SAMHD1 Promotes the Antiretroviral Adaptive Immune Response in Mice Exposed to Lipopolysaccharide.

SAMHD1 is a potent HIV-1 restriction factor that blocks reverse transcription in monocytes, dendritic cells and resting CD4+ T cells by decreasing intracellular dNTP pools. However, SAMHD1 may diminish innate immune sensing and Ag presentation, resulting in a weaker adaptive immune response. To date, the role of SAMHD1 on antiretroviral immunity remains unclear, as mouse SAMHD1 had no impact on murine retrovirus replication in prior in vivo studies. Here, we show that SAMHD1 significantly inhibits acute Friend retrovirus infection in mice. Pretreatment with LPS, a significant driver of inflammation during HIV-1 infection, further unmasked a role for SAMHD1 in influencing immune responses. LPS treatment in vivo doubled the intracellular dNTP levels in immune compartments of SAMHD1 knockout but not wild-type mice. SAMHD1 knockout mice exhibited higher plasma infectious viremia and proviral DNA loads than wild-type mice at 7 d postinfection (dpi), and proviral loads inversely correlated with a stronger CD8+ T cell response. SAMHD1 deficiency was also associated with weaker NK, CD4+ T and CD8+ T cell responses by 14 dpi and weaker neutralizing Ab responses by 28 dpi. Intriguingly, SAMHD1 influenced these cell-mediated immune (14 dpi) and neutralizing Ab (28 dpi) responses in male but not female mice. Our findings formally demonstrate SAMHD1 as an antiretroviral factor in vivo that could promote adaptive immune responses in a sex-dependent manner. The requirement for LPS to unravel the SAMHD1 immunological phenotype suggests that comorbidities associated with a "leaky" gut barrier may influence the antiviral function of SAMHD1 in vivo.

Infection of B Cell Follicle-Resident Cells by Friend Retrovirus Occurs during Acute Infection and Is Maintained during Viral Persistence.

B cell follicles of the spleen and lymph nodes are immune privileged sites and serve as sanctuaries for infected CD4+ cells in HIV infection. It is assumed that CD8+ T cell responses promote the establishment of the reservoir, as B cell follicles do not permit CD8+ T cell entry. Here we analyzed the infected cell population in the Friend retrovirus (FV) infection and investigated whether FV can similarly infect follicular cells. For analysis of FV-infected cells, we constructed a recombinant FV encoding the bright fluorescent protein mWasabi and performed flow cytometry with cells isolated from spleens, lymph nodes and bone marrow of FV-mWasabi-infected mice. Using t-stochastic neighbor embedding for data exploration, we demonstrate how the target cell population changes during the course of infection. While FV was widely distributed in erythrocytes, myeloid cells, B cells, and CD4+ T cells in the acute phase of infection, the bulk viral load in the late phase was carried by macrophages and follicular B and CD4+ T cells, suggesting that FV persists in cells that are protected from CD8+ T cell killing. Importantly, seeding into follicular cells was equally observed in CD8+ T cell-depleted mice and in highly FV-susceptible mice that mount a very weak immune response, demonstrating that infection of follicular cells is not driven by immune pressure. Our data demonstrate that infection of cells in the B cell follicle is a characteristic of the FV infection, making this murine retrovirus an even more valuable model for development of retrovirus immunotherapy approaches.IMPORTANCE Human immunodeficiency virus is notorious for its ability to avoid clearance by therapeutic interventions, which is partly attributed to the establishment of reservoirs in latently infected cells and cells that reside in immunologically privileged B cell follicles. In the work presented here, we show that cells of the B cell follicle are equally infected by a simple mouse gammaretrovirus. Using fluorescently labeled Friend retrovirus, we found that B cells and T cells in the B cell follicle, while not carrying the bulk of the virus load, were indeed infected by Friend virus in the early acute phase of the infection and persisted in the chronic infection. Our results suggest that infection of follicular cells may be a shared property of lymphotropic viruses and propose the FV infection of mice as a useful model to study strategies for follicular reservoir elimination.

Sequences in gibbon ape leukemia virus envelope that confer sensitivity to HIV-1 accessory protein Vpu.

