Mouse hepatitis virus 3 replication in T and B lymphocytes correlate with viral pathogenicity

Viral pathogenicity may be regulated by host defense mechanisms at the virus-immune cell interaction level. The immune system plays an important role in the outcome of acute disease induced by the mouse hepatitis virus type 3 (MHV3) virus. The lymphoid cells act as effectors in the virus elimination as well as targets for viral replication. In order to demonstrate a correlation between MHV3 pathogenicity and viral replication in lymphocytes, genetically-determined resistant A/J and susceptible C57BL/6 mice were infected with pathogenic (L2-MHV3) or nonpathogenic (YAC-MHV3) viral strains. Pathogenicity and histopathologic studies have revealed that lymphoid organs such as thymus and spleen, showed injuries or atrophy in susceptible mice infected with L2-MHV3. No histopathologic lesions in the lymphoid organs occurred in C57BL/6 mice infected with YAC-MHV3 or A/J mice infected with both viruses. The mechanisms involved in the lymphoid injuries were studied regarding viral replication in the lymphoid organs and cells in infected mice. Results indicate that cell depletion in lymphoid organs is caused by a complete viral replication in lymphoid cells. Thy1.2+ and surface IgM+ lymphoid cells from susceptible C57BL/6 mice infected with L2-MHV3 were permissive to viral replication and to subsequent cell lysis. No cell lysis, however, occurred in lymphoid cells from C57BL/6 mice infected with YAC-MHV3 and A/J mice infected with both virus strains. In vitro studies, with purified T and B cell populations were performed to determine the mechanism effecting susceptibility or resistance to viral-induced cell lysis occurring in such cells. A blockade, probably occurring at the viral RNA polymerase activity level, prevents viral replication in resistant cells between the stages of fixation of the virus at the cell-surface receptor and the viral protein translation. These experiments indicate that an intrinsic virus-specific resistant mechanism occurs in lymphoid cells that plays a major role in the viral pathogenicity.     

Antigenic stimulation specifically reactivates the replication of archived simian immunodeficiency virus genomes in chronically infected macaques

Human immunodeficiency virus/simian immunodeficiency virus (SIV) diversification is a direct consequence of viral replication and occurs principally in secondary lymphoid organs where CD4(+) T cells are activated and proliferate. However, the evolution of viral quasispecies may also be driven by various nonexclusive mechanisms, including adaptation to specific immune responses and modification of viral fitness. Analysis of viral quasispecies in SIV-infected macaques subjected to repeated antigenic stimulations allowed us to demonstrate transient expansions of SIV populations that were highly dependent upon activation of antigen-specific T cells. T-cell clones expanded in response to a particular antigen were infected by a specific viral population and persisted for prolonged periods. Upon a second stimulation by encounter with the same antigen, these specific genomes were at the origin of a new burst of replication, leading to rapid but transient replacement of the viral quasispecies in blood. Finally, longitudinal analysis of SIV sequence variation during and between antigenic stimulations revealed that viral evolution is mostly constrained to periods of strong immunological activity.     

Viruses Roll the Dice: The Stochastic Behavior of Viral Genome Molecules Accelerates Viral Adaptation at the Cell and Tissue Levels

Recent studies on evolutionarily distant viral groups have shown that the number of viral genomes that establish cell infection after cell-to-cell transmission is unexpectedly small (1–20 genomes). This aspect of viral infection appears to be important for the adaptation and survival of viruses. To clarify how the number of viral genomes that establish cell infection is determined, we developed a simulation model of cell infection for tomato mosaic virus (ToMV), a positive-strand RNA virus. The model showed that stochastic processes that govern the replication or degradation of individual genomes result in the infection by a small number of genomes, while a large number of infectious genomes are introduced in the cell. It also predicted two interesting characteristics regarding cell infection patterns: stochastic variation among cells in the number of viral genomes that establish infection and stochastic inequality in the accumulation of their progenies in each cell. Both characteristics were validated experimentally by inoculating tobacco cells with a library of nucleotide sequence–tagged ToMV and analyzing the viral genomes that accumulated in each cell using a high-throughput sequencer. An additional simulation model revealed that these two characteristics enhance selection during tissue infection. The cell infection model also predicted a mechanism that enhances selection at the cellular level: a small difference in the replication abilities of coinfected variants results in a large difference in individual accumulation via the multiple-round formation of the replication complex (i.e., the replication machinery). Importantly, this predicted effect was observed in vivo. The cell infection model was robust to changes in the parameter values, suggesting that other viruses could adopt similar adaptation mechanisms. Taken together, these data reveal a comprehensive picture of viral infection processes including replication, cell-to-cell transmission, and evolution, which are based on the stochastic behavior of the viral genome molecules in each cell.     

