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PubMed2004

Multiple V1/V2 env variants are frequently present during primary infection with human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1) exists as a complex population of multiple genotypic variants in persons with chronic infection. However, acute HIV-1 infection via sexual transmission is a low-probability event in which there is thought to be low genetic complexity in the initial inoculum. In order to assess the viral complexity present during primary HIV-1 infection, the V1/V2 and V3 variable regions of the env gene were examined by using a heteroduplex tracking assay (HTA) capable of resolving these genotypic variants. Blood plasma samples from 26 primary HIV-1-infected subjects were analyzed for their level of diversity. Half of the subjects had more than one V1/V2 viral variant during primary infection, indicating the frequent transmission of multiple variants. This observation is inconsistent with the idea of infrequent transmission based on a small transmitting inoculum of cell-free virus. In chronically infected subjects, the complexity of the viral populations was even greater in both the V1/V2 and the V3 regions than in acutely infected subjects, indicating that in spite of the presence of multiple variants in acute infection, the virus does pass through a genetic bottleneck during transmission. We also examined how well the infecting virus penetrated different anatomical compartments by using the HTA. Viral variants detected in blood plasma were compared to those detected in seminal plasma and/or cerebral spinal fluid of six individuals. The virus in each of these compartments was to a large extent identical to virus in blood plasma, a finding consistent with rapid penetration of the infecting variant(s). The low-probability transmission of multiple variants could be the result of transient periods of hyperinfectiousness or hypersusceptibility. Alternatively, the inefficient transfer of a multiply infected cell could account for both the low probability of transmission and the transfer of multiple variants.

Acute Disease↗

PubMed2005

Effects of a temperature sensitivity mutation in the J1R protein component of a complex required for vaccinia virus assembly.

Vaccinia virus J1R protein is required for virion morphogenesis (W. L. Chiu and W. Chang, J. Virol. 76:9575-9587, 2002). In this work, we further characterized the J1R protein of wild-type vaccinia virus and compared it with the protein encoded by the temperature-sensitive mutant virus Cts45. The mutant Cts45 was found to contain a Pro-to-Ser substitution at residue 132 of the J1R open reading frame, which is responsible for a loss-of-function phenotype. The half-life of the J1R-P132S mutant protein was comparable at both 31 and 39 degrees C, indicating that the P132S mutation did not affect the stability of the J1R protein. We also showed that the J1R protein interacts with itself in the virus-infected cells. The N-terminal region of the J1R protein, amino acids (aa) 1 to 77, interacted with the C-terminal region, aa 84 to 153, and the P132 mutation did not abolish this interaction, as determined by two-hybrid analysis. Furthermore, we demonstrated that J1R protein is part of a viral complex containing the A30L, G7L, and F10L proteins in virus-infected cells. In immunofluorescence analyses, wild-type J1R protein colocalized with the A30L, G7L, and F10L proteins in virus-infected cells but the loss-of-function P132 mutant did not. Furthermore, without a functional J1R protein, rapid degradation of A30L and the 15-kDa forms of the G7L and F10L proteins was observed in cells infected with Cts45 at 39 degrees C. This study thus demonstrated the importance of the J1R protein in the formation of a viral assembly complex required for morphogenesis.

Amino Acid Sequence↗

PubMed2004

Accurate representation of the hepatitis C virus quasispecies in 5.2-kilobase amplicons.

