Genetic analysis of the enhancer requirements for polyomavirus DNA replication in mice.

In this report, we describe the first systematic analysis of the genetic requirements for polyomavirus (Py) enhancer-activated viral DNA replication during the acute phase of infection in mice. Four mutants were made which substituted XhoI sites for conserved enhancer consensus sequences (adenovirus type 5 E1A, c-fos, simian virus 40, and a glucocorticoidlike consensus sequence). Viral DNA replication in infected mouse organs was measured by DNA blot analysis. Only the loss of the glucocorticoidlike consensus sequence element significantly reduced Py DNA replication in the kidneys, the primary target organ for viral replication. The loss of the c-fos, adenovirus type 5 E1A, or simian virus 40 consensus sequences, however, expanded organ-specific viral DNA replication, relative to wild-type Py, by allowing high-level replication in the pancreas or heart or both. Analysis of Py variants selected for replication in undifferentiated embryonal carcinoma cell lines (PyF441, PyF111) showed that there was little change in levels of viral DNA replication in kidneys and other organs as compared with those in the wild-type virus. If the entire B enhancer is deleted, only low overall levels of viral replication are observed. Wild-type levels of replication in the kidneys can be reconstituted by addition of a single domain from within the A enhancer (nucleotides 5094 to 5132) to the B enhancer deletion virus, suggesting that a single domain from the A enhancer can functionally substitute for the entire B enhancer. This also indicates that the determinants for kidney-specific replication are not found in the B enhancer.     

The primary site of replication alters the eventual site of persistent infection by polyomavirus in mice.

Using DNA blot analysis, we monitored the course of polyomavirus infection in mice receiving an intranasal inoculation and compared this with the course of infection in mice receiving an intraperitoneal inoculation. Intranasal infection was characterized by an initial primary replication phase in the respiratory tract, followed by a systemic infection of the visceral organs. At 12 days postinfection, there was partial clearing of viral DNA in all organs; by 22 days postinfection, viral DNA persisted only in the lungs and kidneys, and the level of DNA slowly decreased during the next 3 months. Lungs have been a previously unrecognized site for polyomavirus persistent infection. In contrast to intranasal infection, intraperitoneal infection of mice was characterized by only three phases: an initial systemic phase in which viral DNA was found in the same respiratory and visceral organs as during intranasal infection, clearing of the virus from the organs, and ultimately, a persistent infection in the kidneys but not in the lungs. Thus, different organs became persistently infected when mice were inoculated via these different routes.     

Adult mouse kidneys become permissive to acute polyomavirus infection and reactivate persistent infections in response to cellular damage and regeneration.

Kidneys of newborn (but not adult) mice are normally high permissive for polyomavirus (Py) infection and readily establish persistent infections. We have proposed that ongoing cellular differentiation, which occurs in newborn mice, may be necessary for a high level of in vivo Py replication (R. Rochford, J. P. Moreno, M. L. Peake, and L. P. Villarreal, J. Virol. 66:3287-3297, 1992). This cellular differentiation requirement may also be necessary for the reactivation of a persistent Py kidney infection and could provide an alternative to the accepted view that reactivation results from immunosuppression. To examine this proposal, the ability of adult BALB/c mouse kidneys to support primary acute Py infection or to reactivate previously established persistent Py infections after kidney-specific damage was investigated. Kidney damage was induced by both chemical (glycerol, cisplatin, or methotrexate) and mechanical (through renal artery clamping to produce unilateral renal ischemia) treatments. We also examined the effects of epidermal growth factor (EGF), which enhances the rate of kidney regeneration, on Py replication. Using histopathologic techniques, in situ hybridization for Py DNA, and immunofluorescence for Py VP1 production, we established that both chemical damage and damage through renal artery clamping of adult kidneys promoted high levels of primary Py replication in these normally nonpermissive cells. This damage also promoted the efficient reactivation of Py replication from persistently infected kidneys, in the absence of immunosuppression. EGF treatment significantly increased acute Py replication and also reactivation in damaged kidneys. These results support the view that ongoing cellular division and differentiation may be needed both for high levels of acute Py replication and for reactivation of persistent infections in vivo.     

