Loss Of Viral Replication Research Articles

A Novel Leu92 Mutant of HIV-1 Reverse Transcriptase with a Selective Deficiency in Strand Transfer Causes a Loss of Viral Replication

The process of reverse transcription (RTN) in retroviruses is essential to the viral life cycle. This key process is catalyzed exclusively by the viral reverse transcriptase (RT) that copies the viral RNA into DNA by its DNA polymerase activity, while concomitantly removing the original RNA template by its RNase H activity. During RTN, the combination between DNA synthesis and RNA hydrolysis leads to strand transfers (or template switches) that are critical for the completion of RTN. The balance between these RT-driven activities was considered to be the sole reason for strand transfers. Nevertheless, we show here that a specific mutation in HIV-1 RT (L92P) that does not affect the DNA polymerase and RNase H activities abolishes strand transfer. There is also a good correlation between this complete loss of the RT's strand transfer to the loss of the DNA clamp activity of the RT, discovered recently by us. This finding indicates a mechanistic linkage between these two functions and that they are both direct and unique functions of the RT (apart from DNA synthesis and RNA degradation). Furthermore, when the RT's L92P mutant was introduced into an infectious HIV-1 clone, it lost viral replication, due to inefficient intracellular strand transfers during RTN, thus supporting the in vitro data. As far as we know, this is the first report on RT mutants that specifically and directly impair RT-associated strand transfers. Therefore, targeting residue Leu92 may be helpful in selectively blocking this RT activity and consequently HIV-1 infectivity and pathogenesis. Reverse transcription in retroviruses is essential for the viral life cycle. This multistep process is catalyzed by viral reverse transcriptase, which copies the viral RNA into DNA by its DNA polymerase activity (while concomitantly removing the RNA template by its RNase H activity). The combination and balance between synthesis and hydrolysis lead to strand transfers that are critical for reverse transcription completion. We show here for the first time that a single mutation in HIV-1 reverse transcriptase (L92P) selectively abolishes strand transfers without affecting the enzyme's DNA polymerase and RNase H functions. When this mutation was introduced into an infectious HIV-1 clone, viral replication was lost due to an impaired intracellular strand transfer, thus supporting the in vitro data. Therefore, finding novel drugs that target HIV-1 reverse transcriptase Leu92 may be beneficial for developing new potent and selective inhibitors of retroviral reverse transcription that will obstruct HIV-1 infectivity.

E1 Molecular and Cellular Correlates of the CIITA-Mediated Inhibition of HTLV-2 Tax-2 Transactivator Function Resulting in Loss of Viral Replication

MHC class II transactivator CIITA inhibits the function of HTLV-2 Tax-2 viral transactivator and, consequently, the replication of the virus in infected cells. By co-immunoprecipitation of lysates from 293T cells cotransfected with CIITA or fragments of it, and Tax-2 it was found that the two factors interact in vivo. At least two CIITA regions, at the 1–252 N-term and at the 410–1130 C-term, respectively, interacted with Tax-2. Interestingly only the 1–252 N-term region mediated Tax-2 functional inhibition. CIITA and Tax-2 localized both in the cytoplasm and in the nucleus, when separately expressed. Instead, when coexpressed, most of Tax-2 colocalized with CIITA in cytoplasm around the nuclear membrane. Interestingly, when CIITA nucleus-cytoplasm shuttling were blocked by leptomycin B treatment, most of the Tax-2 molecules were also blocked and co-localized with CIITA in the nucleus, suggesting that CIITA-Tax-2 binding does not preclude Tax-2 entry into the nucleus. Finally, the nuclear factor NF-YB, also strongly binds to Tax-2. Notably, although endogenous NF-YB does not inhibit Tax-2-dependent HTLV-2 LTR transactivation, it still binds to Tax-2, and in presence of CIITA, this binding seems to increase. These results strongly suggest that CIITA inhibit Tax-2 by binding the viral transactivator both directly or through a tripartite interaction with NF-YB. CIITA is therefore a viral restriction factor for HTLV-2 and this open the possibility to control HTLV-2 viral replication and spreading by the controlled induction of CIITA in infected cells.