Hepatitis C virus (HCV) exists as a swarm of genetically distinct but related variants, or a quasispecies, whose complexity and sequence evolution are critical to studies of viral pathogenesis. Because most studies of the HCV quasispecies have focused on a relatively small genomic segment, the first hypervariable region of the E2 gene, it is possible that viral complexity is occasionally underestimated (due to primer mismatch) and that sequence evolution is misperceived due to unrecognized covariation. This report describes a sensitive and reproducible method to amplify most of the HCV genome as a single 5.2-kb amplicon by using primers directed at relatively conserved genomic segments. Using 52 specimens obtained during acute infection over a range of viral RNA concentrations, the overall rate of successful amplification was 94% and varied in a concentration-dependent manner, with successful amplification in 26 of 26 (100%) specimens at greater than 10(5) IU/ml, 15 of 16 (94%) at 10(4) to 10(5) IU/ml, 6 of 7 (86%) at 10(3) to 10(4) IU/ml, and 2 of 3 (67%) at less than 10(3) IU/ml. Quasispecies complexity, determined by using this novel long-amplicon method followed by heteroduplex mobility assay combined with single-stranded conformational polymorphism (HDA+SSCP) analysis, was very high, even during acute HCV infection, when 10 to 21 (median, 16) different HDA+SSCP patterns were detected among 33 cDNA clones examined. Replicate analyses indicate that this diversity is not dominated by random errors generated during amplification. Therefore, the HCV quasispecies is highly complex even during acute infection and is accurately represented in amplicons representing more than half of the viral genome.

Amino Acid Sequence↗

PubMed1992

Crystal structures of two viral peptides in complex with murine MHC class I H-2Kb.

The x-ray structures of a murine MHC class I molecule (H-2Kb) were determined in complex with two different viral peptides, derived from the vesicular stomatitis virus nucleoprotein (52-59), VSV-8, and the Sendai virus nucleoprotein (324-332), SEV-9. The H-2Kb complexes were refined at 2.3 A for VSV-8 and 2.5 A for SEV-9. The structure of H-2Kb exhibits a high degree of similarity with human HLA class I, although the individual domains can have slightly altered dispositions. Both peptides bind in extended conformations with most of their surfaces buried in the H-2Kb binding groove. The nonamer peptide maintains the same amino- and carboxyl-terminal interactions as the octamer primarily by the insertion of a bulge in the center of an otherwise beta conformation. Most of the specific interactions are between side-chain atoms of H-2Kb and main-chain atoms of peptide. This binding scheme accounts in large part for the enormous diversity of peptide sequences that bind with high affinity to class I molecules. Small but significant conformational changes in H-2Kb are associated with peptide binding, and these synergistic movements may be an integral part of the T cell receptor recognition process.

Amino Acid Sequence↗

PubMed2000

Formation of the poliovirus replication complex requires coupled viral translation, vesicle production, and viral RNA synthesis.

Poliovirus (PV) infection induces the rearrangement of intracellular membranes into characteristic vesicles which assemble into an RNA replication complex. To investigate this transformation, endoplasmic reticulum (ER) membranes in HeLa cells were modified by the expression of different cellular or viral membrane-binding proteins. The membrane-binding proteins induced two types of membrane alterations, i.e., extended membrane sheets and vesicles similar to those found during a PV infection. Cells expressing membrane-binding proteins were superinfected with PV and then analyzed for virus replication, location of membranes, viral protein, and RNA by immunofluorescence and fluorescent in situ hybridization. Cultures expressing cellular or viral membrane-binding proteins, but not those expressing soluble proteins, showed a markedly reduced ability to support PV replication as a consequence of the modification of ER membranes. The altered membranes, regardless of their morphology, were not used for the formation of viral replication complexes during a subsequent PV infection. Specifically, membrane sheets were not substrates for PV-induced vesicle formation, and, surprisingly, vesicles induced by and carrying one or all of the PV replication proteins did not contribute to replication complexes formed by the superinfecting PV. The formation of replication complexes required active viral RNA replication. The extensive alterations induced by membrane-binding proteins in the ER resulted in reduced viral protein synthesis, thus affecting the number of cells supporting PV multiplication. Our data suggest that a functional replication complex is formed in cis, in a coupled process involving viral translation, membrane modification and vesicle budding, and viral RNA synthesis.

Coated Vesicles↗

PubMed1992

On the cellular localization of the components of the herpes simplex virus type 1 helicase-primase complex and the viral origin-binding protein.