Gamma interferon controls mouse polyomavirus infection in vivo

Human polyomaviruses are associated with substantial morbidity in immunocompromised patients, including those with HIV/AIDS, recipients of bone marrow and kidney transplants, and individuals receiving immunomodulatory agents for autoimmune and inflammatory diseases. No effective antipolyomavirus agents are currently available, and no host determinants have been identified to predict susceptibility to polyomavirus-associated diseases. Using the mouse polyomavirus (MPyV) infection model, we recently demonstrated that perforin-granzyme exocytosis, tumor necrosis factor alpha (TNF-α), and Fas did not contribute to control of infection or virus-induced tumors. Gamma interferon (IFN-γ) was recently shown to inhibit replication by human BK polyomavirus in primary cultures of renal tubular epithelial cells. In this study, we provide evidence that IFN-γ is an important component of the host defense against MPyV infection and tumorigenesis. In immortalized and primary cells, IFN-γ reduces expression of MPyV proteins and impairs viral replication. Mice deficient for the IFN-γ receptor (IFN-γR(-/-)) maintain higher viral loads during MPyV infection and are susceptible to MPyV-induced tumors; this increased viral load is not associated with a defective MPyV-specific CD8(+) T cell response. Using an acute MPyV infection kidney transplant model, we further show that IFN-γR(-/-) donor kidneys harbor higher MPyV levels than donor kidneys from wild-type mice. Finally, administration of IFN-γ to persistently infected mice significantly reduces MPyV levels in multiple organs, including the kidney, a major reservoir for persistent mouse and human polyomavirus infections. These findings demonstrate that IFN-γ is an antiviral effector molecule for MPyV infection.     

Pattern of polyomavirus replication from infection until tumor formation in the organs of athymic nu/nu mice.

Inbred athymic nu/nu BALB/c mice were injected subcutaneously with the highly oncogenic polyomavirus A2 strain, and the sites of viral DNA replication were determined by whole mouse section hybridization (T. W. Dubensky, E. A. Murphy, and L. P. Villareal, J. Virol. 50:779-783, 1984) and Southern blot analysis. We show that infection is persistent in some epithelial tissues (skin, mammary, and salivary glands), in lymphoid organs (spleen and nodes), and in mesenchymal bone tissue. Only mammary glands and bones were targets for tumor formation. Although the same pattern of infection was observed in males and females, mammary adenocarcinomas were induced exclusively in females, while the frequency of osteosarcomas was similar in both sexes. No viral DNA or lytic lesion was detected in kidney, liver, or lung tissue. The restricted targeting of polyomavirus oncogenicity in nude mice, compared with newborn immunocompetent animals, inoculated via the same route with the same virus strain, therefore does not reflect selective tissue targeting of virus replication. These results further document the influence of the age, immunological status, and genetic background of the host on the pattern of viral infection and tumor formation.     

In vivo replication of the hamster polyomavirus genome and generation of specific deletions in the process of lymphomagenesis.

Hamster polyomavirus (HaPV) causes lymphomas when injected into newborn hamsters. These tumors are virus-free but accumulate large amounts of deleted extrachromosomal viral genomes. In order to identify the major sites of virus replication in animals, we have monitored the HaPV DNA present in different organs at various times after injection. The data demonstrate that viral replication preferentially occurs in lymphoid organs. Lymphoma-associated viral genomes display specific deletions. PCR analysis shows that such viral genomes are the only variants detectable in infected animals, suggesting that they are generated by a specific cellular mechanism. We have tested the possible role of the lymphoid cell-specific V(D)J recombination activity in the generation of these specific variants. Our results indicate that this mechanism is not solely responsible for the viral genome rearrangement, if involved at all.     