HIV-1 efficiently forms pseudotyped particles with many gammaretrovirus glycoproteins, such as Friend murine leukemia virus (F-MLV) Env, but not with the related gibbon ape leukemia virus (GaLV) Env or with a chimeric F-MLV Env with a GaLV cytoplasmic tail domain (CTD). This incompatibility is modulated by the HIV-1 accessory protein Vpu. Because the GaLV Env CTD does not resemble tetherin or CD4, the well-studied targets of Vpu, we sought to characterize the modular sequence in the GaLV Env CTD required for this restriction in the presence of Vpu. Using a systematic mutagenesis scan, we determined that the motif that makes GaLV Env sensitive to Vpu is INxxIxxVKxxVxRxK. This region in the CTD of GaLV Env is predicted to form a helix. Mutations in the CTD that would break this helix abolish sensitivity to Vpu. Although many of these positions can be replaced with amino acids with similar biophysical properties without disrupting the Vpu sensitivity, the final lysine residue is required. This Vpu sensitivity sequence appears to be modular, as the unrelated Rous sarcoma virus (RSV) Env can be made Vpu sensitive by replacing its CTD with the GaLV Env CTD. In addition, F-MLV Env can be made Vpu sensitive by mutating two amino acids in its cytoplasmic tail to make it resemble more closely the Vpu sensitivity motif. Surprisingly, the core components of this Vpu sensitivity sequence are also present in the host surface protein CD4, which is also targeted by Vpu through its CTD.

Brainstem 1H nuclear magnetic resonance (NMR) spectroscopy: marker of demyelination and repair in spinal cord.

Measuring in vivo spinal cord injury and repair remains elusive. Using magnetic resonance spectroscopy (MRS) we examined brainstem N-acetyl-aspartate (NAA) as a surrogate for spinal cord injury in two mouse strains with different reparative phenotypes following virus-induced demyelination. Swiss Jim Lambert (SJL) and Friend Virus B (FVB) mice progressively demyelinate with axonal loss. FVB mice demyelinate similarly but eventually remyelinate coincident with functional recovery. Brainstem NAA levels drop in both but recover in FVB mice. Chronically infected SJL mice lost 30.5% of spinal cord axons compared to FVB mice (7.3%). In remyelination-enhancing or axon-preserving clinical trials, brainstem MRS may be a viable endpoint to represent overall spinal cord dysfunction.

Cross-species infectivity and pathogenesis of the Friend murine leukemia virus complex in Syrian hamsters.

To investigate cross-species infectivity and pathogenesis of Friend murine leukemia virus (MuLV) in hamsters, we infected newborn Syrian hamsters with ecotropic Friend MuLV that was passaged in BALB/c mice for approximately 30 years. During acute infection, 39.5% of newborn Syrian hamsters developed severe growth interruption and weight loss, spleen atrophy, severe lymphocyte depletion, and massive viral antigen loads in the spleen. The lymph nodes and thymuses were observed in all diseased hamsters. Ecotropic Friend MuLV was rescued from the sera and spleen and heart extracts of the diseased hamsters, and the same disease was confirmed by induction of erythroleukemia in BALB/c mice. Our results demonstrate that an ecotropic MuLV after extended passage in vivo to infect hamster cells that are resistant to infection by wild type MuLV, causing pathologic lesions and a wasting syndrome in newborn hamsters in vivo. This may occur with variants of Friend MuLV that have lower infectivity in hamster cells and are not cleared by the immune system of newborn hamsters. These findings suggest the potential danger of the interspecies transmission and pathogenesis of heterologous retroviruses in humans.

Cerebral metabolism in brains of rats infected with neuropathogenic murine leukemia viruses.