Variant upstream regulatory region sequences differentially regulate human papillomavirus type 16 DNA replication throughout the viral life cycle

While the central role of the viral upstream regulatory region (URR) in the human papillomavirus (HPV) life cycle has been well established, its effects on viral replication factor expression and plasmid replication of HPV type 16 (HPV16) remain unclear. Some nonprototypic variants of HPV16 contain altered URR sequences and are considered to increase the oncogenic risk of infections. To determine the relationship between viral replication and variant URRs, hybrid viral genomes were constructed with the replication-competent HPV16 prototype W12 and analyzed in assays which recapitulate the different phases of normal viral replication. The establishment efficiencies of hybrid HPV16 genomes differed about 20-fold among European prototypes and variants from Africa and America. Generally, European and African genomes exhibited the lowest replication efficiencies. The high replication levels observed with American variants were primarily attributable to their efficient expression of the replication factors E1 and E2. The maintenance levels of these viral genomes varied about fivefold, which correlated with their respective establishment phenotypes and published P(97) activities. Vegetative DNA amplification could also be observed with replicating HPV16 genomes. These results indicate that efficient E1/E2 expression and elevated plasmid replication levels during the persistent stage of infection may comprise a risk factor in HPV16-mediated oncogenesis.     

Multiple variants of the archaeal DNA rudivirus SIRV1 in a single host and a novel mechanism of genomic variation

The DNA rudivirus SIRV1 of the hyperthermophilic archaeon Sulfolobus shows exceptional properties. Viral isolates invariably contain a population of variants with different but closely related genomes. Upon propagation in a given host strain, one or more genomes dominate in the viral population. However, upon passage into a new host strain the viral population undergoes changes and other dominant variants are selected. Sequencing and analysis of the variant genomes revealed that major differences occur in gene order, gene size and gene content at localized genomic sites. A previously unknown mechanism of genomic rearrangement involving putative 12 bp archaeal introns appears to facilitate alteration of the variant genomes. Inter-genomic recombination between the different variants also occurs. The variant genomes exhibit signature tetranucleotide sequences near their putative sites for replication initiation.     

Oncogenesis of mammary glands, skin, and bones by polyomavirus correlates with viral persistence and prolonged genome replication potential.

A correlation between polyomavirus-induced oncogenesis and viral persistence on the one hand and/or prolonged genome replication potential on the other was established with respect to their respective organ distributions. Prolonged replication potential is defined as the capacity of a genome to replicate in a given organ from the time of infection up to the onset of oncogenesis. This conclusion was derived following intraperitoneal infection of BALB/c mice with wild-type strain A2. Viral genomes were used as parameters of persistence and replication and were detected by Southern blotting and PCR analysis. The major tumor target organs (mammary gland, skin, and bone), which have not been previously analyzed for persistence, were compared with other, non-tumor-prone organs (kidney, liver, lung, spleen, and salivary gland). A progressive loss of viral genomes was observed in all tissues as a function of time postinfection; however, genomes were shown to persist through 20 weeks postinfection in the mammary glands, skin, and bones to an extent similar to that in the previously described kidneys (D. J. McCance, J. Virol. 39:958-962, 1981; W. P. Rowe, J. W. Hartley, J. D. Estes, and R. J. Huebner, Natl. Cancer Inst. Monogr. 4:189-209, 1960). Thus, tumors arise among organs that sustain a persistent infection, but not all such organs develop tumors (e.g., the kidney). The capacity of organs to support de novo replication at various ages, including the age reached when the first tumors are detected, was also determined using a 3-day infection period for ages between 0 and 7 weeks. For all organs tested, a higher level of genomes was observed in organs of mice infected as neonates than in those infected after the age of 3 weeks. However, marked organ-specific differences were seen in the degree and timing of loss of replication. In particular, viral genome replication, although reduced, was maintained in the mammary glands, skin, and bones of adult animals, in contrast to the kidneys. We conclude that organ-specific oncogenesis correlates with two organ-specific parameters: persistence of viral genomes and prolonged viral genome replication potential. This may reflect a requirement for continued viral genome replication and/or gene expression for tumorigenesis. In turn, these parameters may be linked to the tissue-specific continued capacity for cellular division.     

