Accumulation Of Unintegrated Viral DNA Research Articles

An HIV-1 Replication Pathway Utilizing Reverse Transcription Products That Fail To Integrate

Integration is a central event in the replication of retroviruses, yet ≥ 90% of HIV-1 reverse transcripts fail to integrate, resulting in accumulation of unintegrated viral DNA in cells. However, understanding what role, if any, unintegrated viral DNA plays in the natural history of HIV-1 has remained elusive. Unintegrated HIV-1 DNA is reported to possess a limited capacity for gene expression restricted to early gene products and is considered a replicative dead end. Although the majority of peripheral blood CD4(+) T cells are refractory to infection, nonactivated CD4 T cells present in lymphoid and mucosal tissues are major targets for infection. Treatment with cytokine interleukin-2 (IL-2), IL-4, IL-7, or IL-15 renders CD4(+) T cells permissive to HIV-1 infection in the absence of cell activation and proliferation and provides a useful model for infection of resting CD4(+) T cells. We found that infection of cytokine-treated resting CD4(+) T cells in the presence of raltegravir or with integrase active-site mutant HIV-1 yielded de novo virus production following subsequent T cell activation. Infection with integration-competent HIV-1 naturally generated a population of cells generating virus from unintegrated DNA. Latent infection persisted for several weeks and could be activated to virus production by a combination of a histone deacetylase inhibitor and a protein kinase C activator or by T cell activation. HIV-1 Vpr was essential for unintegrated HIV-1 gene expression and de novo virus production in this system. Bypassing integration by this mechanism may allow the preservation of genetic information that otherwise would be lost.

Role of receptor polymorphism and glycosylation in syncytium induction and host range variation of ecotropic mouse gammaretroviruses

BackgroundWe previously identified unusual variants of Moloney and Friend ecotropic mouse gammaretroviruses that have altered host range and are cytopathic in cells of the wild mouse species Mus dunni. Cytopathicity was attributed to different amino acid substitutions at the same critical env residue involved in receptor interaction: S82F in the Moloney variant Spl574, and S84A in the Friend mouse leukemia virus F-S MLV. Because M. dunni cells carry a variant CAT-1 cell surface virus receptor (dCAT-1), we examined the role of this receptor variant in cytopathicity and host range.ResultsWe expressed dCAT-1 or mCAT-1 of NIH 3T3 origin in cells that are not normally infectible with ecotropic MLVs and evaluated the transfectants for susceptibility to virus infection and to virus-induced syncytium formation. The dCAT-1 transfectants, but not the mCAT-1 transfectants, were susceptible to virus-induced cytopathicity, and this cytopathic response was accompanied by the accumulation of unintegrated viral DNA. The dCAT-1 transfectants, however, did not also reproduce the relative resistance of M. dunni cells to Moloney MLV, and the mCAT-1 transfectants did not show the relative resistance of NIH 3T3 cells to Spl574. Western analysis, use of glycosylation inhibitors and mutagenesis to remove receptor glycosylation sites identified a possible role for cell-specific glycosylation in the modulation of virus entry.ConclusionVirus entry and virus-induced syncytium formation using the CAT-1 receptor are mediated by a small number of critical amino acid residues in receptor and virus Env. Virus entry is modulated by glycosylation of cellular proteins, and this effect is cell and virus-specific.

High susceptibility of U937-derived subclones to human immunodeficiency virus type 1 infection correlates with accumulation of unintegrated circular viral DNA.

Our previous report showed that U937-derived subclones were differentiated into at least three types (high, middle, and low types), even in the subclones expressing similar levels of surface CD4, in terms of the kinetics of the appearance of viral antigens and virus production after infection with human immunodeficiency virus type 1 (HIV-1). Here we showed the evidence that high susceptibility to HIV-1 infection, which was confirmed by the profound expression of viral messages and antigens, was exclusively associated with a high number of the unintegrated extrachromosomal form of viral DNA, but not with the amounts of adsorbed virus RNA nor those of integrated DNA form. The difference in the amounts of extrachromosomal form of viral DNA was also observed in the culture with 3'-azido-3'-deoxythymidine (AZT), indicating that the susceptibility is essentially unrelated to multiple infection events. Thus, the susceptibility of U937-derived subclones to HIV-1 infection seems to be affected by the occurrence of specific events involved in the accumulation of unintegrated viral DNA after viral adsorption.