Friend murine leukemia virus A8 and PVC211 cause spongiform neurodegeneration in rat brains. Glutamate is an important neurotransmitter synthesized from alpha-ketoglutaric acid, an intermediate product of the citric acid cycle, and glutamine is synthesized from glutamate. To examine the brain metabolism of rats infected with neuropathogenic viruses, the amount of glutamate and glutamine in the brains of rats infected with A8, PVC211, and non-neuropathogenic 57 was measured using high performance liquid chromatography, and the (13)C-label incorporation into the C4 position of glutamate and glutamine from [1-(13)C] glucose was measured with (13)C nuclear magnetic resonance. In the cerebral hemisphere and region containing the brain stem and basal ganglia of rats infected with A8 and PVC211 at 8-9 weeks post-infection (wpi), the amount of glutamine was decreased compared with the 57-infected rats. The amount of glutamate was decreased in the cerebral hemisphere of the A8-infected rats and the region containing the brain stem and basal ganglia of PVC211-infected rats at 8-9 wpi. The amount of [4-(13)C] glutamine and [4-(13)C] glutamate in the cerebral hemisphere and region containing the brain stem and basal ganglia of rats infected with A8 and PVC211 at 8-9 wpi was equivalent to that of the 57-infected rats. These results suggest that in the brains of rats infected with neuropathogenic viruses, de novo synthesis of glutamate and glutamine is not decreased, but the ability to maintain quantitative levels of glutamate and glutamine is decreased compared with the brains of rats infected with non-neuropathogenic virus.

Independent roles of perforin, granzymes, and Fas in the control of Friend retrovirus infection.

Cytotoxic T-cells (CTL) play a central role in the recovery of mammalian hosts from retroviral infections. However, the molecular pathways that mediate the antiretroviral activity of CTL are still elusive. Here we explore the protective role of the two main cytolytic pathways of CTL, that is, granule exocytosis and Fas/Fas ligand (FasL), in acute and persistent Friend retrovirus (FV) infection of mice. For this purpose, we have used mutant mouse strains with targeted gene defects in one or more components of the two cytolytic pathways including perforin, granzyme A, granzyme B, Fas, and FasL. The important function of CTL in resistance of C57BL/6 (B6) mice to FV is emphasized by the finding that depletion of CD8+ T-cells prior to virus infection resulted in severe splenomegaly and high viral loads in blood and spleen tissue. Analysis of primary FV infection in knockout mice revealed that acute infection was readily controlled in the absence of functional Fas. Most notably in the presence of Fas/FasL each of the three effector molecules of the exocytosis pathway (i.e., perforin, granzyme A, and granzyme B) was capable on its own to mediate suppression of virus replication and protection from leukemia. However, triple knockout mice lacking perforin and the two granzymes were fully susceptible to FV-induced leukemia. In contrast to acute infection the Fas/FasL pathway was mandatory for effective control of FV replication during persistent infection. These findings suggest novel pathways of CTL-mediated viral defense and contribute towards a better understanding of the molecular mechanisms of CTL activity in retroviral infections.

Effects of tacrolimus on infection of Friend murine leukemia virus to Fv-4 gene heterozygous mice.

AIM: To investigate the effect of tacrolimus (FK506) on the infection of Friend murine leukemia virus (Friend MuLV) in vivo. METHODS: Three kinds of mice were used including Friend MuLV-sensitive BALB/c mice, Friend MuLV-resistant Fv-4 gene-homozygous mice (Fv-4 mice), and Friend MuLV-resistant Fv-4 gene-heterozygous mice (F1 mice). Tacrolimus was administrated i.p. to those mice in every 2 d. Those treated mice were inoculated i.p. with Friend MuLV once on d 3. The symptoms and viral proliferations in those mice were observed to recognize the Friend MuLV infection. The expression and genotype of Fv-4 gene that resistant against the infection of Friend MuLV were analyzed to confirm the genomic background and related mechanism of the resistance. RESULTS: BALB/c mice and F1 mice, but not Fv-4 mice, appeared obvious early death, spleenomegaly, and viral proliferation after both treatments of viral inoculation and tacrolimus administration, whereas the expression and genotype of Fv-4 gene was not changed in F1 mice and Fv-4 mice with treatment of tacrolimus. Compared to the virus-inoculated control, the Friend MuLV-sensitivity of tacrolimus-treated BALB/c mice and the Friend MuLV-resistance of tacrolimus-treated Fv-4 mice were the same as the controls, but only F1 mice became the symptoms and viral proliferation after both treatments. It suggested the Friend MuLV-resistant F1 mice could be converted to be Friend MuLV-sensitive by treatment of tacrolimus, and this conversion was not depended on the expression and genotype of Fv-4 gene. CONCLUSION: Tacrolimus could not inhibit the infection of Friend MuLV in all mice, furthermore, it could enhance the infection of Friend MuLV in F1 mice. The enhancement may be related to the immunosuppressive effect of tacrolimus.