In vitro and in vivo infectivity and pathogenicity of the lymphoid cell-derived woodchuck hepatitis virus

Woodchuck hepatitis virus (WHV) and human hepatitis B virus are closely related, highly hepatotropic mammalian DNA viruses that also replicate in the lymphatic system. The infectivity and pathogenicity of hepadnaviruses propagating in lymphoid cells are under debate. In this study, hepato- and lymphotropism of WHV produced by naturally infected lymphoid cells was examined in specifically established woodchuck hepatocyte and lymphoid cell cultures and coculture systems, and virus pathogenicity was tested in susceptible animals. Applying PCR-based assays discriminating between the total pool of WHV genomes and covalently closed circular DNA (cccDNA), combined with enzymatic elimination of extracellular viral sequences potentially associated with the cell surface, our study documents that virus replicating in woodchuck lymphoid cells is infectious to homologous hepatocytes and lymphoid cells in vitro. The productive replication of WHV from lymphoid cells in cultured hepatocytes was evidenced by the appearance of virus-specific DNA, cccDNA, and antigens, transmissibility of the virus through multiple passages in hepatocyte cultures, and the ability of the passaged virus to infect virus-naive animals. The data also revealed that WHV from lymphoid cells can initiate classical acute viral hepatitis in susceptible animals, albeit small quantities (approximately 10(3) virions) caused immunovirologically undetectable (occult) WHV infection that engaged the lymphatic system but not the liver. Our results provide direct in vitro and in vivo evidence that lymphoid cells in the infected host support propagation of infectious hepadnavirus that has the potential to induce hepatitis. They also emphasize a principal role of the lymphatic system in the maintenance and dissemination of hepadnavirus infection, particularly when infection is induced by low virus doses.     

Identification and characterization of the hamster polyomavirus middle T antigen.

Hamster polyomavirus (HaPV) is associated with lymphoid and hair follicle tumors in Syrian hamsters. The early region of HaPV has the potential to encode three polypeptides (which are related to the mouse polyomavirus early proteins) and can transform fibroblasts in vitro. We identified the HaPV middle T antigen (HamT) as a 45-kDa protein. Like its murine counterpart, HamT was associated with serine/threonine phosphatase, phosphatidylinositol-3 kinase, and protein tyrosine kinase activities. However, whereas mouse middle T antigen associates predominantly with pp60c-src and pp62c-yes, HamT was associated with a different tyrosine kinase, p59fyn. The ability of HaPV to cause lymphoid tumors may therefore reside in its ability to associate with p59fyn, a potentially important tyrosine kinase in lymphocytes.     

Viral load in tissues during the early and chronic phase of non-pathogenic SIVagm infection

African green monkeys (AGMs) persistently infected with SIVagm do not develop AIDS, although their plasma viremia levels can reach those reported for pathogenic HIV-1 and SIVmac infections. In contrast, the viral burden in lymph nodes in SIVagm-infected AGMs is generally lower in comparison with HIV/SIVmac pathogenic infections, at least during the chronic phase of SIVagm infection. We searched for the primary targets of viral replication, which might account for the high viremias in SIVagm-infected AGMs. We evaluated for the first time during primary infection SIVagm dissemination in various lymphoid and non-lymphoid tissues. Sixteen distinct organs at a time point corresponding to maximal virus production were analyzed for viral RNA and DNA load. At days 8 and 9 p.i., viral RNA could be detected in a wide range of tissues, such as jejunum, spleen, mesenteric lymph nodes, thymus and lung. Quantification of viral DNA and RNA as well as of productively infected cells revealed that viral replication during this early phase takes place mainly in secondary lymphoid organs and in the gut (5 x 10(4)-5 x 10(8) RNA copies/10(6) cells). By 4 years p.i., RNA copy numbers were below detection level in thymus and lung. Secondary lymphoid organs displayed 6 x 10(2)-2 x 10(6) RNA copies/10(6) cells, while some tissue fragments of ileum and jejunum still showed high viral loads (up to 10(9) copies/10(6) cells). Altogether, these results indicate a rapid dissemination of SIVagm into lymphoid tissues, including the small intestine. The latter, despite showing marked regional variations, most likely contributes significantly to the high levels of viremia observed during SIVagm infection.     