We constructed recombinant viruses based on the herpes simplex virus type 1 mutant tsK which individually were able to express the products of four viral DNA replication genes (UL5, UL8, UL9 and UL52) in the absence of any of the other proteins required for viral DNA synthesis. These viruses were used in immunofluorescence experiments to investigate the cellular localization of the four replication proteins expressed. The results demonstrated that all three components of the viral helicase-primase complex (UL5, UL8 and UL52 proteins) must be co-expressed to allow their efficient localization to the nucleus. Since the UL5 and UL52 proteins together form a complex which is enzymatically indistinguishable from a complex formed from all three proteins, a possible role of the UL8 protein may be in facilitating nuclear uptake. The UL9 protein (origin-binding protein) efficiently entered the cell nucleus when expressed alone. Both UL9 protein and the tripartite helicase-primase complex exhibited patterns of fluorescence which resembled the 'pre-replicative sites' described previously.

Cell Nucleus↗

PubMed1996

Protection and defence mechanisms in HCV infection.

The immune response triggered by hepatitis C virus (HCV) infection is still poorly documented and its underlying mechanisms are still to be elucidated. Following infection, both humoral and cellular responses are directed at multiple determinants (polyclonal) involving most of the viral antigens. These responses apparently involve neutralizing antibodies as well as peripheral and liver infiltrating cytotoxic T lymphocytes (CD8+) and proliferative reaction associated with CD4+. In most cases these responses cannot control infection. In addition, re-infection following an episode of self-limited infection or during chronic carrier condition has been described in chimpanzees and in man. Among the factors that may influence immune response by the host are the possible existence of extra-hepatic sites capable of supporting viral replication (in particular of haematopoietic origin) as well as the existence of complexed viral particles (such as with immunoglobulins and lipid components).

Animals↗

PubMed2002

Identification of specific HIV-1 reverse transcriptase contacts to the viral RNA:tRNA complex by mass spectrometry and a primary amine selective reagent.

We have devised a high-resolution protein footprinting methodology to dissect HIV-1 reverse transcriptase (RT) contacts to the viral RNA:tRNA complex. The experimental strategy included modification of surface-exposed lysines in RT and RT-viral RNA:tRNA complexes by the primary amine selective reagent NHS-biotin, SDSPAGE separation of p66 and p51 polypeptides, in gel proteolysis, and comparative mass spectrometric analysis of peptide fragments. The lysines modified in free RT but protected from biotinylation in the nucleoprotein complex were readily revealed by this approach. Results of a control experiment examining the RT-DNA:DNA complex were in excellent agreement with the crystal structure data on the identical complex. Probing the RT-viral RNA:tRNA complex revealed that a majority of protein contacts are located in the primer-template binding cleft in common with the RT-DNA:DNA and RT-RNA:DNA species. However, our footprinting data indicate that the p66 fingers subdomain makes additional contacts to the viral RNA:tRNA specific for this complex and not detected with DNA:DNA. The protein footprinting method described herein has a generic application for high-resolution solution structural studies of multiprotein-nucleic acid contacts.

Amino Acid Sequence↗

PubMed1994

Viruses and schizophrenia.

A viral hypothesis for the pathogenesis of schizophrenia has been under serious consideration for more than 70 years. To date, attempts have failed to identify a specific virus which contributes to the aetiology of the disorder. There has, however, been a recent resurgence of interest in a possible relationship between viral illness and schizophrenia. This renewed attention is the result of epidemiological evidence suggesting an excess of winter births in patients with schizophrenia, indications of foetal insults in persons who develop schizophrenia and an association between foetal exposure to the influenza virus and the subsequent development of schizophrenia. Advances in our understanding of the pathophysiology of viral diseases and the development of sophisticated techniques to study them have resulted in more complex viral hypotheses of schizophrenic aetiology, such as viral disruption of normal neurodevelopment, viral induced autoimmunity and retroviral integration. These hypotheses are now beginning to be tested experimentally.

Autoimmunity↗

PubMed1999

Kaposi's sarcoma: breeding ground of herpesviridae - A tour de force over viral evolution (review).