Inhibition of neurotropic mouse retrovirus replication in glial cells by synthetic oligo(2′-O-methyl)ribonucleoside phosphorothioates

Synthetic oligo(2′-O-methyl)ribonucleoside phosphorothioate, FS-25, which is complementary to the splicing acceptor site of neurotropic mouse retrovirus (FrC6 virus), and non-complementary analogs including 2′-O-methylinosine homo oligomer (MIS-25), both inhibited viral infection in glial cells. In addition, FS-25 and MIS-25 partially suppressed viral production of glial cells persistently infected with FrC6 virus. Both FS-25 and MIS-25 potently inhibited reverse transcriptase activity of the FrC6 virus in a cell-free system. Addition of these compounds before or after second-round infection of the FrC6 virus inhibited the accumulation of unintegrated viral DNA. These results indicate that these compounds fundamentally inhibit retrovirus production in glial cells in the same manner in which they inhibit HIV production, by blocking several viral replication pathways including fresh infection, second-round infection, and reverse transcription of the viral genome. Our novel neurotropic retrovirus is a useful experimental model for the development of drugs against HIV infection.

4 Haematological disorders associated with feline retrovirus infections

SummaryFeline oncornavirus and lentivirus infections have provided useful models to characterize the virus and host cell factors involved in a variety of marrow suppressive disorders and haematological malignancies. Exciting recent progress has been made in the characterization of the viral genotypic features involved in FeLV-associated diseases. Molecular studies have clearly defined the causal role of variant FeLV env gene determinants in two disorders: the T-lymphocyte cytopathicity and the clinical acute immuno-suppression induced by the FeLV-FAIDS variant and the pure red cell aplasia induced by FeLV-C/Sarma. Variant or enFeLV env sequences also appear to play a role in FeLV-associated lymphomas. Additional studies are required to determine the host cell processes that are perturbed by these variant env gene products. In the case of the FeLV-FAIDS variant, the aberrant env gene products appear to impair superinfection interference, resulting in accumulation of unintegrated viral DNA and cell death. In other cases it is likely that the viral env proteins interact with host products that are important in cell viability and/or proliferation. Understanding of these mechanisms will therefore provide insights to factors involved in normalThe haematological consequences of FIV are quite similar to those associated with HIV. As with HIV, FIV does not appear to directly infect myeloid or erythroid precursors, and the mechanisms of marrow suppression likely involve virus, viral antigen, and/or infected accessory cells in the marrow microenvironment. Studies using in vitro experimental models are required to define the effects of each of these microenvironmental elements on haematopoietic progenitors. As little is known about the molecular mechanisms of FIV pathogenesis, additional studies of disease-inducing FIV strains are needed to identify the genotypic features that correlate with virulent phenotypic features. Finally, experimental FIV infection in cats provides the opportunity to correlate in vivo virological and haematological changes with in vitro observations in a large animal model that closely mimics HIV infection in man.

