A Potent Postentry Restriction to Primate Lentiviruses in a Yinpterochiropteran Bat.

Bats are primary reservoirs for multiple lethal human viruses, such as Ebola, Nipah, Hendra, rabies, severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome-related coronavirus (MERS-CoV), and, most recently, SARS-CoV-2. The innate immune systems of these immensely abundant, anciently diverged mammals remain insufficiently characterized. While bat genomes contain many endogenous retroviral elements indicative of past exogenous infections, little is known about restrictions to extant retroviruses. Here, we describe a major postentry restriction in cells of the yinpterochiropteran bat Pteropus alecto Primate lentiviruses (HIV-1, SIVmac) were potently blocked at early life cycle steps, with up to 1,000-fold decreases in infectivity. The block was specific, because nonprimate lentiviruses such as equine infectious anemia virus and feline immunodeficiency virus were unimpaired, as were foamy retroviruses. Interspecies heterokaryons demonstrated a dominant block consistent with restriction of incoming viruses. Several features suggested potential TRIM5 (tripartite motif 5) or myxovirus resistance protein 2 (MX2) protein restriction, including postentry action, cyclosporine sensitivity, and reversal by capsid cyclophilin A (CypA) binding loop mutations. Viral nuclear import was significantly reduced, and this deficit was substantially rescued by cyclosporine treatment. However, saturation with HIV-1 virus-like particles did not relieve the restriction at all. P. alecto TRIM5 was inactive against HIV-1 although it blocked the gammaretrovirus N-tropic murine leukemia virus. Despite major divergence in a critical N-terminal motif required for human MX2 activity, P. alecto MX2 had anti-HIV activity. However, this did not quantitatively account for the restriction and was independent of and synergistic with an additional CypA-dependent restriction. These results reveal a novel, specific restriction to primate lentiviruses in the Pteropodidae and advance understanding of bat innate immunity.IMPORTANCE The COVID-19 pandemic suggests that bat innate immune systems are insufficiently characterized relative to the medical importance of these animals. Retroviruses, e.g., HIV-1, can be severe pathogens when they cross species barriers, and bat restrictions corresponding to retroviruses are comparatively unstudied. Here, we compared the abilities of retroviruses from three genera (Lentivirus, Gammaretrovirus, and Spumavirus) to infect cells of the large fruit-eating bat P. alecto and other mammals. We identified a major, specific postentry restriction to primate lentiviruses. HIV-1 and SIVmac are potently blocked at early life cycle steps, but nonprimate lentiviruses and foamy retroviruses are entirely unrestricted. Despite acting postentry and in a CypA-dependent manner with features reminiscent of antiretroviral factors from other mammals, this restriction was not saturable with virus-like particles and was independent of P. alecto TRIM5, TRIM21, TRIM22, TRIM34, and MX2. These results identify a novel restriction and highlight cyclophilin-capsid interactions as ancient species-specific determinants of retroviral infection.

Mutational analysis and glycosylation sensitivity of restrictive XPR1 gammaretrovirus receptors in six mammalian species.

Most viruses infect only a few hosts, but the xenotropic and polytropic mouse leukemia viruses (X/P-MLVs) are broadly infectious in mammalian species. X/P-MLVs use the XPR1 receptor for cell entry, and tropism differences are due to polymorphisms in XPR1 and the viral envelope. To characterize these receptor variants and identify blocks to cross-species transmission, we examined the XPR1 receptors in six mammalian species that restrict different subsets of X/P-MLVs. These restrictive receptors have replacement mutations in regions implicated in receptor function, and some entry restrictions can be relieved by glycosylation inhibitors. Mutation of the cow and hamster XPR1 genes identified a shared, previously unrecognized receptor-critical site. This G/Q503N replacement dramatically improves receptor function. While this substitution introduces an N-linked glycosylation site, XPR1 receptors are not glycosylated indicating that this replacement alters the virus-receptor interface independently of glycosylation. Our data also suggest that an unidentified glycosylated cofactor may influence X/P-MLV entry.

Co-option of an endogenous retrovirus envelope for host defense in hominid ancestors.