Woodchuck hepatitis virus X protein is required for viral infection in vivo.

The X gene of the mammalian hepadnaviruses is believed to encode a protein of 17 kDa which has been shown to transactivate a wide range of viral and cellular promoters. The necessity for X gene expression during the viral life cycle in vivo has recently been suggested (H.-S. Chen, S. Kaneko, R. Girones, R. W. Anderson, W. E. Hornbuckle, B. C. Tennant, P. J. Cote, J. L. Gerin, R. H. Purcell, and R. H. Miller, J. Virol. 67:1218-1226, 1993). We have independently constructed two variants of woodchuck hepatitis virus (WHV) with mutations in the X coding region. Transient transfection of two different hepatoma cell lines showed that these WHV X gene mutants were competent for virus replication in vitro. To determine whether X expression was required for viral replication in vivo, we injected mutant and wild-type genomes into the livers of susceptible woodchucks. While the wild-type WHV genomes were infectious in all animals examined, the mutant genomes did not initiate a WHV infection in woodchucks. These results indicate that the X gene of the hepadnaviruses plays a major role in viral replication in vivo.     

Development and migration of protective CD8+ T cells into the nervous system following ocular herpes simplex virus-1 infection

After infection of epithelial surfaces, HSV-1 elicits a multifaceted antiviral response that controls the virus and limits it to latency in sensory ganglia. That response encompasses the CD8(+) T cells, whose precise role(s) is still being defined; immune surveillance in the ganglia and control of viral spread to the brain were proposed as the key roles. We tracked the kinetics of the CD8(+) T cell response across lymphoid and extralymphoid tissues after ocular infection. HSV-1-specific CD8(+) T cells first appeared in the draining (submandibular) lymph node on day 5 and were detectable in both nondraining lymphoid and extralymphoid tissues starting on day 6. However, although lymphoid organs contained both resting (CD43(low)CFSE(high)) and virus-specific cells at different stages of proliferation and activation, extralymphoid sites (eye, trigeminal ganglion, and brain) contained only activated cells that underwent more than eight proliferations (CD43(high)CFSE(neg)) and promptly secreted IFN-gamma upon contact with viral Ags. Regardless of the state of activation, these cells appeared too late to prevent HSV-1 spread, which was seen in the eye (from day 1), trigeminal ganglia (from day 2), and brain (from day 3) well before the onset of a detectable CD8(+) T cell response. However, CD8(+) T cells were critical in reducing viral replication starting on day 6 and for its abrogation between days 8 and 10; CD8-deficient animals failed to control the virus, exhibited persisting high viral titers in the brain after day 6, and died of viral encephalitis between days 7 and 12. Thus, CD8(+) T cells do not control HSV-1 spread from primary to tertiary tissues, but, rather, attack the virus in infected organs and control its replication in situ.     

Monitoring Insect Transposable Elements in Large Double-Stranded DNA Viruses Reveals Host-to-Virus and Virus-to-Virus Transposition

The mechanisms by which transposable elements (TEs) can be horizontally transferred between animals are unknown, but viruses are possible candidate vectors. Here, we surveyed the presence of host-derived TEs in viral genomes in 35 deep sequencing data sets produced from 11 host–virus systems, encompassing nine arthropod host species (five lepidopterans, two dipterans, and two crustaceans) and six different double-stranded (ds) DNA viruses (four baculoviruses and two iridoviruses). We found evidence of viral-borne TEs in 14 data sets, with frequencies of viral genomes carrying a TE ranging from 0.01% to 26.33% for baculoviruses and from 0.45% to 7.36% for iridoviruses. The analysis of viral populations separated by a single replication cycle revealed that viral-borne TEs originating from an initial host species can be retrieved after viral replication in another host species, sometimes at higher frequencies. Furthermore, we detected a strong increase in the number of integrations in a viral population for a TE absent from the hosts’ genomes, indicating that this TE has undergone intense transposition within the viral population. Finally, we provide evidence that many TEs found integrated in viral genomes (15/41) have been horizontally transferred in insects. Altogether, our results indicate that multiple large dsDNA viruses have the capacity to shuttle TEs in insects and they underline the potential of viruses to act as vectors of horizontal transfer of TEs. Furthermore, the finding that TEs can transpose between viral genomes of a viral species sets viruses as possible new niches in which TEs can persist and evolve.     

