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Results for “viral complexity”

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PubMed2001

Changes in anti-viral effectiveness of interferon after dose reduction in chronic hepatitis C patients: a case control study.

BACKGROUND: High dose interferon induction treatment of hepatitis C viral infection blocks viral production over 95%. Since dose reduction is often performed due to clinical considerations, the effect of dose reduction on hepatitis C virus kinetics was studied. METHODS: A new model that allowed longitudinal changes in the parameters of viral dynamics was used in a group of genotype-1 patients (N = 15) with dose reduction from 10 to 3 million units of interferon daily in combination with ribavirin, in comparison to a control group (N = 9) with no dose reduction. RESULTS: Dose reduction gave rise to a complex viral kinetic pattern, which could be only explained by a decrease in interferon effectiveness in blocking virion production. The benefit of the rapid initial viral decline following the high induction dose is lost after dose reduction. In addition, in some patients also the second phase viral decline slope, which is highly predictive of success of treatment, was impaired by the dose reduction resulting in smaller percentage of viral clearance in the dose reduction group. CONCLUSIONS: These findings, while explaining the failure of many induction schedules, suggest that for genotype-1 patients induction therapy should be continued till HCVRNA negativity in serum in order to increase the sustained response rate for chronic hepatitis C.

Antiviral Agents↗

PubMed1985

The molecular biology of Epstein-Barr virus.

Epstein-Barr virus (EBV) was discovered in continuously growing tumor cells derived from African patients with Burkitt's lymphoma. In the intervening twenty years, much biological and biochemical information has been accumulated. The virus infects B lymphocytes and occupies a unique position among human herpesviruses in that it is the only one which is capable of forming a latent infection whereby complete copies of the virus genome persist in growth transformed cells. Since there are no fully permissive cell systems of virus replications, only the established B cell lines are available for study of the molecular events of EB virus in infected cells. A viral cycle consists of four stages, latent, early replicative, middle replicative and late replicative stages. In the latent state, only small parts of the viral genome are transcribed and express transformation proteins: nuclear antigens (EBNAs) and lymphocyte determined membrane antigen (LYDMA). After reactivation of viral genome and during a productive cycle, more than 50 RNAs are expressed and over 30 viral-specified polypeptides are detectable by immunoprecipitation with a high titer human anti-EBV serum. During the early replicative stage, early antigens (EA) and DNA enzymes, both necessary for DNA synthesis, are synthesized. In the late replicative stage, about 30-40 mRNA are transcribed and two major late antigen complexes, viral capsid antigens (VCA) and membrane antigens (MA), are identified. These antigens are indispensable for the formation of virions.

Animals↗

PubMed2006

Binding of phosphinate and phosphonate inhibitors to aspartic proteases: a first-principles study.

Phosphinate and phosphonate derivatives are potent inhibitors of aspartic proteases (APs). The affinity for the enzyme might be caused by the presence of low barrier hydrogen bonds between the ligand and the catalytic Asp dyad in the cleavage site. We have used density functional theory calculations along with hybrid quantum mechanics/molecular mechanics Car-Parrinello molecular dynamics simulations to investigate the hydrogen-bonding pattern at the binding site of the complexes of human immunodeficiency virus type-1 AP and the eukaryotic endothiapepsin and penicillopepsin. Our calculations are in fair agreement with the NMR data available for endothiapepsin (Coates et al. J. Mol. Biol. 2002, 318, 1405-1415) and show that the most stable active site configuration is the diprotonated, negatively charged form. In the viral complex both protons are located at the catalytic Asp dyad, while in the eukaryotic complexes the proton shared by the closest oxygen atoms is located at the phosphinic/phosphonic group.

Aspartic Acid Endopeptidases↗

PubMed2002

HIV type 1 variants transmitted to women in Kenya require the CCR5 coreceptor for entry, regardless of the genetic complexity of the infecting virus.