High-level polyomavirus JC viruria following long-term steroid therapy

C virus is a highly seroprevalent ubiquitous polyomavirus which is acquired at an early age through respiratory or oral route, Thereafter JCV establishes persistent, but mainly asymptomatic, infections in various tissues, including the genitourinary tract and brain. In individuals with altered immunity, viral replication can occur leading to serious organ diseases. In contrast to polyomavirus BK, that is found infrequently in the urine of healthy adults, JC viruria occurs universally. Herein we describe a case of an otherwise immunocompetent patient who presented a very high-level asymptomatic polyomavirus JC viruria following long-term steroid therapy.     

Polyomavirus DNA replication in the pancreas and in a transformed pancreas cell line has distinct enhancer requirements.

The regulatory DNA (enhancer) of polyomavirus (Py) is a major determinant of tissue-specific DNA replication during acute infection of newborn mice. Previously, we reported that the combination of one of the two Py enhancers (A enhancer) and the repeated Moloney murine leukemia virus (Mo-MuLV) enhancer gave a chimeric Py genome (Py-MuLV) that replicates predominantly in the acinar cells of the pancreas, a tissue not permissive for wild-type PyA2 replication (R. Rochford, B. A. Campbell, and L. P. Villareal. Proc. Nat. Acad. Sci. USA 84:449-453,1987). In this report, we further examine the combined enhancer requirements for acinar cell-specific Py replication. We also compare enhancer requirements for Py replication in the acinar cells of the pancreas with those of a transformed acinar cell line (266-6 cells). The deletion of sequences within the A enhancer of Py-MuLV (nucleotides 5098 to 5132) results in a virus with 10-fold-reduced levels of pancreas-specific replication. The deletion, however, of one of the 72-bp repeated Mo-MuLV enhancer sequences from Py-MuLV results in a complete loss of pancreas-specific DNA replication. Thus, the Py A enhancer is required for efficient replication of Py in the pancreas without otherwise altering organ specificity, but both of the repeated copies of the Mo-MuLV enhancer are essential for pancreas-specific Py replication. In contrast to the enhancer requirements for in vivo pancreas replication, in transformed acinar cells (266-6), PyA2 wild-type replicated efficiently and the Py-MuLV recombinant replicated inefficiently. These data suggest that the cell-specific control of DNA replication is different between normal pancreas cells and their transformed cell line counterparts and that this difference is apparent in the enhancer requirement of cell-specific Py DNA replication.     

Stimulation of BK virus DNA replication by NFI family transcription factors

BK polyomavirus (BKV) establishes persistent, low-level, and asymptomatic infections in most humans and causes polyomavirus-associated nephropathy (PVAN) and other pathologies in some individuals. The activation of BKV replication following kidney transplantation, leading to viruria, viremia, and, ultimately, PVAN, is associated with immune suppression as well as inflammation and stress from ischemia-reperfusion injury of the allograft, but the stimuli and molecular mechanisms leading to these pathologies are not well defined. The replication of BKV DNA in cell cultures is regulated by the viral noncoding control region (NCCR) comprising the core origin and flanking sequences, to which BKV T antigen (Tag), cellular proteins, and small regulatory RNAs bind. Six nuclear factor I (NFI) binding sites occur in sequences flanking the late side of the core origin (the enhancer) of the archetype virus, and their mutation, either individually or in toto, reduces BKV DNA replication when placed in competition with templates containing intact BKV NCCRs. NFI family members interacted with the helicase domain of BKV Tag in pulldown assays, suggesting that NFI helps recruit Tag to the viral core origin and may modulate its function. However, Tag may not be the sole target of the replication-modulatory activities of NFI: the NFIC/CTF1 isotype stimulates BKV template replication in vitro at low concentrations of DNA polymerase-α primase (Pol-primase), and the p58 subunit of Pol-primase associates with NFIC/CTF1, suggesting that NFI also recruits Pol-primase to the NCCR. These results suggest that NFI proteins (and the signaling pathways that target them) activate BKV replication and contribute to the consequent pathologies caused by acute infection.     

Persistence of polyomavirus in mice infected as adults differs from that observed in mice infected as newborns.

