Amino-acid inserts of HIV-1 capsid (CA) induce CA degradation and abrogate viral infectivity: Insights for the dynamics and mechanisms of HIV-1 CA decomposition.

Accumulation of amino acid (AA) insertions/substitutions are observed in the Gag-protein of HIV-1 variants resistant to HIV-1 protease inhibitors. Here, we found that HIV-1 carrying AA insertions in capsid protein (CA) undergoes aberrant CA degradation. When we generated recombinant HIV-1s (rHIV-1s) containing 19-AAs in Gag, such insertions caused significant CA degradation, which initiated in CA's C-terminal. Such rHIV-1s had remarkable morphological abnormality, decreased infectivity, and no replicative ability, which correlated with levels of CA degradation. The CA degradation observed was energy-independent and had no association with cellular/viral proteolytic mechanisms, suggesting that the CA degradation occurs due to conformational/structural incompatibility caused by the 19-AA insertions. The incorporation of degradation-prone CA into the wild-type CA resulted in significant disruption of replication competence in "chimeric" virions. The data should allow better understanding of the dynamics and mechanisms of CA decomposition/degradation and retroviral uncoating, which may lead to new approach for antiretroviral modalities.

A case of hepatitis C-associated osteosclerosis: accelerated bone turnover controlled by pulse steroid therapy.

Hepatitis C-associated osteosclerosis (HCAO), a very rare disorder in which an extremely rapid bone turnover occurs and results in osteosclerosis, was acknowledged in 1990s as a new clinical entity with the unique bone disorder and definite link to chronic type C hepatitis, although the pathogenesis still remains unknown. Affected patients suffer from excruciating deep bone pains. We report the 19th case of HCAO with diagnosis confirmed by bone biopsy, and treated initially with a bisphosphonate, next with corticosteroids and finally with direct acting antivirals (DAA: sofosbuvir and ribavirin) for HCV infection. Risedronate, 17.5 mg/day for 38 days, did not improve the patient's symptoms or extremely elevated levels of bone markers, which indicated hyper-bone-formation and coexisting hyper-bone-resorption in the patient. Next, intravenous methylprednisolone pulse therapy followed by high-dose oral administration of prednisolone evidently improved them. DAA therapy initiated after steroid therapy successfully achieved sustained virological response, but no additional therapeutic effect on them was observed. Our results strongly suggested that the underlying immunological alteration is the crucial key to clarify the pathogenesis of HCAO. Bone mineral density of lumbar vertebrae of the patient was increased by 14% in four-month period of observation. Clarification of the mechanisms that develop osteosclerosis in HCAO might lead to a new therapeutic perspective for osteoporosis. LEARNING POINTS: HCAO is an extremely rare bone disorder, which occurs exclusively in patients affected with HCV, of which only 18 cases have been reported since 1992 and pathogenesis still remains unclear.Pathophysiology of HCAO is highly accelerated rates of both bone formation and bone resorption, with higher rate of formation than that of resorption, which occur in general skeletal leading to the diffuse osteosclerosis with severe bone pains.Steroid therapy including intravenous pulse administration in our patient evidently ameliorated his bone pains and reduced elevated values of bone markers. This was the first successful treatment for HCAO among cases reported so far and seemed to propose a key to solve the question for its pathogenesis.The speed of increase in the bone mineral content of the patient was very high, suggesting that clarification of the mechanism(s) might lead to the development of a novel therapy for osteoporosis.

[A case of solitary fibrous malignant tumor with multiple metastases].

An 80-year-old woman visited her family physician because of back pain. A chest X-ray film showed a mass in the left middle lung field. She was referred to our hospital for further examinations. A computed tomography-guided lung biopsy revealed a solitary fibrous tumor, and a whole body examination demonstrated multiple metastases, including in the spine, ribs, femurs and pubis. Considering her age, chemotherapy was not given, but we administered radiotherapy for the metastatic lesions. Subsequently detected metastases of the left orbit, liver, scapula and humerus were also then irradiated, but she died 11 months after the initial diagnosis due to the complication of bacterial pneumonia. A case of a solitary malignant fibrous tumor with multiple metastases was reported.

[A case of Legionella pneumonia with diabetic ketoacidosis rescued despite the development of acute renal failure and ARDS].

A 57-year-old man was admitted to our hospital complaining of general fatigue and appetite loss. The initial chest radiograph showed infiltration in the left upper lung field. Cefozopran was administered. Concomitant diabetic ketoacidosis was treated with hydration and rapid-acting insulin. However he was intubated and ventilated because of deteriorated respiratory condition and ketoacidosis on the 3rd hospital day. Urinary legionella antigen was detected the same day, therefore ciprofloxacin and imipenem were initiated. On the 4th hospital day, he developed acute renal failure and was treated with continuous hemodiafiltration. In addition, he developed adult respiratory distress syndrome on the 6th hospital day, therefore siveletat sodium was given. The patient gradually began to improve and was extubated on the 17th hospital day. After that he was transferred to the metabolic ward on the 24th hospital day for control of his diabetes mellitus. Despite the severe complications in his clinical course, including diabetic ketoacidosis, acute renal failure and ARDS, detection of Legionella pneumophila by a urinary antigen test, Gimenez stain and sputum culture made prompt and proper administration of antibiotics possible, finally yielding a desirable outcome.

Emergence of human immunodeficiency virus type 1 variants containing the Q151M complex in children receiving long-term antiretroviral chemotherapy.