Although nearly half of the HIV-1-infected adults in the world are women, little is known about the virologic determinants of transmission to women. Studies suggest that women are frequently infected with multiple HIV-1 genotypes, whereas men are infected with a single genotype. In the current study, we assessed whether the diverse HIV-1 genomes present at the time of infection in women encode viruses that have diverse coreceptor specificities. For this purpose, we defined the coreceptor requirements of viruses found in recently infected Kenyan women, three of whom had multiple viral genotypes and the remaining two of whom had a single genotype. Full-length envelope clones were amplified directly from blood and the dominant genotypes were identified. Envelope clones derived from all five women were able to pseudotype infectious particles competent to infect cells expressing CCR5, but not cells expressing only CXCR4. Thus, regardless of viral complexity at the time of infection, the viruses present at early stages of HIV-1 infection in women use CCR5, suggesting that cells expressing CCR5 are important targets for heterosexual HIV-1 transmission to women.

Base Sequence↗

PubMed2000

Biochemical mechanisms involved in overcoming HIV resistance to nucleoside inhibitors of reverse transcriptase.

The development of drug combinations that act effectively against both wild-type and mutated resistant forms of HIV-1 reverse transcriptase (RT) is a major goal in management of HIV disease. Recent studies have shown that resistance to different nucleoside analog RT inhibitors (NRTIs), an important class of anti-viral drugs, can result in different amino acid substitutions in close proximity to the dNTP binding pocket of the enzyme. Some of these mutations have been shown to cause cross- or multiple resistance among various members of this family of inhibitors. In contrast, certain combinations of amino acid substitutions can sometimes lead to increased drug susceptibility and may also result in resensitization of formerly resistant viruses. A biochemical understanding of these complex viral phenotypes may be of major importance in regard to development of novel chemotherapeutic strategies that can act at the level of drug-resistant mutated enzymes. In this review, we discuss several principles that help to explain the increased susceptibility and resensitization to some antiviral agents used in the context of combination treatment. The conclusions are largely based on our current understanding of mechanisms involved in drug-resistance to 3TC and AZT. Copyright 2000 Harcourt Publishers Ltd.

Journal Article↗

PubMed2006

Papillomavirus genome structure, expression, and post-transcriptional regulation.

Papillomaviruses are a group of small non-enveloped DNA tumor viruses whose infection usually causes benign epithelial lesions (warts). Certain types of HPVs, such as HPV-16, HPV-18, and HPV-31, have been recognized as causative agents of cervical cancer and anal cancer and their infections, which arise via sexual transmission, are associated with more than 95% of cervical cancer. Papillomaviruses infect keratinocytes in the basal layer of stratified squamous epithelia and replicate in the nucleus of infected keratinocytes in a differentiation-dependent manner. Viral gene expression in infected cells depends on cell differentiation and is tightly regulated at the transcriptional and post-transcriptional levels. A noteworthy feature of all papillomavirus transcripts is that they are transcribed as a bicistronic or polycistronic form containing two or more ORFs and are polyadenylated at either an early or late poly(A) site. In the past ten years, remarkable progress has been made in understanding how this complex viral gene expression is regulated at the level of transcription (such as via DNA methylation) and particularly post-transcription (including RNA splicing, polyadenylation, and translation). Current knowledge of papillomavirus mRNA structure and RNA processing has provided some clues on how to control viral oncogene expression. However, we still have little knowledge about which mRNAs are used to translate each viral protein. Continuing research on post-transcriptional regulation of papillomavirus infection will remain as a future focus to provide more insights into papillomavirus-host interactions, the virus life-cycle, and viral oncogenesis.

DNA Methylation↗

PubMed2005

In-depth, longitudinal analysis of viral quasispecies from an individual triply infected with late-stage human immunodeficiency virus type 1, using a multiple PCR primer approach.