Molecular and cellular correlates of the CIITA-mediated inhibition of HTLV-2 Tax-2 transactivator function resulting in loss of viral replication

BackgroundMHC class II transactivator CIITA inhibits the function of HTLV-2 Tax-2 viral transactivator and, consequently, the replication of the virus in infected cells. Moreover overexpression of the nuclear factor NF-YB, that cooperates with CIITA for the expression of MHC class II genes, results also in inhibition of Tax-2 transactivation. The purpose of this investigation was to assess the cellular and molecular basis of the CIITA-mediated inhibition on Tax-2, and the relative role of NF-YB in this phenomenon.MethodsBy co-immunoprecipitation of lysates from 293T cells cotransfected with CIITA or fragments of it, and Tax-2 it was assessed whether the two factors interact in vivo. A similar approach was used to assess Tax-2-NF-YB interaction. In parallel, deletion fragments of CIITA were tested for the inhibition of Tax-2-dependent HTLV-2 LTR-luciferase transactivation. Subcellular localization of CIITA and Tax-2 was investigated by immunofluorescence and confocal microscopy.ResultsCIITA and Tax-2 interact in vivo through at least two independent regions, at the 1-252 N-term and at the 410-1130 C-term, respectively. Interestingly only the 1-252 N-term region mediates Tax-2 functional inhibition. CIITA and Tax-2 are localized both in the cytoplasm and in the nucleus, when separately expressed. Instead, when coexpressed, most of Tax-2 colocalize with CIITA in cytoplasm and around the nuclear membrane. The Tax-2 minor remaining nuclear portion also co-localizes with CIITA. Interestingly, when CIITA nucleus-cytoplasm shuttling is blocked by leptomycin B treatment, most of the Tax-2 molecules are also blocked and co-localize with CIITA in the nucleus, suggesting that CIITA-Tax-2 binding does not preclude Tax-2 entry into the nucleus.Finally, the nuclear factor NF-YB, also strongly binds to Tax-2. Notably, although endogenous NF-YB does not inhibit Tax-2-dependent HTLV-2 LTR transactivation, it still binds to Tax-2, and in presence of CIITA, this binding seems to increase.ConclusionsThese results strongly suggest that CIITA inhibit Tax-2 by binding the viral transactivator both directly or through a tripartite interaction with NF-YB in. CIITA is therefore a viral restriction factor for HTLV-2 and this open the possibility to control HTLV-2 viral replication and spreading by the controlled induction of CIITA in infected cells

Clinical, histological and immunohistochemical study of feline viral plaques and bowenoid in situ carcinomas

Feline viral plaques (FVP) induced by papillomavirus (PV) are often hyperpigmented and flat warts. The fact that up to 47% of bowenoid in situ carcinomas (BISC), which also usually occur in the form of hyperpigmented plaques, are positive for PV antigen in immunochemistry suggests that BISC could evolve from FVP. The relationship between the presence of PV antigens and the clinical and histological features of 26 cases of feline dermatoses (clinically described as pigmented plaques and with histological diagnosis of FVP and/or BISC) was therefore determined. The cases were classified into one of the three following groups: FVP, FVP + BISC or BISC. Immunohistological detection of papillomavirus group-specific antigen was performed using a polyclonal rabbit antibovine papillomavirus antiserum. Of the seven cases in the FVP group, six were deemed positive by immunohistology as were all 10 cats in the FVP + BISC group. On the other hand, only one of the nine BISC cats was positive. The presence of both FVP and BISC lesions in some cats and the high detection rate of PV antigens in the FVP and FVP + BISC groups suggest that both conditions might have the same viral cause and that some BISC may evolve from FVP. The low rate of viral antigen detection in the BISC group indicates another cause or a loss of viral replication during the cancerogenesis.

The cellular HIV-1 Rev cofactor hRIP is required for viral replication

An important goal of contemporary HIV type 1 (HIV-1) research is to identify cellular cofactors required for viral replication. The HIV-1 Rev protein facilitates the cytoplasmic accumulation of the intron-containing viral gag-pol and env mRNAs and is required for viral replication. We have previously shown that a cellular protein, human Rev-interacting protein (hRIP), is an essential Rev cofactor that promotes the release of incompletely spliced HIV-1 RNAs from the perinuclear region. Here, we use complementary genetic approaches to ablate hRIP activity and analyze HIV-1 replication and viral RNA localization. We find that ablation of hRIP activity by a dominant-negative mutant or RNA interference inhibits virus production by mislocalizing Rev-directed RNAs to the nuclear periphery. We further show that depletion of endogenous hRIP by RNA interference results in the loss of viral replication in human cell lines and primary macrophages; virus production was restored to wild-type levels after reintroduction of hRIP protein. Taken together, our results indicate that hRIP is an essential cellular cofactor for Rev function and HIV-1 replication. Because hRIP is not required for cell viability, it may be an attractive target for the development of new antiviral strategies.