Molecular breeding of viruses.

Genetic recombination is a major force driving the evolution of many viruses. Recombination between two copackaged retroviral genomes may occur at rates as high as 40% per replication cycle. This enables genetic information to be shuffled rapidly, leading to recombinants with new patterns of mutations and phenotypes. The in vitro process of DNA shuffling (molecular breeding) mimics this mechanism on a vastly parallel and accelerated scale. Multiple homologous parental sequences are recombined in parallel, leading to a diverse library of complex recombinants from which desired improvements can be selected. Different proteins and enzymes have been improved using DNA shuffling. We report here the first application of molecular breeding to viruses. A single round of shuffling envelope sequences from six murine leukaemia viruses (MLV) followed by selection yielded a chimaeric clone with a completely new tropism for Chinese Hamster Ovary (CHOK1) cells. The composition and properties of the selected clone indicated that this particular permutation of parental sequences cannot be readily attained by natural retroviral recombination. This example demonstrates that molecular breeding can enhance the inherently high evolutionary potential of retroviruses to obtain desired phenotypes. It can be an effective tool, when information is limited, to optimize viruses for gene therapy and vaccine applications when multiple complex functions must be simultaneously balanced.

Contribution of virus-receptor interaction to distinct viral proliferation of neuropathogenic and nonneuropathogenic murine leukemia viruses in rat glial cells.

The efficiency of receptor-mediated entry of pseudotyped virus carrying the surface protein (SU) of clone A8, a neuropathogenic variant of Friend murine leukemia virus (FrMLV), to rat glial cell line F10 was 1 order of magnitude greater than that of pseudotyped virus carrying SU of nonneuropathogenic FrMLV clone 57. Introduction of the gene coding for ecotropic MLV receptor on F10 cells (F10-ecoR) into SIRC cells, which are naturally resistant to FrMLV infection, also revealed the difference in receptor recognition between the A8 and the 57 viruses. Our results show that the difference in receptor utilization between A8-SU and 57-SU only partially explains the 3-order-of-magnitude difference in proliferation between A8 and 57 viruses in F10 cells.

Inhibition of Friend virus replication by a compound that reacts with the nucleocapsid zinc finger: anti-retroviral effect demonstrated in vivo.

The zinc finger structure that is found in the nucleocapsid protein of nearly all retroviruses has been proposed as a target for antiviral therapy. Since compounds that chemically attack the cysteines of the finger have been shown to inactivate both human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MuLV) in vitro, 14 of these compounds were tested in an MuLV-induced Friend disease model to assess their ability to inhibit retroviral replication in vivo. Of the 14 compounds tested, only Aldrithiol-2 clearly exhibited anti-retroviral activity as measured indirectly by the delay of Friend disease onset (P < 0.05). These results were confirmed by quantitative competitive polymerase chain reaction studies which monitored viral spread by measuring the level of viral DNA in the peripheral blood mononuclear cells of treated mice. Comparison of treated mice with untreated mice revealed that Aldrithiol-2 produced a greater than 2-log reduction in virus levels. These results functionally demonstrate that a zinc finger-attacking compound can inhibit viral replication in vivo. Since only 1 of the 14 compounds studied was effective, this study also shows the importance of in vivo testing of these types of antiviral compounds in an animal model. Given the strict conservation of the metal-coordinating cysteine structure within HIV-1 and MuLV zinc fingers, our results support the proposal that anti-retroviral drugs which target the nucleocapsid zinc finger may be clinically useful against HIV-1.

Efficient transduction of hemopoietic CD34+ progenitors of human origin using an original retroviral vector derived from Fr-MuLV-FB29: in vitro assessment.