We examined 28 children with HIV-1 infection who were not responding to existing antiviral regimens and were enrolled into clinical trials conducted at the National Cancer Institute to receive salvage therapy. In 3 of the 28 patients (10.7%), the Q151M complex amino acid substitutions were identified. The three patients had received nucleoside reverse transcriptase inhibitor (NRTI) monotherapy and/or combination regimens with multiple NRTIs for 4.3-8.6 years prior to the study. Recombinant infectious clones generated by incorporating the RT-encoding region of HIV-1 isolated from patients' plasma samples were highly resistant to zidovudine, didanosine and stavudine, while they were moderately resistant to lamivudine and tenofovir disoproxil fumarate (TDF). TDF-containing regimens reduced HIV-1 viremia in two of the three children carrying the Q151M complex. These data suggest that the Q151M could be prevalent in pediatric patients with long-term NRTI monotherapy and/or dual NRTI regimens and that HAART regimens containing TDF may be meritorious in such patients.

Preferential selection of human T-cell leukemia virus type 1 provirus lacking the 5' long terminal repeat during oncogenesis.

In adult T-cell leukemia (ATL) cells, a defective human T-cell leukemia virus type 1 (HTLV-1) provirus lacking the 5' long terminal repeat (LTR), designated type 2 defective provirus, is frequently observed. To investigate the mechanism underlying the generation of the defective provirus, we sequenced HTLV-1 provirus integration sites from cases of ATL. In HTLV-1 proviruses retaining both LTRs, 6-bp repeat sequences were adjacent to the 5' and 3' LTRs. In 8 of 12 cases with type 2 defective provirus, 6-bp repeats were identified at both ends. In five of these cases, a short repeat was bound to CA dinucleotides of the pol and env genes at the 5' end, suggesting that these type 2 defective proviruses were formed before integration. In four cases lacking the 6-bp repeat, short (6- to 26-bp) deletions in the host genome were identified, indicating that these defective proviruses were generated after integration. Quantification indicated frequencies of type 2 defective provirus of less than 3.9% for two carriers, which are much lower than those seen for ATL cases (27.8%). In type 2 defective proviruses, the second exons of the tax, rex, and p30 genes were frequently deleted, leaving Tax unable to activate NF-kappaB and CREB pathways. The HTLV-1 bZIP factor gene, located on the minus strand, is expressed in ATL cells with this defective provirus, and its coding sequences are intact, suggesting its significance in oncogenesis.

9-{[3-fluoro-2-(hydroxymethyl)cyclopropylidene]methyl}adenines and -guanines. Synthesis and antiviral activity of all stereoisomers1.

All stereoisomers of adenine and guanine methylene-3-fluoromethylenecyclopropane analogues of nucleosides 9a, 9b, 10a, 10b, 11a, 11b, 12a, and 12b were synthesized and their antiviral activities were evaluated. A highly convergent approach permitted the synthesis of all these analogues using a single intermediate 15. Reaction of aldehyde 13 with fluorotrichloromethane and tri-n-butylphosphine gave fluoroalkenes 14a+14b (83:17). Addition of carbene derived from ethyl diazoacetate gave cyclopropane 15 as the major product. Reduction (19), bromination (20), and phenylselenenylation (21), followed by Se oxidation and beta-elimination gave cis-methylenecyclopropane 22. Addition of bromine provided the reagent 23 for alkylation-elimination. Reaction of 23 with adenine led to an isomeric mixture 25a+26a that after deprotection afforded analogues 9a and 10a. The 2-amino-6-chloropurine furnished 25e+26e and after deblocking (9e and 10e) and hydrolysis gave targets 9b and 10b. Intermediate 15 provided, after debenzylation (27), 2-nitrophenylselenenylation (28), reduction (29), benzylation (30), and oxidation-elimination trans-methylenecyclopropane 31. Addition of bromine gave reagent 32. Further transformations followed the sequence outlined for analogues 9a, 9b, 10a, and 10b. Analogue 9b was effective against human cytomegalovirus (HCMV; Towne) with EC50 2.9 microM. The trans-isomer 10b inhibited AD169 strain of HCMV (EC50 15 microM) and the murine virus MCMV (EC50 2.5 microM). Compound 12a was effective against Epstein-Barr virus (EC50<0.03 microM). Analogue 9a inhibited varicella zoster virus (EC50 5.9 microM) and human immunodeficiency virus type 1 (EC50 5.2 microM). Analogues 9a, 10a, and 11a are moderate substrates for adenosine deaminase. The structure-activity relationships will be discussed in context with other methylenecyclopropane analogues.

Amino acid insertions near Gag cleavage sites restore the otherwise compromised replication of human immunodeficiency virus type 1 variants resistant to protease inhibitors.

A variety of amino acid substitutions in the protease and Gag proteins have been reported to contribute to the development of human immunodeficiency virus type 1 (HIV-1) resistance to protease inhibitors. In the present study, full-length molecular infectious HIV-1 clones were generated by using HIV-1 variants isolated from heavily drug-experienced and therapy-failed AIDS patients. Of six full-length infectious clones generated, four were found to have unique insertions (TGNS, SQVN, AQQA, SRPE, APP, and/or PTAPPA) near the p17/p24 and p1/p6 Gag cleavage sites, in addition to the known resistance-related multiple amino acid substitutions within the protease. The addition of such Gag inserts mostly compromised the replication of wild-type HIV-1, whereas the primary multidrug-resistant HIV infectious clones containing inserts replicated significantly better than those modified to lack the inserts. Western blot analyses revealed that the processing of Gag proteins by wild-type protease was impaired by the presence of the inserts, whereas that by mutant protease was substantially improved. The present study represents the first report clearly demonstrating that the inserts seen in the proximity of the Gag cleavage sites in highly multi-PI resistant HIV-1 variants restore the otherwise compromised enzymatic activity of mutant protease, enabling the multi-PI-resistant HIV-1 variants to remain replication competent.