By using the polymerase chain reaction (PCR) technique, a technique more sensitive than Southern analysis, which allows detection of polyomavirus DNA only in newborn and nude adult mice, it has now been possible to monitor the persistence pattern of polyomavirus DNA after infection of normal adult CBA mice for the first time. Viral signs appeared gradually, showing variations in time course and organ distribution between mice, and reached a peak activity after 2 to 3 weeks, when they could be found in bone, heart, gonads, lymph node, and skin, but disappeared by 2 to 5 months. No virus DNA was detected in the kidneys or lungs, which is in contrast to what is observed after infection of newborn mice. This finding suggests that the persistence pattern of polyomavirus is age dependent.     

Multiple domains in the polyomavirus B enhancer are required for productive infection of F9 embryonal carcinoma cells.

A point mutation at nucleotide 5258 in the B enhancer of the polyomavirus host range mutant F441 leads to productive infection of F9 embryonal carcinoma cells, which are refractory to infection by wild-type polyomavirus. Specific oligonucleotides were used to construct mutations in two other potentially important domains within the B enhancer of F441 DNA. One of these domains is the binding site for a factor present in nuclear extracts of F9 cells, and the other is a region that has sequence similarity to putative core sequences observed in a number of different viral enhancers. Mutation within either of these two domains, even in the presence of the F441 mutation, was detrimental to polyomavirus enhancer activity in F9 cells, as determined by both transfection and infection assays.     

The Role of Mouse Adenovirus Type 1 Early Region 1A in Acute and Persistent Infections in Mice

Mouse adenovirus type 1 (MAV-1) early region 1A (E1A) viral mutants were used to determine the importance of this region in pathogenesis and establishment of a persistent infection in the natural host. Lethal dose analysis with adult male Swiss outbred mice revealed a significant reduction in virulence for all of the E1A mutants. During acute infections with 10⁵ PFU of virus, an E1A null mutant, pmE109, was found in the same organs (brain, spleen, and spinal cord) and the same cell types (endothelial cells and mononuclear cells in lymphoid tissue) as wild-type virus. Another null mutant, pmE112, was detected in the same organs but in lower numbers. However, when mice were given a lower dose, 1 PFU, pmE109 and pmE112 reached none of the target organs analyzed by 14 days postinfection (p.i.). The absence of E1A did not hinder the ability of MAV-1 to establish a persistent infection. Viral nucleic acid was detected by PCR amplification or in situ hybridization in the kidneys, brains, spleens, and prefemoral lymph nodes of mice infected with wild-type or mutant virus up to 55 weeks p.i. The brain, spleen, and lymph node are recognized sites of acute viral infection but are previously unrecognized sites for MAV-1 persistence. Evidence for the potential reactivation of persistent MAV-1 infections is also presented.     

Natural biology of polyomavirus middle T antigen.

"It has been commented by someone that 'polyoma' is an adjective composed of a prefix and suffix, with no root between--a meatless linguistic sandwich" (C. J. Dawe). The very name "polyomavirus" is a vague mantel: a name given before our understanding of these viral agents was clear but implying a clear tumor life-style, as noted by the late C. J. Dawe. However, polyomavirus are not by nature tumor-inducing agents. Since it is the purpose of this review to consider the natural function of middle T antigen (MT), encoded by one of the seemingly crucial transforming genes of polyomavirus, we will reconsider and redefine the virus and its MT gene in the context of its natural biology and function. This review was motivated by our recent in vivo analysis of MT function. Using intranasal inoculation of adult SCID mice, we have shown that polyomavirus can replicate with an MT lacking all functions associated with transformation to similar levels to wild-type virus. These observations, along with an almost indistinguishable replication of all MT mutants with respect to wild-type viruses in adult competent mice, illustrate that MT can have a play subtle role in acute replication and persistence. The most notable effect of MT mutants was in infections of newborns, indicating that polyomavirus may be highly adapted to replication in newborn lungs. It is from this context that our current understanding of this well-studied virus and gene is presented.