Accumulation of unintegrated circular viral DNA in monocytes and growth-arrested T cells following infection with HIV-1

Cytocidal retrovirus infection is characterized by rapid accumulation of unintegrated viral DNA forms. These are thought to be generated by multiple rounds of reinfection and have been suggested to play a central role in cytopathogenesis. Here we have reviewed the work done in this area with HIV-1, mostly using acutely and chronically infected T cell and monocytic cell lines and in some cases T cells blocked at S phase of the cell cycle by aphidicolin treatment. To these studies, we have compared our findings with HIV-1 infected primary peripheral blood monocyte-derived macrophages and untreated and growth-arrested MT-2 cells, two biologically disparate cell populations. Using 1- and 2-long terminal repeat (LTR) circular forms as indicators of unintegrated viral DNA, we found similar rapid accumulation in both untreated and growth-arrested MT-2 cells. In contrast, we found much lower levels in monocyte/macrophages. Our findings suggest that accumulation of unintegrated viral DNA does not require virus production and reinfection in growth-arrested T cells. The significantly lower levels found in monocyte/macrophages may reflect superinfection resistance, allowing the maintenance of a persistent infection.

Accumulation of unintegrated viral DNA in rat glial cells infected with neurotropic Friend murine leukemia virus

FrC6 virus isolated from Friend murine leukemia virus (FLV) as a neurotropic virus clone induced a high-degree of accumulation of unintegrated viral DNA during infection of rat glial cells by 72 h post-infection. When anti-FLV neutralizing antibody, dextran sulfate, or 3'-azido-3'-deoxythymidine (AZT) was added to the culture within 24 h after infection, the accumulation of unintegrated viral DNA was inhibited. However, after 30-36 h post-infection, addition of anti-FLV antibody or dextran sulfate scarcely inhibited the accumulation of the unintegrated viral DNA, while addition of AZT at 30-36 h post-infection still reduced the amount of unintegrated viral DNA. Our results demonstrate that after 30-36 h post-infection, when second-round infection had already taken place, further superinfection with cell-free virus to glial cells was not required for the accumulation of unintegrated viral DNA. Possible mechanisms are discussed.

The cytopathic effect of human immunodeficiency virus is independent of high levels of unintegrated viral DNA accumulated in response to superinfection of cells.

Large quantities of genome-sized viral DNA are detected in the nucleoplasm of CD4+ T cells infected with human immunodeficiency virus type 1 (HIV-1). This unintegrated HIV DNA is in the form of both circular and linear species. Accumulation of such DNA occurs gradually during a 5 day HIV infection and is correlated with the proportion of cells involved in the production of HIV proteins. To pinpoint the stage in a synchronized HIV infection during which accumulation of HIV DNA occurs, high titres of HIV were employed to infect CEM cells to infect the majority of cells by the input virus. By this latter infection, more than 95% of cells became producers of HIV proteins at 48 h post-infection (p.i.) concomitantly with the development of the c.p.e. of HIV, manifested by formation of syncytia and induction of cell death by apoptosis. Addition of azidothymidine (AZT) or neutralizing anti-gp120 monoclonal antibodies at 8 h p.i. did not alter the course of virus infection nor the amount of virus produced at 48 h p.i. but the accumulation of unintegrated HIV DNA was drastically reduced. These results indicate that viral DNA accumulates as a result of superinfection of cells late in the virus cycle. The development of the c.p.e. of HIV was inhibited in the presence of neutralizing antibodies, whereas in the presence of AZT the accumulation of unintegrated HIV DNA was completely blocked without apparent effect on the c.p.e. These observations indicate that the c.p.e. of the HIV infection, which is manifested by syncytium formation and apoptosis, does not require superinfection of cells or accumulation of unintegrated viral DNA.

Unintegrated human immunodeficiency virus type 1 DNA in chronically infected cell lines is not correlated with surface CD4 expression

Using a polymerase chain reaction-based assay on total cell lysates, we have detected unintegrated human immunodeficiency virus type 1 (HIV-1) DNA in chronically infected T-lymphocytic (ACH-2, J1) and promyelocytic (OM-10.1) cell lines. Treatment with 3'-azido-3'-deoxythymidine (AZT) or soluble CD4 inhibited accumulation of unintegrated viral DNA about 10-fold within 72 h; removal of AZT permitted recovery to pretreatment levels within 72 h. Our results indicate that unintegrated HIV-1 DNA is unstable in these cell lines and originates from a continuous process of reinfection. OM-10.1 cells had relatively high levels of surface CD4 by flow cytometry and high levels of unintegrated viral DNA by polymerase chain reaction. ACH-2 cells had very low levels of both surface CD4 and unintegrated viral DNA. However, J1 cells, with surface CD4 below the level of detection of flow cytometry had a high level of unintegrated viral DNA similar to that of OM-10.1 cells. This implies that the number of CD4 receptors is not rate limiting for reinfection.