Endogenous retroviral sequences provide a molecular fossil record of ancient infections whose analysis might illuminate mechanisms of viral extinction. A close relative of gammaretroviruses, HERV-T, circulated in primates for ~25 million years (MY) before apparent extinction within the past ~8 MY. Construction of a near-complete catalog of HERV-T fossils in primate genomes allowed us to estimate a ~32 MY old ancestral sequence and reconstruct a functional envelope protein (ancHTenv) that could support infection of a pseudotyped modern gammaretrovirus. Using ancHTenv, we identify monocarboxylate transporter-1 (MCT-1) as a receptor used by HERV-T for attachment and infection. A single HERV-T provirus in hominid genomes includes an env gene (hsaHTenv) that has been uniquely preserved. This apparently exapted HERV-T env could not support virion infection but could block ancHTenv mediated infection, by causing MCT-1 depletion from cell surfaces. Thus, hsaHTenv may have contributed to HERV-T extinction, and could also potentially regulate cellular metabolism.

Phenotypic changes and loss of melanoma-specific endogenous C-type retroviruses in BL6 melanoma cells transfected with the H-2Kb gene.

The murine B16 melanoma and its sublines are low or totally deficient in expression of the H-2Kb class I major histocompatibility complex (MHC) gene. In clones derived from the B16F10BL6 subline, expression of the transfected or endogenous H-2Kb gene resulted in alterations of various phenotypic properties of these melanoma cells, among which was the loss of melanoma-associated antigen (MAA) expression. Because our previous immunoelectron microscopy studies showed that MAA was associated with a C-type ecotropic retrovirus specific for melanomas of C57BL/6 origin, we examined the effect of class I H-2Kb as well as class II H-2IAk gene expression on retrovirus production in subclones of BL6 melanoma cells. Here we have shown that expression of the transfected or endogenous H-2Kb gene resulted in the loss of production of budding, MAA-specific, C-type retrovirus particles. Northern blot analysis demonstrated expression of ecotropic retroviral mRNAs in both particle-producing and non-producing BL6 melanoma clones. Southern blot analysis of high-molecular-weight cellular DNAs using an ecotropic env-specific DNA probe indicated that the parental BL6-8 melanoma cells contained the C57BL/6 endogenous ecotropic MuLV (Emv-2) and at least three additional, novel ecotropic retroviral DNAs. Restriction enzyme analysis of the proviral DNA suggests that the loss of retrovirus production in the H-2Kb-expressing melanoma clones is the result of multiple rearrangements in the novel proviral DNAs. Thus, phenotypic changes observed in the H-2Kb gene-transfected BL6 melanoma cells were associated with loss of endogenous melanoma-specific ecotropic retrovirus production.

Microglial infection by a neurovirulent murine retrovirus results in defective processing of envelope protein and intracellular budding of virus particles.

The observation of murine retrovirus infection of microglial cells in brain regions expressing spongiform neurodegenerative changes suggests that these cells may play an important role in pathogenesis. To evaluate this potential in vitro, murine microglial cells were infected in mixed glial cultures with the highly neurovirulent murine retrovirus, FrCasE. The microglia were then isolated from the mixed cultures on the basis of their differential adherence and shown to be approximately 98% pure. The infected microglia expressed viral envelope protein at the plasma membrane, while viral budding was primarily intracellular. Evaluation of the viral envelope protein by immunoblotting indicated that the immunoreactive species produced was exclusively a 90-kDa precursor protein. Very little of the envelope protein was associated with particles released from these cells, and viral titers in the culture supernatant were low. Interestingly, these cells were still capable of infecting permissive target cells when seeded as infectious centers. This partially defective infection of microglial cells suggests a potential cellular means by which a neurovirulent retrovirus could disrupt normal microglia and in turn central nervous system motor system functioning.

BALB/c myeloma retroviruses have mink cell focus-inducing activity.

We have determined the in vitro host range of the cloned MO-21 and FL-1 murine myeloma retroviruses grown in SC-1 cells that were originally isolated from cloned MOPC-21 and FLOPC-1 BALB/c plasmacytoma cell lines. These viruses are able to replicate in murine (BALB/3T3, NIH/3T3) as well as numerous heterologous cell lines. These myeloma retroviruses also exhibit mink cell focus-inducing activity. MO-21 and FL-1 shared interference patterns with each other, but their replication was not interfered with by ecotropic, xenotropic, or amphotropic viruses. The lack of cross-interference with ecotropic or xenotropic viruses distinguishes these isolates from other mink cell focus-inducing viruses.