Co-infections with more than one human immunodeficiency virus type 1 (HIV-1) subtype appear to be the source of new recombinant strains and may be commonplace in high-risk cohorts exposed to multiple subtypes. Many potential dual infections have been identified during the HIV Superinfection Study in Mbeya, Tanzania, where 600 female bar workers who are highly exposed to subtypes A, C, and D have been evaluated every 3 months for over 3 years by use of the MHAacd HIV-1 genotyping assay. Here we describe an in-depth, longitudinal analysis of the viral quasispecies in a woman who was triply infected with HIV-1 and who developed AIDS and passed away 15 months after enrollment. The MHA results obtained at 0, 3, 6, 9, and 12 months revealed dual-probe reactivities and shifts in subtype over time, indicating a potential dual infection and prompting further investigation. The multiple infection was confirmed by PCR amplification of three genome regions by a multiple primer approach, followed by molecular cloning and sequencing. A highly complex viral quasispecies was found, including several recombinant forms, with vpu/gp120 being the most diverse region. A significant fluctuation in molecular forms over time was observed, showing that the serial sample format is highly desirable, if not essential, for the identification of multiple infections. In a separate experiment, we confirmed that the detection of co-infections is more efficient with the use of multiple amplification primers to overcome the primer bias that results from the enormous diversity in the HIV-1 genome.

Acquired Immunodeficiency Syndrome↗

PubMed2006

tRNAs promote nuclear import of HIV-1 intracellular reverse transcription complexes.

Infection of non-dividing cells is a biological property of HIV-1 crucial for virus transmission and AIDS pathogenesis. This property depends on nuclear import of the intracellular reverse transcription and pre-integration complexes (RTCs/PICs). To identify cellular factors involved in nuclear import of HIV-1 RTCs, cytosolic extracts were fractionated by chromatography and import activity examined by the nuclear import assay. A near-homogeneous fraction was obtained, which was active in inducing nuclear import of purified and labeled RTCs. The active fraction contained tRNAs, mostly with defective 3' CCA ends. Such tRNAs promoted HIV-1 RTC nuclear import when synthesized in vitro. Active tRNAs were incorporated into and recovered from virus particles. Mutational analyses indicated that the anticodon loop mediated binding to the viral complex whereas the T-arm may interact with cellular factors involved in nuclear import. These tRNA species efficiently accumulated into the nucleus on their own in a energy- and temperature-dependent way. An HIV-1 mutant containing MLV gag did not incorporate tRNA species capable of inducing HIV-1 RTC nuclear import and failed to infect cell cycle-arrested cells. Here we provide evidence that at least some tRNA species can be imported into the nucleus of human cells and promote HIV-1 nuclear import.

Active Transport, Cell Nucleus↗

PubMed2004

Human apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G) is incorporated into HIV-1 virions through interactions with viral and nonviral RNAs.

Apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G) is a host cytidine deaminase that is packaged into virions and confers resistance to retroviral infection. APOBEC3G deaminates deoxycytidines in minus strand DNA to deoxyuridines, resulting in G to A hypermutation and viral inactivation. Human immunodeficiency virus type 1 (HIV-1) virion infectivity factor counteracts the antiviral activity of APOBEC3G by inducing its proteosomal degradation and preventing virion incorporation. To elucidate the mechanism of viral suppression by APOBEC3G, we developed a sensitive cytidine deamination assay and analyzed APOBEC3G virion incorporation in a series of HIV-1 deletion mutants. Virus-like particles derived from constructs in which pol, env, and most of gag were deleted still contained high levels of cytidine deaminase activity; in addition, coimmunoprecipitation of APOBEC3G and HIV-1 Gag in the presence and absence of RNase A indicated that the two proteins do not interact directly but form an RNase-sensitive complex. Viral particles lacking HIV-1 genomic RNA which were generated from the gag-pol expression constructs pC-Help and pSYNGP packaged APOBEC3G at 30-40% of the wild-type level, indicating that interactions with viral RNA are not necessary for incorporation. In addition, viral particles produced from an nucleocapsid zinc finger mutant contained approximately 1% of the viral genomic RNA but approximately 30% of the cytidine deaminase activity. The reduction in APOBEC3G incorporation was equivalent to the reduction in the total RNA present in the nucleocapsid mutant virions. These results indicate that interactions with viral proteins or viral genomic RNA are not essential for APOBEC3G incorporation and suggest that APOBEC3G interactions with viral and nonviral RNAs that are packaged into viral particles are sufficient for APOBEC3G virion incorporation.