After reviewing the molecular biological basis of prominent theories for the integration of viruses into the earliest forms of living matter, an account is given on the immunoevasive strategies viruses have had to acquire in order to secure their existence against the most sophisticated anti-viral defensive mechanisms evolving in their hosts. Herpes-viridae and Kaposi's sarcoma illustrate the complexity of host-virus relationship. In following the evolutionary steps of simians and hominoids to Homo, it becomes evident that: a) Epstein-Barr virus evolved in Africa and its ancestral viruses are present in cercopithecines and hominoids; b) human herpes-virus-8-related viruses are present in macaques, in S. American primates and in Homo but such isolates from the great apes are missing. Thus interspecies transfer occurred from lower monkeys to Homo but when and at what geographical location? The human retrolentiviruses also jumped species barriers: this occurred recently in Africa, from great apes (chimpanzee and bonobo) to Homo sapiens (except when HIV-2 was transferred to mankind from sooty mangabeys). The matter is further complicated by the long coevolutionary cooperative interactions between herpes- and retrolentiviruses. Of pathological entities suspected to be etiologically affected by such complex viral cooperation, the origin of Reed-Sternberg cells of Hodgkin's disease is singled out for critical analysis. In this article the senior author summarizes his own 52 years of studentship in virology.

Animals↗

PubMed1986

Further characterization of a morphogenetic mutant of the foot-and-mouth disease virus.

In this paper we describe further characterization of a foot-and-mouth disease virus (FMDV) temperature-sensitive mutant, ts 139. This mutant was very sensitive to heat inactivation, suggesting that its viral particles are somehow altered. The electrophoretic analysis of ts 139 structural proteins indicated that VP2 has an altered mobility. Furthermore, two known protein precursors of VP2, VP0 and p88, were shown to be altered, as was p64, which supports a VP2 precursor role for p64. The ts 139 viral particle assembly pathway was analyzed during viral replication. The empty-capsid to complete-viral-particle ratio was clearly increased compared to that found for the wt strain, indicating an alteration in the morphogenetic process. This encouraged us to search for the presence of possible viral precursors of low sedimentation coefficient which have not been described previously in aphthovirus-infected cells. Analysis of the viral morphogenetic process in ts 139-infected cells demonstrated the presence of viral complexes formed by VP0, VP1 and VP3 which sedimented slightly faster than a 12 S marker. The protein composition, the sedimentation coefficient of this complex and the pulse-chase results strongly suggest that it is a morphogenetic precursor of the mature viral particle.

Animals↗

PubMed1982

Effect of toyocamycin on oncornaviral production by acutely infected cells.

The adenosine analogue toyocamycin incorporates into the RNA species of mammalia cells and abolishes at low concentrations of the processing of 45S preribosomal nucleolar RNA into the mature 28 and 18S cytoplasmic ribosomal RNAs. We have previously shown that toyocamycin depresses the production of the Friend leukemia viral complex by chronically infected cells. In this article, we report the study of the action of the drug on viral RNA in acutely infected cells. We found that, although abolishing viral production, the incorporation of toyocamycin does not inhibit the formation of mature viral messenger RNAs nor prevent the synthesis of specific viral proteins. These results are obtained at concentrations of analogue sufficient to abolish the appearance of mature cytoplasmic ribosomal RNA.

Animals↗

PubMed1996

AIDS patient monocytes target CD4 T cells for cellular conjugate formation and deletion through the membrane expression of HIV-1 envelope molecules.

The human immunodeficiency virus (HIV) causes in humans the acquired immunodeficiency syndrome (AIDS). It replicates at a high rate in lymphoid organs even before it causes clinical symptoms. It binds to CD4 cell surface markers and destroys T lymphocytes that express the receptor. The immune system replenishes CD4 T cells at a formidable rate but, unable to keep up with the losses, allows the CD4 T cell compartment to disintegrate gradually. The net loss of CD4 T cells is an indicator for disease progression. How the virus destroys CD4 T cells and whether their loss accounts for the ensuing immunodeficiency have not been fully explained. We have reported evidence, and confirm here, that HIV-infected subjects deposit on monocytes immune complexes containing the virus or its envelope molecule gp120. Armed with these immune complexes monocytes form specific cellular conjugates with CD4 T cells and kill them. The destruction of normal CD4 T cells by monocytes from AIDS patients can be blocked by soluble CD4 and by free gp120. Normal monocytes and macrophages can be armed with CD4-binding gp120, and so induced to destroy CD4 T cells, by incubating them with gp120 and gp120-specific antibody. CD4-reactive HIV-1 components have a short half-life on the phagocyte surface. Removed from the HIV-infected environment, monocytes clear their surfaces of antibody-complexed viral components within hours, which abrogates their ability to destroy CD4 T cells. Rearming the monocytes with gp120-anti-gp120 complexes restores their capacity to destroy CD4 T cells. The data imply that for uninterrupted deletion of CD4 T cells, monocytes require a continued productive HIV-1 infection of their host.