Quantitative electron microscopy of intracytoplasmic type A particles at kinetochores of metaphase chromosomes isolated from Chinese hamster and murine cell lines.

We have successfully isolated and spread individual chromosomes of CHO-KI cells for electron microscopic karyotyping. Controlled preparation permitted a quantitative evaluation of the association between endogenous intracytoplasmic type A virus precursor particles and the centromeric region (kinetochores) of isolated chromosomes at prophase and metaphase. Our results suggest the transfer of type A particles from the cytoplasmic to the centromeric regions during early metaphase in conjunction with microtubule assembly at a time when the kinetochores are structurally mature and capable of binding microtubules. Preliminary comparable studies of the endogenous M432 virus propagated in murine cells support these findings. Our results are discussed with respect to mechanisms of intracellular movement of virus precursor particles and the interference with components of both the cytoskeleton and the mitotic apparatus.

An interferon-sensitive early step in the establishment of infection of murine cells by murine sarcoma/leukaemia virus.

NIH 3T3 mouse cells were infected at very high multiplicity with murine sarcoma/leukaemia virus (MSV/MLV) and then cloned. All of the 48 clones obtained were morphologically transformed, all but one showed anchorage-independence of growth, typical of MSV-transformed NIH 3T3 cells and most (91%) produced MSV/MLV. When cells which had been pre-treated with 10(4) units/ml of purified interferon (IF) were infected under the same conditions and then cloned in the presence of the same amount of IF, only 6 of a total of 63 clones were morphologically transformed. All but these 6 showed a degree of anchorage-independence typical of the uninfected parental cells and very few (2.4%) produced virus. Furthermore, the MSV genome could not be rescued in any of the 23 clones tested and only 1 out of 10 clones produced tumours. The properties of these clones remained stable over a period of 10 to 20 passages in the absence of interferon. We conclude that interferon can irreversibly block an early step in the MSV/MLV infectious process.

Rescue and transmission of a replication-defective variant of moloney murine leukemia virus.

We have described a clone of mouse cells, termed "8A," which appears to be infected with a replication-defective variant of Moloney murine leukemia virus (MuLV) (Rein et al., J. Virol. 25:146-156, 1978). Clone 8A cells release virus particles which do not form plaques in the standard XC test. However, approximately 10(2) particles per ml of clone 8A supernatant do form plaques in a modified XC test (the "complementation plaque assay"), in which the assay cells are coinfected with the XC-negative, nondefective amphotropic MuLV as well as the test virus. Superinfection of clone 8A cells themselves with amphotropic MuLV results in the production of approximately 10(5), rather than approximately 10(2), particles per ml which register in the complementation plaque assay. This increase is due to the rescue of replication-defective ecotropic MuLV from clone 8A cells by amphotropic MuLV since (i) this ecotropic MuLV can only form XC plaques in cells which are coinfected with amphotropic MuLV; and (ii) it is possible to transmit this defective variant, rescued from superinfected clone 8A cells, to a fresh clone of normal mouse cells. The time course of production of the rescued MuLV particles by superinfected clone 8A cells is virtually identical to that of rescue from these cells of murine sarcoma virus. Amphotropic MuLV superinfection of "NP-N" cells, which contain a "non-plaque-forming" variant of N-tropic MuLV (Hopkins and Jolicoeur, J. Virol. 16:991-999, 1975), also increases the titer of particles registering in the complementation plaque assay; thus, NP-N cells, like clone 8A cells, contain a rescuable defective variant of ecotropic MuLV.

Phosphorylation of chromatin- and ribosome-associated proteins in cells transformed by adenovirus, murine sarcoma virus, and methylcholanthrene.