Reinfection results in accumulation of unintegrated viral DNA in cytopathic and persistent human immunodeficiency virus type 1 infection of CEM cells.

High levels of unintegrated viral DNA accumulate during human immunodeficiency virus type 1 (HIV-1) infection of CEM T cells. Reinfection of already infected cells is required to attain these levels and reinfection also promotes the development of HIV-induced cytopathology. Rates of virus production, however, are independent of the accumulation of unintegrated viral DNA. Neutralizing antibody added soon after infection reduced viral DNA levels without appreciably affecting the production of cell-free viral p24 antigen or reverse transcriptase activity. Only 50 pM AZT were required to reduce the accumulation of unintegrated viral DNA by 50% in contrast to the 25 nM required to inhibit virus production by 50%. Cytopathology, as measured by number of syncytia in infected cell cultures, was correlated with highly elevated levels of unintegrated viral DNA. The minimal levels of unintegrated viral DNA present constitutively in the persistently infected HCEM cell line were consonant with the absence of cytopathic effects in these cells. These data demonstrate that inhibiting the reinfection of already infected cells modulates cytopathic HIV-1 infection to a form that is persistent and noncytopathic.

Unintegrated DNA in cells infected in vitro with human immunodeficiency virus (HIV): a new approach to suppression of virus release

Retroviral RNA is copied into DNA by reverse transcriptase when the viral genome enters into its life cycle. In the case of human immunodeficiency virus (HIV), massive amounts of unintegrated viral DNA reportedly appear in the early phase of primary infection. However, the relationship between the accumulation of this DNA and the cytopathic effect (CPE) remains obscure. In an attempt to delineate this association, we examined the appearance of the unintegrated viral DNA by means of two experimental systems: (1) primary infection of highly susceptible MOLT-4#8 cells and (2) induction of CPE by cell-fusion of persistently infected MOLT-4#8 cells. A correlation was observed between the accumulation of unintegrated viral DNA and the appearance of CPE, both when MOLT-4#8 cells were infected with cell-free virus and when persistently infected MOLT-4#8 cells were co-cultured with uninfected cells. Persistently infected cells did not fuse spontaneously in culture, because they lack the CD4-molecule on their surfaces. However, when treated with polyethylene glycol (PEG), the cells fused, exhibited ballooning degeneration, and released fewer viruses. After PEG treatment, unintegrated viral DNA also appeared. Since such DNA is generally not detected in persistently infected cells, it is possible that some cellular mechanism exists to suppress the synthesis of viral DNA and that the fusion induced by PEG treatment cancels the suppression. Treatment of persistently infected cells with Ca 2+ ionophore and Ca 2+ antagonist also resulted in the accumulation of unintegrated viral DNA and inhibited virus release. These findings suggest that the induction of unintegrated HIV DNA may be an effective strategy for reducing the release of the virus.

Sequences near the 5' long terminal repeat of avian leukosis viruses determine the ability to induce osteopetrosis

Avian leukosis virus (ALV)-induced osteopetrosis is associated with the accumulation of unintegrated viral DNA in osteoblasts. Viruses constructed from the DNAs of an osteopetrosis-inducing ALV (Br21) and a non-osteopetrosis-inducing ALV (RAV-0) have been used to test for the role of viral genes in the induction of osteopetrosis. Our results map osteopetrotic potential to a 1,400-base-pair region near the 5' long terminal repeat. This region contains signals for the splicing, translation, and packaging of viral RNAs and coding sequences for the gag proteins p19 and p10 and the N terminus of p27.