The infectivities of turnip yellow mosaic virus genomes with altered tRNA mimicry are not dependent on compensating mutations in the viral replication protein

Five highly infectious turnip yellow mosaic virus (TYMV) genomes with sequence changes in their 3'-terminal regions that result in altered aminoacylation and eEF1A binding have been studied. These genomes were derived from cloned parental RNAs of low infectivity by sequential passaging in plants. Three of these genomes that are incapable of aminoacylation have been reported previously (J. B. Goodwin, J. M. Skuzeski, and T. W. Dreher, Virology 230:113-124, 1997). We now demonstrate by subcloning the 3' untranslated regions into wild-type TYMV RNA that the high infectivities and replication rates of these genomes compared to their progenitors are mostly due to a small number of mutations acquired in the 3' tRNA-like structure during passaging. Mutations in other parts of the genome, including the replication protein coding region, are not required for high infectivity but probably do play a role in optimizing viral amplification and spread in plants. Two other TYMV RNA variants of suboptimal infectivities, one that accepts methionine instead of the usual valine and one that interacts less tightly with eEF1A, were sequentially passaged to produce highly infectious genomes. The improved infectivities of these RNAs were not associated with increased replication in protoplasts, and no mutations were acquired in their 3' tRNA-like structures. Complete sequencing of one genome identified two mutations that result in amino acid changes in the movement protein gene, suggesting that improved infectivity may be a function of improved viral dissemination in plants. Our results show that the wild-type TYMV replication proteins are able to amplify genomes with 3' termini of variable sequence and tRNA mimicry. These and previous results have led to a model in which the binding of eEF1A to the 3' end to antagonize minus-strand initiation is a major role of the tRNA-like structure.     

Viral genomes maintained extrachromosomally in hamster polyomavirus-induced lymphomas display a cell-specific replication in vitro.

Hamster polyomavirus causes lymphomas when injected into newborn Syrian hamsters. Large amounts of extrachromosomal viral genomes are accumulated in the lymphoma cells. These genomes are characterized by deletions affecting the late coding region as well as a specific part of the noncoding regulatory region. By contrast with wild-type genomes, lymphoma-associated genomes replicate in a lymphoblastoid cell line but not in a fibroblastic cell line. The deletion acts in a cis-dominant manner and is the primary determinant of this host-range effect on replication. The boundaries of the regulatory region necessary for viral DNA replication in the two cell contexts have been defined. The regulatory region can be functionally divided in two domains: one domain (distal from the origin of replication) is necessary for viral genome replication in fibroblasts, whereas the other domain (proximal to the origin of replication) is functional only in the lymphoblastoid cell context and contains the sequence specifically conserved in the lymphoma-associated genomes. This sequence harbors a motif recognized by a lymphoblastoid cell-specific trans-acting factor.     

The gut mucosal viral reservoir in HIV-infected patients is not the major source of rebound plasma viremia following interruption of highly active antiretroviral therapy