Endogenous protein phosphorylation in chromatin and ribosomes of monkey, mouse, and rat cells transformed by DNA, RNA tumor viruses, and a chemical carcinogen revealed the association of a protein of approximately 90,000 daltons (90K), which is highly phosphorylated in vitro. Peptide map analysis showed that the 90K proteins associated with these organelles of various transformed cells are similar irrespective of the species of cells and transforming agents. This species of protein could not be detected, or was scarcely detected, by phosphorylation in chromatin and ribosomes of untransformed cells and in revertants of transformed cells. These results suggest that the alteration in the pattern of endogenous protein phosphorylation in these organelles is closely related to the transformed state of cells.

Origin and biological properties of a new BALB/c mouse sarcoma virus.

A focus-forming virus previously isolated from a BALB/c mouse hemangiosarcoma has been shown to be replication defective. Analysis of individual BALB/c mouse sarcoma virus (BALB-MSV) nonproducer transformants for expression of helper virus-coded proteins revealed genetically stable variants that expressed two, three, or all four gag gene products in the absence of detectable helper viral env gene expression. The type-specific antigenic determinants of helper viral proteins encoded by the BALB-MSV genome and by the B-tropic virus isolated from the BALB-MSV stock were demonstrated to be indistinguishable from those of BALB:virus-1, a known endogenous virus of BALB/c cells. These findings imply that a BALB/c endogenous virus was involved in the generation of BALB-MSV. By the same immunological approach, the presence of at least a portion of the Moloney-MuLV gag gene has been identified in two other transforming viruses--Moloney-MSV and Abelson lymphosarcoma virus--previously isolated from the BALB/c strain. The tissue culture properties of cells transformed by these defective viruses were also shown to be distinguishable. These findings indicate that transforming virus isolates of the same inbred strain differ in their transforming activities as well as in the helper viral sequences stably associated with their genomes.

FBJ osteosarcoma virus in tissue culture. III. Isolation and characterization of non-virus-producing FBJ-transformed cells.

Hamster and rat cell lines have been established that have been transformed by FBJ murine sarcoma virus (FBJ-MuSV) but that do not produce virus. The hamster cell line originated from an osteosarcoma that appeared in a hamster inoculated at birth with an extract of a CFNo1 mouse FBJ-osteosarcoma. The rat cell line was obtained by transferring the FBJ-MuSV genome to normal rat kidney cells in the absence of the FBJ type C virus (FBJ-MuLV), which, usually in high concentration, accompanies the FBJ-MuSV. Both transformed hamster and rat cell lines contain the FBJ-MuSV genome, which can be rescued by ecotropic and xenotropic murine type C viruses. This rescued genome produces characteristic FBJ-MuSV foci in tissue culture and, in appropriate animal hosts, induces osteosarcomas typical of those induced by FBJ-MuSV. FBJ-MuSV was isolated originally from a parosteal osteosarcoma that occurred naturally in a mouse. Since there was no previous history of passage of the agent through any other animal species, these non-virus-producing hamster and rat cells transformed by FBJ-MuSV should be very helpful in molecular studies examining the origin of spontaneous sarcoma genomes in mice.

Retrovirus purification: method that conserves envelope glycoprotein and maximizes infectivity.

A Sepharose 4B chromatographic method for purification of retroviruses is described which was less time consuming, increased purified virus yields, conserved viral glycoprotein, and increased recovery of biological infectivity in comparison with conventional sucrose gradient ultracentrifugation techniques.

Correlation between polyion effect on cell susceptibility to in vitro infection with murine C-type viruses and polyion effect on some membrane-related functions.

Polyions were tested for effects on some membrane-related functions. Both polycations investigated reduced the negative surface charge of assay cells and enhanced in vitro infectivity of murine C-type viruses, but had no influence on leukemia-virus-induced XC cell syncytia formation. Three polyanions increased the net outer cell charge, while only one of four inhibited infectivity and two of three impeded syncytia formation. Polyions had a slight, probably toxic, effect on the transmembrane potential, independent of their charge. Cells treated with fluorescent DEAE-dextran showed diffuse staining, which 4 h later had been modified into a granular fluorescence with unstained areas now present. This change correlated with a loss of enhancement of viral infectivity. The only polyanion which inhibited viral infectivity had a strong antihyaluronidase activity, and hyaluronidase and Ca++ both increased viral infectivity. It is suggested, therefore, that polyions may in part work on virus-cell membrane interactions by influencing membrane enzymes and not necessarily by simply changing the net outer cell surface charge.