Acquired Immunodeficiency Syndrome↗

PubMedDate not supplied

[Specific markers for the detection of circulation of Tahyna, Inko and Batai viruses (Bunyaviridae, Bunyavirus) in humans, mosquitoes, ticks and cattle of the Ul'ianovsk region].

Comprehensive examinations of the population, sanguivorous arthropoda and domestic animals were for the first time held in the territory of the Ulyanovsk region. An active circulation of certain arboviruses was detected and a predominantly ecological relation of California encephalitis serogroup viruses with Aedes mosquitoes, on the one hand, and of Batai viruses with Anopheles mosquitoes, on the other hand, was established. The most reliable detection of antibodies to California encephalitis viral complex was pointed out.

Animals↗

PubMed2005

Broadly targeted human cytomegalovirus-specific CD4+ and CD8+ T cells dominate the memory compartments of exposed subjects.

Human cytomegalovirus (HCMV) infections of immunocompetent hosts are characterized by a dynamic, life-long interaction in which host immune responses, particularly of T cells, restrain viral replication and prevent disease but do not eliminate the virus or preclude transmission. Because HCMV is among the largest and most complex of known viruses, the T cell resources committed to maintaining this balance have never been characterized completely. Here, using cytokine flow cytometry and 13,687 overlapping 15mer peptides comprising 213 HCMV open reading frames (ORFs), we found that 151 HCMV ORFs were immunogenic for CD4(+) and/or CD8(+) T cells, and that ORF immunogenicity was influenced only modestly by ORF expression kinetics and function. We further documented that total HCMV-specific T cell responses in seropositive subjects were enormous, comprising on average approximately 10% of both the CD4(+) and CD8(+) memory compartments in blood, whereas cross-reactive recognition of HCMV proteins in seronegative individuals was limited to CD8(+) T cells and was rare. These data provide the first glimpse of the total human T cell response to a complex infectious agent and will provide insight into the rules governing immunodominance and cross-reactivity in complex viral infections of humans.

Adult↗

PubMed1984

Transcription of overlapping sets of RNAs from the genome of Autographa californica nuclear polyhedrosis virus: a novel method for mapping RNAs.

The insect baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) contains a double-stranded, supercoiled circular genome of 126 to 129 kilobase pairs in length. In cultured Spodoptera frugiperda insect cells the virus replications, and early and late phases of viral genome expression are discernible. We previously mapped 5 early and at least 32 late different viral polypeptides on the viral genome (H. Esche, H. Lübbert, B. Siegmann, and W. Doerfler, EMBO J. 1:1629-1633, 1982). However, at the same time we located 11 early and more than 90 late different size-classes of viral RNA on the AcNPV genome. Evidence for extensive RNA splicing in this virus system has not been adduced, although tiny splices cannot yet be ruled out (H. Lübbert and W. Doerfler, J. Virol. 50:497-506, 1984). The large number of AcNPV transcripts and the apparent lack of splicing have raised tantalizing questions about the mechanisms involved in the expression of AcNPV DNA and its regulation. It is also unknown how the widely differing numbers of RNAs and polypeptides can be correlated. For this reason, we have started to analyze in detail the map locations of some of the RNA size-classes in three different segments of the viral genome. For this purpose a novel method has been devised which will prove useful for the analyses of transcriptional patterns in complex viral genomes. The EcoRI fragments J, O through F, and Q, comprising viral DNA segments between 81.8 and 86.4, 32.6 and 41.0, and 88.2 and 89.7 map units, respectively, were investigated. Surprisingly, overlapping sets of viral RNAs of various lengths and with apparently common 3' termini in EcoRI fragments J (seven size-classes) and O through F (four size-classes) or with common 5' termini in EcoRI fragment Q (two size-classes) have been detected. At present, the functional significance of this mode of transcription is unknown. EcoRI fragment Q of AcNPV DNA encodes a 10,000-molecular-weight polypeptide which is expressed abundantly late after infection. The function of this protein has not yet been elucidated. The promoter and 5' part of the gene for the 10,000-molecular-weight polypeptide have been sequenced, and we have shown that at least two RNAs of different lengths are transcribed in this region and initiated at one site of three nucleotides. Studies on the expression of the AcNPV genome have revealed interesting properties not commonly found in other eucaryotic systems.