Lethal mutagenesis of HIV by mutagenic ribonucleoside analogs.

The extraordinarily high mutation rate of HIV suggests that the viral population exists near the threshold for viral viability. An increase in mutation could mediate a lethal increase in the already high proportion of defective viruses. We initially tested this hypothesis by culturing HIV-infected cells in mutagenic deoxyribonucleoside analogs. In the presence of 5-hydroxydeoxycytidine, there was a loss of viral replication after sequential passages of HIV in human CEM cells (Loeb LA, et al.: Proc Natl Acad Sci USA 1999;96:14921497). We now propose to use mutagenic ribonucleosides to induce lethal mutagenesis. The incorporation of mutagenic analogs into the HIV genome by the host cell RNA polymerase, rather than by the viral reverse transcriptase, augurs well for the avoidance of resistant viruses.

Lethal mutagenesis of HIV with mutagenic nucleoside analogs.

The human immunodeficiency virus (HIV) replicates its genome and mutates at exceptionally high rates. As a result, the virus is able to evade immunological and chemical antiviral agents. We tested the hypothesis that a further increase in the mutation rate by promutagenic nucleoside analogs would abolish viral replication. We evaluated deoxynucleoside analogs for lack of toxicity to human cells, incorporation by HIV reverse transcriptase, resistance to repair when incorporated into the DNA strand of an RNA.DNA hybrid, and mispairing at high frequency. Among the candidates tested, 5-hydroxydeoxycytidine (5-OH-dC) fulfilled these criteria. In seven of nine experiments, the presence of this analog resulted in the loss of viral replicative potential after 9-24 sequential passages of HIV in human CEM cells. In contrast, loss of viral replication was not observed in 28 control cultures passaged in the absence of the nucleoside analog, nor with other analogs tested. Sequence analysis of a portion of the HIV reverse transcriptase gene demonstrated a disproportionate increase in G --> A substitutions, mutations predicted to result from misincorporation of 5-OH-dC into the cDNA during reverse transcription. Thus, "lethal mutagenesis" driven by the class of deoxynucleoside analogs represented by 5-OH-dC could provide a new approach to treating HIV infections and, potentially, other viral infections.

Pharmacological Management of Chronic Viral Hepatitis

Chronic viral hepatitis is a worldwide health problem. Complications associated with chronic viral hepatitis include liver failure, cirrhosis and hepatocellular carcinoma. The disease often is asymptomatic, but malaise, weakness and fatigue can occur. Alfa-inter-feron (α-IFN) is the only treatment with proven benefit in chronic viral hepatitis. Common adverse physical and psychiatric effects of therapy are described. The efficacy, however, varies widely. In chronic hepatitis B, α-IFN improves liver aminotransferases and histology, and leads to loss of viral replication in 25–40% of patients. Nucleoside analogues are being evaluated as an alternative treatment. In chronic hepatitis C, α-IFN leads to improvement in liver aminotransferases and histology, and clearance of HCV RNA in 10–25% of patients. The new treatment under investigation is a combination of α-IFN and ribavirin. In chronic hepatitis D, prolonged treatment using similar dosing regimens of α-IFN in chronic hepatitis B result in clearance of HD...