APOBEC-3G Deaminase↗

PubMed2003

Global screening for human viral pathogens.

We propose a system for continuing surveillance of viral pathogens circulating in large human populations. We base this system on the physical isolation of viruses from large pooled samples of human serum and plasma (e.g., discarded specimens from diagnostic laboratories), followed by shotgun sequencing of the resulting genomes. The technology for concentrating virions from 100-L volumes was developed previously at Oak Ridge National Laboratory, and the means for purifying and concentrating virions from volumes in microliters have been developed recently. At the same time, marine virologists have developed efficient methods for concentrating, amplifying, and sequencing complex viral mixtures obtained from the ocean. Given this existing technology base, we believe an integrated, automated, and contained system for surveillance of the human "virome" can be implemented within 1 to 2 years. Such a system could monitor the levels of known viruses in human populations, rapidly detect outbreaks, and systematically discover novel or variant human viruses.

Automation↗

PubMed1994

A case of viral encephalitis with hyperthermia and status epilepticus: efficacy of dantrolene treatment.

A case of viral encephalitis is described. A 55-year-old female developed hyperthermia and status epilepticus. Herpes simplex virus was regarded as a factor since viral complex titres were elevated and decreased. In addition to acyclovir and anticonvulsive agents, dantrolene (150 mg/day) was administered orally through a nasogastric tube for heat stroke. Convulsions and hyperthermia gradually decreased and intravenous agents were replaced by oral agents. Dantrolene may be a useful adjuvant in hyperthermia and convulsions associated with encephalitis.

Anticonvulsants↗

PubMed2006

Genetic diversity analyses of grapevine Rupestris stem pitting-associated virus reveal distinct population structures in scion versus rootstock varieties.

Grapevine Rupestris stem pitting-associated virus (GRSPaV) is a member of the genus Foveavirus within the family Flexiviridae. GRSPaV is closely associated with the disease Rupestris stem pitting and is frequently detected in grapevines worldwide. Previous research in several laboratories suggests that GRSPaV consists of a family of sequence variants. However, the genetic composition of GRSPaV variants in viral isolates from scion and rootstock varieties has not been studied extensively. In this report, the genetic diversity and population structure of GRSPaV isolates from scion and rootstock varieties were analysed using two pairs of primers targeting two different genomic regions encoding the helicase domain of the replicase and the capsid protein. In total, 190 cDNA clones derived from 24 isolates were sequenced and analysed. At least four major groups of GRSPaV variants were found to exist in grapevines. Interestingly, the majority of the scion varieties (9/10) that were analysed, regardless of their genetic background and geographical origin, harboured complex viral populations composed of two to four distinct viral variants. In contrast, the viral populations in isolates from rootstock varieties were homogeneous and comprised a single variant. The practice of grafting between scion and rootstock varieties commonly used in modern viticulture, coupled with the frequent regional and international exchange of propagating materials, may have played a major role in the ubiquitous distribution and mixed infections of distinct GRSPaV variants among scion varieties. The possible origin and evolution of GRSPaV are also discussed.

Cloning, Molecular↗

PubMed1980

Subgenomic fragment of molecular cloned Friend murine leukemia virus DNA contains the gene(s) responsible for Friend murine leukemia virus-induced disease.