A novel retroviral vector has been designed based on a Friend-murine leukemia virus (Fr-MuLV) FB29 strain. The latter has been selected according to characteristics of pathogenicity in mice where it induces a disease of the haemopoietic system affecting all lineages. Higher infectivity has also been demonstrated as compared to other strains. In accordance with these findings, the amphotropic producer clone used in this study carrying along the neomycine resistance gene (FOCH-Neo), harbors viral titers over 10(7) cfu/ml. To investigate the potential of genetically engineering hematopoietic precursors, CD34+ progenitors were selected from cord blood, bone marrow, and peripheral blood mobilized stem cells (patients + solid tumors) and transduced with FOCH-Neo. High transduction rates were achieved using virus supernatant and minimal doses of hematopoietic growth factors during pretransduction and transduction steps. A polymerase chain reaction (PCR) assay investigating the presence of both neomycin-encoding and viral vector sequences tested positive in 45-90% of granulocyte-macrophage colony-forming units (CFU-GM) generating cells (bone marrow and peripheral blood derived cells) following transduction. An average of 35% colonies showed resistance to G418. Such levels of transduction proved reproducible using only supernatants harboring over 10(7) cfu/ml. In those experiments where long-term in vitro cultures could be maintained over 5 weeks (all cord blood and 5 among 23 PBSC), efficient transduction of long-term culture initiating cell (LTC-IC) hematopoietic progenitors was demonstrated on the basis of both resistance to G418 and virus integration. In the latter case, the PCR assay tested positive in as much as 35-60% of late unselected CFU-colonies. This novel retroviral vector harbors interesting features toward genetic modification of hematopoietic progenitors.

Susceptibility of nude mice carrying the Fv-4 gene to Friend murine leukemia virus infection.

Fv-4 is a mouse gene that dominantly confers resistance to infection with Friend murine leukemia virus (F-MuLV) (S. Suzuki, Jpn. J. Exp. Med. 45:473-478, 1975). Despite complete resistance to ecotropic MuLV infection in mice carrying the Fv-4 gene, it is known that cells carrying the resistance gene in tissue culture do not always show resistance as extensive as that in vivo (H. Yoshikura and T. Odaka, JNCI 61:461-463, 1978). To investigate the immunological effect on resistance in vivo, we introduced the Fv-4 gene into BALB/c nude mice (Fv-4-/- nude[nu/nu]) by mating them with Fv-4 congenic BALB/c mice (Fv-4r/r nude+/+) and examined the susceptibility of the F2 progeny to F-MuLV. All BALB/c nude mice without the Fv-4 gene (Fv-4-/- nude[nu/nu]) were permissive to F-MuLV and developed erythroleukemia within 2 weeks after virus inoculation. The BALB/c nude mice with the Fv-4 gene (Fv-4r/r nude[nu/nu]) did not develop leukemia, and no or little virus was detected in the spleen 7 weeks after virus inoculation. The resistance to F-MuLV was dominant in (Fv-4 congenic BALB/c x BALB/c nude) F1 mice with the Fv-4r/- nude(nu/+) genotype as strictly as in (Fv-4 congenic BALB/c x BALB/c) F1 mice with the Fv-4r/- nude+/+ genotype. However, almost all BALB/c nude mice with the Fv-4r/- nude(nu/nu) genotype developed the disease within 7 weeks, and the virus was detected in all of their spleens even in the mice without leukemia. These results show that the resistance caused by the Fv-4 gene is recessive in nude mice and dominant in BALB/c mice. Some immunological effects, perhaps cell-mediated immunity, may play important roles in the resistance to F-MuLV infection in vivo in addition to the dosage effect of the Fv-4 product.

Interaction between the dominant negative mutant and the wild-type envelope proteins of Friend murine leukemia virus.

Interaction between the previously obtained dominant negative mutant, referred to as fcr (T. Matano, T. Odawara, M. Ohshima, H. Yoshikura, and A. Iwamoto, J. Virol. 67:2026-2033, 1993), and the wild-type envelope proteins (Env) of Friend murine leukemia virus was examined. The wild-type Env was bound to the fcr mutant Env and trapped in the endoplasmic reticulum. The virus receptor was not involved in this interaction.

Transgenic Fv-4 mice resistant to Friend virus.