THERAPY FOR HEPATITIS B VIRUS INFECTION

Major advances have been made in the therapy of chronic viral hepatitis B during the past several years. This period has witnessed the publication of large, multicenter trials of recombinant interferon alfa for chronic hepatitis B in the United States and the completion of several similarly designed studies in North America, Europe and Asia. These studies have defined an initial response rate of approximately 40% to 50%. In contrast to the experience with chronic hepatitis C, loss of viral replication is generally sustained. Repeat courses of therapy are likely to result in the same type of response as that observed initially. Quantitative assessment of viral replication (HBV DNA, HBeAg) is important in predicting the likelihood of response and in monitoring patients during therapy. Perhaps the most compelling reason to treat chronic hepatitis B with interferon is the disappearance of circulating HBsAg in one third of responders with a further increase in frequency of this phenomenon as follow-up continues. Another important advantage to treatment is the striking degree of histologic improvement that is frequently observed years after a response has been achieved. Although a substantial number of patients do not respond to interferon, several promising agents that should allow for a greater degree of success, when used either alone or in combination with interferon, are under study. A short course of corticosteroids prior to interferon appears to improve response rates in patients who have low ALT levels at baseline and has been the preferred approach for these patients at our medical center. Progress is being made in the development of safer interferon regimens for patients with mild-to-moderate hepatic decompensation. Nonetheless, even patients with marginal synthetic function, as reflected by albumin levels within the low-normal range, appear to be at greater risk for complications during therapy and should preferably be referred for inclusion in research programs. Appropriate patient selection remains a critical step for maximizing the safety as well as efficacy of interferon treatment.

Virucidal effects of glucose oxidase and peroxidase or their protein conjugates on human immunodeficiency virus type 1

Glucose oxidase and peroxidase (lactoperoxidase or myeloperoxidase) are virucidal to human immunodeficiency virus type 1 (HIV-1) in the presence of sodium iodide, as assessed by the loss of viral replication in a syncytium-forming assay or by the inhibition of cytopathic effects on infected cells. In the presence of low concentrations of sodium iodide, five HIV-1 isolates were equally susceptible to this virucidal system at enzyme concentrations of a few milliunits. The loss of viral replication was linearly related to the time of incubation in the enzyme solutions, with an inactivation rate of 1 log unit every 30 min. These enzymes and this halide were also cytotoxic to chronically infected, but not to uninfected, cultured CEM cells. Protein conjugates were prepared by using the enzymes and murine antibody 105.34, which recognized the V3 loop of HIV-1 LAI isolate surface glycoprotein, or recombinant human CD4. The protein conjugates inactivated free virus at rates similar to those of the free enzymes and were more effective than antibody or recombinant CD4 alone. These in vitro findings demonstrate that the peroxidase-H2O2-halide system provides potent virucidal activity against HIV-1.

Antiviral therapy of chronic hepatitis B: past, present, and future

A wide variety of agents has been used to treat chronic hepatitis B, but none has proved effective with the exception of interferon. Toxicity has been a major problem with some drugs whereas in others a lack of antiviral potency has been demonstrated. Alpha-interferon represents a good compromise because it has both immunomodulatory and antiviral properties; moreover, it is generally well tolerated. Loss of HBeAg and hepatitis B virus DNA may be anticipated in 40-50% of patients who are treated with doses of 5 million units daily or 10 million units thrice weekly for 16 weeks. While drug-related adverse effects occur commonly, the majority of clinically stable patients are able to tolerate this regimen, and withdrawal from drug is necessary in approximately 5% of patients. Unlike the situation with chronic hepatitis C, loss of viral replication tends to be sustained years later. Disappearance of HBsAg only occurs in 10-15% of treated patients within the first year after therapy, but an increasing number of responders demonstrate HBsAg seroconversion upon prolonged follow-up. Hepatitis B virus DNA usually disappears from serum by polymerase chain reaction at the time of HBsAg loss. Low copy numbers of residual viral DNA are still detectable in liver tissue at this time, but this has uncertain significance. Marked improvement in histological features has been observed years after loss of HBsAg. Pre-therapy levels of circulating viral DNA and aminotransferase activity, degree of histologic activity, and HIV status appear to influence the response to therapy.(ABSTRACT TRUNCATED AT 250 WORDS)

Initial results with recombinant interferon alfa-2b in patients with chronic hepatitis C: the United States experience.

Early experience with recombinant interferon alfa-2b in chronic hepatitis C (HCV) has shown that normalisation of serum alanine aminotransferase activities and reduction in hepatic inflammation can be achieved in about 40% of patients. This occurs with loss of viral replication as measured by polymerase chain reaction for HCV-RNA. Further studies are needed to identify patients most likely to respond to treatment and to investigate the issues of non-response and relapse when treatment is stopped. Alternative interferon treatment regimens, as well as other drugs and combinations of agents, need to be investigated.