Friend murine leukemia virus (G-MuLV) is a helper-independent, type C retrovirus isolated from stocks of Friend virus complex (spleen focus-forming virus plus MuLV). In cell culture, F-MuLV has an ecotropic and NB-tropic host range and causes XC cells to fuse. When injected into newborn NIH Swiss mice, F-MuLV produces hepatosplenomegaly, severe anemia, and numerous circulating hematopoietic precursors in the peripheral blood with normal thymus and lymph nodes after 3 to 6 weeks. Recently, we molecularly cloned an 8.5-kilobase pair (kbp) form of F-MuLV DNA from which we could recover the pathogenic F-MuLV virus by DNA transfection of NIH 3T3 cells. From this molecularly cloned F-MuLV DNA, we have now subcloned in pBR322 a 4.1-kbp HindIII fragment which contains in continuity 3.0 kbp from the 3' terminus (env and c region), 0.6 kbp of the terminal repeat sequences, and 0.5 kbp from the 5'terminus of the viral RNA (genome). NIH 3T3 fibroblasts were transfected with this DNA fragment an then infected with the wild mouse amphotropic retrovirus (cl 1504-A). In cell culture, 1504-A is a helper-independent type C virus which has an N-tropic host range and does not cause fusion of XC cells. When injected into newborn NIH Swiss mice, 1504-A does not produce splenomegaly or thymic enlargement in mice held for up to 8 months. The transfection with the F-MuLV fragment and the infection with 1504-A consistently yielded virus preparations that were XC positive. From such virus stocks we were able to isolate both helper-independent and replication-defective XC-positive viruses. The helper-independent virus was shown to be a recombinant virus since it contains a gp70 molecule derived at least in part from F-MuLV and a specific gag precursor derived from 1504-A as determined by radioactive immune precipitation assays. When injected into newborn Swiss mice, the recombinant helper-independent virus caused hepatosplenomegaly in approximately 50% of the mice in 6 to 8 weeks. The histology of the diseased splenic tissue was indistinguishable from that seen in the disease caused by the whole F-MuLV. The replication-defective virus could be pseudotyped with new 1504-A virus, and this viral complex also caused the F-MuLV disease picture when the complex was injected into newborn Swiss mice. We conclude that the genetic information responsible for the pathogenicity of F-MuLV is contained within the 4.1-kbp DNA fragment, which includes env gene sequences, the terminal repeat sequences, and the c region sequences of the F-MuLV genome.

Animals↗

PubMed2006

Fitness landscape of human immunodeficiency virus type 1 protease quasispecies.

Here we show, at a high resolution (1%), the human immunodeficiency virus type 1 (HIV-1) protease gene quasispecies landscape from three infected naïve individuals. A huge range of genetic configurations was found (67%, 71%, and 80% of the nucleotide clones from the three individuals, respectively, were different), and these configurations created a dense net that linked different parts of the viral population. Similarly, a vast diversity of different protease activities was also found. Importantly, 65% of the analyzed enzymes had detectable protease activity, and 11% of the minority individual variants showed similar or better fitness than the master (most abundant) enzyme, suggesting that the viral complexity in this genomic region does not exclusively depend on the enzyme's catalytic efficiency. Several high-fitness minority variants had only one substitution compared to the master sequence, supporting the possibility that the rugged HIV-1 protease quasispecies fitness landscape may be formed by a continuous network that can be traversed by single mutational steps without passing through defective or less-adapted proteins.

Amino Acid Sequence↗

PubMed1977

[Expression of an antigen associated with Gross virus on the surfaces of murine cells producing an oncornavirus from the radioleukemia of C57BL/6 mice].

A leukemogenic viral complex was demonstrated in cultures of 13-3 C cell line derived from a C57BL/6, radiation leukemia virus (RadLV-Rs) induced tumor. Both 13-3C and leukemic cells induced in C57BL/6 mice by 13-3C virus carry a cell surface antigen associated with Gross leukemia virus (GCSAa). These findings point to a close similarity between these antigens and those of murine endogenous ecotropic viruses.

AKR murine leukemia virus↗

PubMed1995

Isolation of new oncogenic forms of the murine c-fms gene.