Interruption of suppressive highly active antiretroviral therapy (HAART) in HIV-infected patients leads to increased HIV replication and viral rebound in peripheral blood. Effects of therapy interruption on gut-associated lymphoid tissue (GALT) have not been well investigated. We evaluated longitudinal changes in viral replication and emergence of viral variants in the context of T cell homeostasis and gene expression in GALT of three HIV-positive patients who initiated HAART during primary HIV infection but opted to interrupt therapy thereafter. Longitudinal viral sequence analysis revealed that a stable proviral reservoir was established in GALT during primary HIV infection that persisted through early HAART and post-therapy interruption. Proviral variants in GALT and peripheral blood mononuclear cells (PBMCs) displayed low levels of genomic diversity at all times. A rapid increase in viral loads with a modest decline of CD4(+) T cells in peripheral blood was observed, while gut mucosal CD4(+) T cell loss was severe following HAART interruption. This was accompanied by increased mucosal gene expression regulating interferon (IFN)-mediated antiviral responses and immune activation, a profile similar to those found in HAART-naive HIV-infected patients. Sequence analysis of rebound virus suggested that GALT was not the major contributor to the postinterruption plasma viremia nor were GALT HIV reservoirs rapidly replaced by HIV rebound variants. Our data suggest an early establishment and persistence of viral reservoirs in GALT with minimal diversity. Early detection of and therapy for HIV infection may be beneficial in controlling viral evolution and limiting establishment of diverse viral reservoirs in the mucosal compartment.     

Trade-offs in resource allocation in the intracellular life-cycle of hepatitis C virus

Positive sense single-stranded RNA viruses undergo three mutually exclusive processes to replicate within a cell. These are translation to produce proteins, replication to produce RNA viral genomes, and packaging to form virions. The allocation of newly synthesised viral genomes to these processes, which can be regarded as life-history traits, may be subject to natural selection for efficient reproduction. Here, we develop a mathematical model of the process of intracellular viral replication to study alternative strategies for the allocation and reallocation of viral genomes to these processes. We explore four cases of the model: (1) Free Movement, in which viral genomes can freely be allocated and reallocated among translation, replication and packaging; (2) Unidirectional Reallocation, in which allocation occurs freely but reallocation can only proceed from translation to replication to packaging; (3) Conveyor Belt, in which viral genomes are first allocated to translation, then passed on to replication and finally to packaging; and (4) Permanent Allocation in which new genomes are allocated to the three processes but not reallocated between them. We apply this model to hepatitis C virus and study changes in the production of virus as the rates of allocation and reallocation are varied. We find that high viral production occurs when allocation and reallocation of the genome are weighted towards the translation and replication processes. The replication process in particular is favoured. The most productive strategy is a form of the Free Movement model in which genomes are allocated entirely to the replication-translation cycle while allowing some genomes to be packaged through reallocation.     

Distinct viral populations differentiate and evolve independently in a single perennial host plant

The complex structure of virus populations has been the object of intensive study in bacteria, animals, and plants for over a decade. While it is clear that tremendous genetic diversity is rapidly generated during viral replication, the distribution of this diversity within a single host remains an obscure area in this field of science. Among animal viruses, only Human immunodeficiency virus and Hepatitis C virus populations have recently been thoroughly investigated at an intrahost level, where they are structured as metapopulations, demonstrating that the host cannot be considered simply as a "bag" containing a homogeneous or unstructured swarm of mutant viral genomes. In plants, a few reports suggested a possible heterogeneous distribution of virus variants at different locations within the host but provided no clues as to how this heterogeneity is structured. Here, we report the most exhaustive study of the structure and evolution of a virus population ever reported at the intrahost level through the analysis of a Prunus tree infected by Plum pox virus for over 13 years following a single inoculation event and by using analysis of molecular variance at different hierarchical levels combined with nested clade analysis. We demonstrate that, following systemic invasion of the host, the virus population differentiates into several distinct populations that are isolated in different branches, where they evolve independently through contiguous range expansion while colonizing newly formed organs. Moreover, we present and discuss evidence that the tree harbors a huge "bank" of viral clones, each isolated in one of the myriad leaves.     

Polyoma genome in hamster BHK-21-C13 cells: integration into cellular DNA and induction of the viral replication.

When grown at 39.5 degrees C, BHK-21 C-13 cells transformed by A gene mutants of polyoma virus contain viral sequences that are predominantly associated with cellular DNA pelleted in the Hirt lysis procedure. At this temperature, in cells that are inducible for viral DNA replication (Folk, 1973), the majority of the viral genomes are covalently joined with cellular DNA's containing repetitious sequences. Upon a shift to 31 degrees C, free viral genomes appear and are replicated. Coupled with the replication of the free viral genomes at 31 degrees C is an increase in the viral genomes associated with cellular DNA.