Fv-4 is a mouse gene that confers resistance to infection with ecotropic retroviruses. A candidate Fv-4 gene was cloned previously and found to resemble the 3' half of a murine leukemia virus (MuLV). To study the effect of this gene in vivo, we generated two transgenic mouse strains carrying the Fv-4 env gene under control of its presumed natural promoter, a cellular sequence unrelated to retroviruses. Transgenic progeny expressed a 3-kb Fv-4 env RNA in all of the organs and tissues examined, as well as an Fv-4 envelope antigen on the surface of thymocytes and spleen cells, similar to mice carrying the natural Fv-4 gene. One of the two transgenic strains (designated Fv4-2) expressed three to nine times as much transgene RNA and protein as the other strain (Fv4-11). When challenged with a Friend virus complex containing up to 10(4) XC PFU of Friend MuLV, Fv4-2 mice were completely resistant to development of splenomegaly and had no detectable ecotropic virus in the spleen or blood, confirming that the cloned Fv-4 gene is responsible for resistance to ecotropic MuLV in vivo. In contrast, Fv4-11 mice were only partially resistant, developing viremia and splenomegaly at the highest inoculum dose but recovering from viremia several weeks after inoculation with 10-fold less virus. The phenotype of recovery from viremia in Fv4-11 mice was unexpected and suggests that low levels of expression of the Fv-4 gene enhance the effectiveness of the immune response.

trans-dominant interference with virus infection at two different stages by a mutant envelope protein of Friend murine leukemia virus.

A dominant negative mutant Friend murine leukemia virus (FMLV) env gene was cloned from an immunoselected Friend erythroleukemia cell. The mutant env had a point mutation which resulted in a Cys-to-Arg substitution at the 361st amino acid in the FMLV envelope protein (Env). The mutant Env was retained in the endoplasmic reticulum (ER) and accumulated because of its slow degradation. The NIH 3T3 cells expressing the mutant env were resistant to ecotropic Moloney MLV (MoMLV) penetration, suggesting that the mutant Env traps the ecotropic MLV receptors in the ER. When the mutant env gene was transfected into and expressed in the cells persistently infected with MoMLV, the wild-type Env was trapped in the ER, and the MoMLV production was suppressed. Thus, the mutant Env accumulating in the ER trans-dominantly and efficiently interfered with the ecotropic MLV infection at both the early and the late stages.

Replication of lactate dehydrogenase-elevating virus in various species cell lines infected with dual-, ampho- and xenotropic murine leukaemia viruses in vitro.

Lactate dehydrogenase-elevating virus (LDV) replicates in mouse macrophages in vivo and in vitro. It has been shown that LDV infects and replicates in motorneurons of the spinal cord of old, immunosuppressed C58 mice, which results in an acute poliomyelitis. In spite of extensive study, cells or cell lines other than macrophages which could support LDV infection and replication in vitro have not yet been detected. We have shown that LDV can replicate in mouse or rat cell lines which were previously infected with ecotropic murine leukaemia virus (MuLV). It was examined in this study whether other types of MuLV (dualtropic, amphotropic and xenotropic viruses) can also render the mouse cells or cells of other species susceptible to LDV infection as well as the ecotropic viruses. LDV infection and replication were seen in mouse cells infected with ecotropic, dual-tropic and amphotropic viruses. These were also seen in mink, rabbit and human cell lines infected with dual-, ampho- and xenotropic viruses. These results suggested that virtually all four classes of MuLV have the ability to elicit, in mouse cells or cells from heterologous species, permissiveness to LDV infection. The percent of LDV-infected cells increased up to approximately 80% in concentrated neurovirulent LDV-C-infected ecotropic MuLV-infected-mouse cells. The susceptibility of the cells gradually declined when they were maintained for more than one month. The LDV antigen-positive cells appeared as early as 6-8 h p.i., when a large amount of LDV and MuLV were added simultaneously. The replication of LDV was inhibited in MuLV-infected cells which had been treated previously with actinomycin D and cycloheximide, but not with zidovudine (AZT). A small percent of mouse cells became susceptible to LDV, when the cells were treated with iododeoxyuridine. This suggested that the induction of endogenous MuLV or part(s) of its genome from mouse chromosomes resulted in cells that were permissive to LDV.