Animals↗

PubMed1985

Alternative excision products originating from a single integration of polyomavirus DNA.

The Cyp cell line consists of mouse cells transformed by a thermosensitive polyomavirus (Py) genome and routinely propagated at 39 degrees C. Cyp cells are readily induced to synthesize free Py DNA by being transferred to 33 degrees C. In one subclone (C12/a1/S48, or S48) of this line, such induction resulted in the intracellular accumulation of three discrete species of cyclic DNA, i.e., genomic Py DNA, RmI, and RmII. RmI and RmII are Py-mouse chimeras, each of which contains a distinct set of sequences originating from the site of integration. Conceivably, genomic Py DNA, RmI, and RmII could persist at 39 degrees C as free replicating plasmids or originate from distinct populations of cells in S48 cultures. The data indicated that all three species arise at 33 degrees C from a genetically homogeneous cell population in which neither RmI nor RmII replicates at 39 degrees C. Examination of the sequence at the viral-cellular junction unique to RmII indicated that this chimera is excised from the host chromosome through a recombination event involving a complex viral sequence and a simple cellular sequence. Therefore, RmII provides another example of precise recombination occurring between nonhomologous sequences in a mammalian cell, as already observed for RmI (B. S. Sylla, D. Huberdeau, D. Bourgaux-Ramoisy, and P. Bourgaux, Cell 37:661-667, 1984).

Animals↗

PubMed2006

[How the bovine viral diarrhea virus outwits the immune system].

The interaction of bovine viral diarrhea virus (BVD virus) with its host has several unique features, most notably the capacity to infect its host either transiently or persistently. The transient infection stimulates an antiviral immune reaction similar to that seen in other transient viral infections. In contrast, being associated with immunotolerance specific for the infecting BVD viral strain, the persistent infection differs fundamentally from other persistent infections like those caused by lentiviruses. Whereas the latter are characterized by complex viral evasion of the host's adaptive immune response by mechanisms such as antigenic drift and interference with presentation of T cell epitopes, BVD virus avoids the immune response altogether by inducing both humoral and cellular immune tolerance. This is made possible by invasion of the fetus at an early stage of development. In addition to adaptive immunity, BVD virus also manipulates key elements of the host's innate immune response. The non-cytopathic biotype of BVD virus, which is capable of persistently infecting its host, fails to induce type I interferon. In addition, persistently infected cells are resistant to the induction of apoptosis by double-stranded RNA and do not produce interferon when treated with this pathogen-associated molecular pattern (PAMP) that signals viral infection. Moreover, when treated with interferon, cells persistently infected with non-cytopathic BVD virus do not clear the virus. Surprisingly, however, despite this lack of effect on persistent infection, interferon readily induces an antiviral state in these cells, as shown by the protection against infection by unrelated viruses. Overall, BVD virus manipulates the host's interferon defense in a manner that optimises its chances of maintaining the persistent infection as well as decreasing the risks that heterologous viral infections may carry for the host. Thus, since not all potential host cells are infected in animals persistently infected with BVD virus, heterologous viruses replicating in cells uninfected with BVD virus will still trigger production of interferon. Interferon produced by such cells will curtail the replication of heterologous viruses only, be that in cells already infected with BVD virus, or in cells in which the heterologous virus may replicate alone. From an evolutionary viewpoint, this strategy clearly enhances the chances of transmission of BVD virus to new hosts, as it attenuates the negative effects that a global immunosuppression would have on the survival of persistently infected animals.

Animals↗