apoptosis. This advantage may allow the cell to escape immune surveillance, providing the opportunity for the development of Th-cell cancer. In this review, we will discuss the impact of Th-cell activation and general immune activation on human retrovirus expression with a focus upon Th-cell function and the progression to disease.     

Frequent simian foamy virus infection in persons occupationally exposed to nonhuman primates

The recognition that AIDS originated as a zoonosis heightens public health concerns associated with human infection by simian retroviruses endemic in nonhuman primates (NHPs). These retroviruses include simian immunodeficiency virus (SIV), simian T-cell lymphotropic virus (STLV), simian type D retrovirus (SRV), and simian foamy virus (SFV). Although occasional infection with SIV, SRV, or SFV in persons occupationally exposed to NHPs has been reported, the characteristics and significance of these zoonotic infections are not fully defined. Surveillance for simian retroviruses at three research centers and two zoos identified no SIV, SRV, or STLV infection in 187 participants. However, 10 of 187 persons (5.3%) tested positive for SFV antibodies by Western blot (WB) analysis. Eight of the 10 were males, and 3 of the 10 worked at zoos. SFV integrase gene (int) and gag sequences were PCR amplified from the peripheral blood lymphocytes available from 9 of the 10 persons. Phylogenetic analysis showed SFV infection originating from chimpanzees (n = 8) and baboons (n = 1). SFV seropositivity for periods of 8 to 26 years (median, 22 years) was documented for six workers for whom archived serum samples were available, demonstrating long-standing SFV infection. All 10 persons reported general good health, and secondary transmission of SFV was not observed in three wives available for WB and PCR testing. Additional phylogenetic analysis of int and gag sequences provided the first direct evidence identifying the source chimpanzees of the SFV infection in two workers. This study documents more frequent infection with SFV than with other simian retroviruses in persons working with NHPs and provides important information on the natural history and species origin of these infections. Our data highlight the importance of studies to better define the public health implications of zoonotic SFV infections.     

Insertional polymorphisms of full-length endogenous retroviruses in humans

Human endogenous retrovirus K (HERV-K) is distinctive among the retroviruses in the human genome in that many HERV-K proviruses were inserted into the human germline after the human and chimpanzee lineages evolutionarily diverged [1, 2]. However, all full-length endogenous retroviruses described to date in humans are sufficiently old that all humans examined were homozygous for their presence [1]. Moreover, none are intact; all have lethal mutations [1, 3, 4]. Here, we describe the first endogenous retroviruses in humans for which both the full-length provirus and the preintegration site alleles are shown to be present in the human population today. One provirus, called HERV-K113, was present in about 30% of tested individuals, while a second, called HERV-K115, was found in about 15%. HERV-K113 has full-length open reading frames (ORFs) for all viral proteins and lacks any nonsynonymous substitutions in amino acid motifs that are well conserved among retroviruses. This is the first such endogenous retrovirus identified in humans. These findings indicate that HERV-K remained capable of reinfecting humans through very recent evolutionary times and that HERV-K113 is an excellent candidate for an endogenous retrovirus that is capable of reinfecting humans today.     

Safety of human blood products: inactivation of retroviruses by heat treatment at 60 degrees C

Acquired immune deficiency syndrome (AIDS) can be transferred to patients by blood transfusions or human blood preparations, such as cryoprecipitates or factor VIII concentrates. Retroviruses have been discussed as infectious AIDS agents and more recently human T-lymphotropic retroviruses designated as HTLV type III and LAV (lymphadenopathy-associated virus) have been isolated from AIDS patients. Whether heat treatment at 60 degrees C (pasteurization) of liquid human plasma protein preparations inactivates retroviruses was therefore investigated. Pasteurization had already been included in the routine manufacturing process of human plasma protein preparations in order to guarantee safety with regard to hepatitis B. Since high titer preparations of human retroviruses were not available, heat inactivation was studied using Rous sarcoma virus added to the various plasma protein preparations tested. This retrovirus which was obtained in preparations of 6.0 log10 FFU/ml was shown to be at least as heat stable as two mammalian retroviruses studied, i.e., feline and simian sarcoma virus. In all of eight different plasma protein preparations tested, Rous sarcoma virus was completely inactivated after a heat treatment lasting no longer than 4 hr. It is thus concluded that pasteurization of liquid plasma protein preparations at 60 degrees C over a period of 10 hr must confer safety to these products with respect to AIDS, provided that the AIDS agents are retroviruses of comparable heat stability as Rous sarcoma virus and the mammalian retroviruses tested.