The c-fms gene encodes the receptor for the macrophage colony-stimulating factor, which plays a key role in the proliferation and differentiation of cells of the myelomonocytic lineage. In order to study the effects of overexpression of the macrophage colony-stimulating factor receptor in hematopoietic cells, a Harvey sarcoma virus-derived retroviral vector containing the murine c-fms cDNA was pseudotyped with Friend murine leukemia virus and inoculated into newborn DBA/2 mice. This viral complex induced monoclonal or oligoclonal leukemias with a shorter latency than that for Friend murine leukemia virus alone. Unexpectedly, 60% of the integrated fms proviruses had deletions at the 5' end of the c-fms gene. Sequence analysis of seven mutant proviruses indicated that the deletions always included the c-fms ligand binding domain and either occurred within the c-fms sequences, leaving the fms open reading frame unchanged, or joined VL30 sequences located at the 5' end of the parental retroviral vector to internal c-fms sequences, resulting in truncated fms proteins devoid of the canonical signal peptide. In contrast to all tyrosine kinase receptors transduced in retroviruses, no helper gag- or env-derived sequences were fused to the rearranged fms sequences. Viral supernatants isolated from hematopoietic tumors with viruses with deletions were able to transform NIH 3T3 cells as efficiently as parental fms virus, indicating that deletions resulted in constitutive activation of the c-fms gene. These oncogenic variants differ from those transduced in the Suzan McDonough strain of feline sarcoma viruses (L. Donner, L. A. Fedele, C. F. Garon, S. J. Anderson, and C. J. Sherr, J. Virol. 41:489-500, 1982). The high rate of c-fms rearrangement and its relevance in the occurrence of hematopoietic tumors are discussed.

3T3 Cells↗

PubMedDate not supplied

Transformation of mononuclear phagocytes in vivo and malignant histiocytosis caused by a novel murine spleen focus-forming virus.

The study of retrovirus-induced leukaemias in mice is a powerful tool for the elucidation of the normal regulation of the haematopoietic system. The acute murine spleen focus-forming viruses (SFFV) can be classified according to the haematopoietic lineage on which they exert their effects in the adult mouse. Here we report a new SFFV isolate, the AF-1 virus, with the novel ability to transform cells of the mononuclear phagocyte lineage. The virus was isolated from sarcomas that were induced on passage of a cloned Friend helper virus (F-MuLV, 643/22F) in newborn BALB/c mice. We have cloned the transforming defective subunit of the AF-1 viral complex in NRK cells and isolated several subclones. Analysis of the proviral genome in two non-producer cell clones reveals that AF-1 virus contains Harvey v-ras-specific sequences (Fig. 1). Thus, AF-1 virus is closely related to Harvey murine sarcoma virus (Ha-MSV), and is, at present, the only tool by which permanent cell lines can be obtained from mononuclear phagocytes in the mouse.

Animals↗

PubMed2000

Measles virus-induced immunosuppression in vitro is independent of complex glycosylation of viral glycoproteins and of hemifusion.

Expression of the measles virus (MV) F/H complex on the surface of viral particles, infected cells, or cells transfected to express these proteins (presenter cells [PC]) is necessary and sufficient to induce proliferative arrest in both human and rodent lymphoid cells (responder cells [RC]). This inhibition was found to occur independent of apoptosis and soluble mediators excluded by a pore size filter of 200 nm released from either PC or RC. We now show that reactive oxygen intermediates which might be released by RC or PC also do not contribute to MV-induced immunosuppression in vitro. Using an inhibitor of Golgi-resident mannosidases (deoxymannojirimycin), we found that complex glycosylation of the F and H proteins is not required for the induction of proliferative arrest of RC. As revealed by our previous studies, proteolytic cleavage of the MV F protein precursor into its F1 and F2 subunits, but not of F/H-mediated cellular fusion, was found to be required, since fusion-inhibitory peptides such as Z-D-Phe-L-Phe-Gly (Z-fFG) did not interfere with the induction of proliferative inhibition. We now show that Z-fFG inhibits cellular fusion at the stage of hemifusion by preventing lipid mixing of the outer membrane layer. These results provide strong evidence for a receptor-mediated signal elicited by the MV F/H complex which can be uncoupled from its fusogenic activity is required for the induction of proliferative arrest of human lymphocytes.

Animals↗