Anti-viral activity of sulfated chitin derivatives against Friend murine leukaemia and herpes simplex type-1 viruses.

Sulfated chitin derivatives, selected for their low toxicity and high inhibitory activity of melanoma metastasis, were examined for anti-viral activity against Friend murine leukaemia, herpes simplex type-1 (HSV) and Sendai viruses. Carboxymethyl chitin with a 7.66% degree of sulfation (SCM-chitin III) showed a significant inhibition of Friend murine leukaemia helper virus (F-MuLV) and HSV, but not of Sendai virus growth in vitro. Sulfated N-deacetylated chitin had a significant but weak activity against F-MuLV and HSV infections. Carboxymethyl chitin showed no effect on these infections in vitro. SCM-chitin III also exhibited anti-viral activity in vivo by suppressing the splenomegaly which was caused by prior infection of mice with FV, a complex of F-MuLV and spleen focus-forming virus.

Approach to a retrovirus vaccine: immunization of mice against Friend virus disease with a replication-defective Friend murine leukemia virus.

In an initial attempt to test the ability of replication-defective retroviruses to immunize against immunologically related pathogenic viruses, we have worked with the erythroleukemogenic Friend retrovirus complex (FV), which consists of a replication-competent helper component, Friend murine leukemia virus (FMuLV), and a related defective pathogenic component, spleen focus-forming virus (SFFV). An 81-base-pair deletion was introduced into the p15E-encoding region of the env gene of an otherwise replication-competent molecular clone of the FMuLV provirus. After transfection of this clone into cells that package the viral RNA in MuLV coats, infectious virus was released into the culture medium. Mouse fibroblasts infected with this virus, here called delta FMuLV, expressed the truncated viral env gene products in their cytoplasm but not on cell surfaces, and culture fluids from these cells did not transmit the infection to fresh mouse fibroblasts. In preliminary experiments, immunization of mice of H-2-congenic BALB/c strains with delta FMuLV conferred levels of immunity to FV disease ranging from weak to relatively strong. Immunized mice developed anti-FV IgM and IgG antibodies and cytotoxic T cells. Mice observed for 15 weeks after the first of two immunizations showed no detectable pathology, but delta FMuLV DNA was detectable in livers of some immunized mice for at least 3-6 weeks. These results suggest that our approach to development of retrovirus vaccines may be a useful one.

Murine retrovirus-induced spongiform encephalomyelopathy: host and viral factors which determine the length of the incubation period.

A molecular clone of wild mouse ecotropic retrovirus CasBrE (clone 15-1) causes a spongiform neurodegenerative disease with a long incubation period, greater than or equal to 6 months. This virus infects the central nervous system (CNS) at low levels. In contrast, a chimeric virus, FrCasE, containing env and 3' pol sequences of 15-1 in a Friend murine leukemia virus background, infects the CNS at high levels and causes a rapid neurodegenerative disease with an incubation period of only 16 days. With both viruses, the induction of neurologic disease is dependent on inoculation during the perinatal period. Since the length of the incubation period of this disease appears to be a function of the relative level of CNS infection, we have attempted to identify the viral and host factors which determine the relative level of virus infection of the CNS. It was previously shown that the CNS is susceptible to infection only during the perinatal period (M. Czub, S. Czub, F. J. McAtee, and J. L. Portis, J. Virol. 65:2539-2544, 1991). Here we have found that the susceptibility of the CNS wanes progressively or gradually as a function of the age of the host, this age-dependent resistance being complete by 12 to 14 days of age. Utilizing a group of chimeric viruses, we found that the relative level of CNS infection achieved after inoculation of mice at 1 day of age was a function of the kinetics of virus replication and spread in peripheral organs. Viruses which reached peak viremia titers early (5 to 7 days of age) infected the CNS at high levels, and viruses which reached peak titers later infected the CNS at lower levels. Among the group of viruses examined in the current study, the kinetics of peripheral virus replication and spread appeared to be influenced primarily by sequences within the R-U5-5' leader region of the viral genome. These results suggested that the relative level of CNS infection was determined very early in life and appeared to be a function of a dynamic balance between the kinetics of virus replication in the periphery and a progressively developing restriction of virus replication in the CNS.