bk polyoma virus allograft nephropathy: ultrastructural features from viral cell entry to lysis

American Journal of Transplantation 2003; 3: 1383–1392Blackwell Munksgaard

Copyright C© Blackwell Munksgaard 2003

ISSN 1600-6135doi: 10.1046/j.1600-6135.2003.00237.x

BK Polyoma Virus Allograft Nephropathy:Ultrastructural Features from Viral Cell Entry to Lysis

Cinthia B. Drachenberga, John C.

Papadimitrioua,∗, Ravinder Walib, Christopher

L. Cubittb,c and Emilio Ramosb

Departments of aPathology and bMedicine, Universityof Maryland School of Medicine, Baltimore, MD, USAcNational Institute of Neurological Disorders and Stroke(NINDS), National Institutes of Health, Bethesda,MD, USA∗Corresponding author: John C. Papadimitriou,[email protected]

BK virions must enter the host cell and target theirgenome to the nucleus in order to complete theirlife cycle. The mechanisms by which the virions ac-complish these tasks are not known. In this mor-phological study we found that BK virions localizedbeneath the host cell cytoplasmic membrane in 60–70-nm, smooth (non-coated) monopinocytotic vesiclessimilar to, or consistent with, caveolae. In the cyto-plasm, the monopinocytotic vesicles carrying virionsappeared to fuse with a system of smooth, vesicles andtubules that communicated with the rough endoplas-mic reticulum and was continuous with the Golgi sys-tem. Membrane-bound single virions and large tubulo-reticular complexes loaded with virions accumulatedin paranuclear locations. Occasional nuclei displayedvirions within the perinuclear cisterna in associationto the perinuclear viral accumulations. Tubular cellswith mature productive infection had large nuclei, dis-tended by daughter virions, whereas they lacked sig-nificant numbers of cytoplasmic virions. In addition tovirally induced cell necrosis, there was extensive tubu-lar cell damage (apoptosis and necrosis) in morpholog-ically non-infected tubules. The observed ultrastruc-tural interactions between the BK virions and host cellsare remarkably similar to viral cell entry and nucleartargeting described for SV40 virus.

Key words: Caveolae, cell injury, cytopathic changes,kidney, SV40, transplantation

Received 9 February 2003, revised and accepted forpublication 20 May 2003

Introduction

Two of the 13 known polyomaviruses, BK and JC, are hu-man pathogens causing nephritis and progressive multi-

focal leukoencephalopathy (PML), respectively. In normalindividuals, subclinical BK and JC primary infections are as-sociated with seroconversion in more than 90% of casesby the age of 20 (1). JC and/or BK viral reactivation is typi-cally observed in immunosuppressed individuals (2). Withthe AIDS epidemic, there has been a marked increase inthe incidence of progressive multifocal leukoencephalopa-thy (2). In recent years, an increasing incidence of BK virusnephropathy has been reported in renal transplant patients(3). In general, polyoma viruses are species-specific. Thesimian polyomavirus (SV40) is endemic in monkeys and inassociation with immunosuppression causes simian PML(4). SV40 has been reported to co-infect renal transplantrecipients presenting with BK allograft nephropathy (5).

SV40 has been extensively studied since 1960, when itwas first reported. These studies have led to extremelyimportant insights into the molecular biology of mammaliancells and oncogenesis (6). In contrast, very little is knownon the pathogenesis of diseases associated with the BKand JC viruses.

Similarities between BK, JC and SV40 at the DNA andprotein level result in identical morphology. These threeviruses are considered to have similar epidemiology andbiological behavior in their respective natural hosts aswell (1,2). Transmission of infection is most likely bythe oral route (1,2). After multiplication at the site ofentry, the viruses reach their target organs. The princi-pal target organ for all three viruses is the kidney (4).The primary infection is followed by latency in the uri-nary tract epithelium, lymphoid cells and central nervoussystem (1).

The polyomaviruses are a family of small, non-enveloped,DNA viruses. The viral capsid is icosahedral and has a di-ameter of 40–44 nm (7). The genome consists of a closedcircular double-stranded DNA molecule with approximately5 kb, that encodes the early (regulatory) and late (structural)proteins (6). For the life-cycle of the virus to be completed,the virions must attach to the host cell plasma membraneand target their genome to the nucleus. In the cell nucleus,the uncoated mini-chromosome is transcribed. Transcrip-tion of the early genes results in the production of the Tantigens that cause quiescent cells to re-enter the cell cy-cle and thus begin replication of cellular DNA. In permissivehost cells the T antigens, acting as regulatory proteins, di-rect the remaining events, resulting in a productive infec-tion (8). The completion of the process consists of viral

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DNA replication and transcription of late genes for theproduction of the structural proteins (VP1,VP2 and VP3)that will constitute the capsid. Viral capsomeres assemblearound the daughter mini-chromosomes in the nucleus, toform stable viral particles (6).

SV40 uses an ‘atypical’ endocytic mechanism to enter thehost cells. This virus is internalized in non-clathrin-coatedvesicles called caveolae. From caveolae the virions reachthe nucleus through an intermediate vesicular and tubularsystem (9–13). This is in contrast to many other viruses thatenter the host cells by the usual endocytic pathway. Thelatter is a constitutive pathway that utilizes clathrin-coatedvesicles targeted to the endosomal/lysosomal system. Inthese organelles the viruses are disassembled throughacid-activated hydrolytic enzymes (10,12). Disassembly ofSV40, however, is not dependent on low pH (14).

Several studies have described the interactions betweenthe murine polyoma virus and the host cells. It appearsthat this virus follows the same pathway as SV40, usingcaveolae to enter the host cell (15–18). On the other hand,one study found that the murine polyoma virus entered thehost cells using non-clathrin-coated, non-caveolar vesicles(19).

For the BK and JC viruses, little or nothing is known aboutthe mechanisms of cellular entry, cytoplasmic transportand nuclear targeting. While there have been no studiesspecifically addressing these aspects of the BK virus infec-tion, a well-conducted study has shown that JC virions usefor cell entry the typical endocytic pathway with clathrin-coated vesicles (20).

Despite their important similarities at the molecular andmorphological level, it appears that the polyoma virusesuse divergent pathways for cellular entry. Understandingthe interactions between virions and host cells at the ultra-structural and molecular level has important implications inthe treatment and prevention of the specific infection. Thisinformation may also be important in the context of genetherapy.

In the current electron microscopic study we describethe morphological findings in patients with BK allograftnephropathy. Our main goals are to: (a) describe the in-teractions between the BK virions and the renal tubularepithelial cells, particularly in relationship to viral entry, cy-toplasmic trafficking and nuclear targeting; (b) characterizethe overall cytopathic changes and the features of cell in-jury and death in the cells showing nuclear accumulation ofviruses; and (c) identify and describe any other additionalultrastructural features directly or indirectly related to theviral infection.

Materials and Methods

Renal transplant biopsies from eight patients and transplant nephrectomiesfrom two patients with active BK virus nephropathy were studied ultrastruc-

turally. The diagnosis was made following previously described criteria (21).By light microscopy, multiple tubular cells showed the typical cytopathic ef-fects of polyoma virus. These have been well described previously (22) andmainly consist of nuclear enlargement with the presence of a basophilic,‘gelatinous’ nuclear inclusion displacing the chromatin. The diagnosis of BKvirus infection was confirmed with urine PCR studies.

PCR amplification

Viral DNA was isolated from a low-speed centrifuged pellet of urine(10–15 mL) using the QIAamp Viral RNA/DNA isolation kit (Qiagen,Valencia, CA, #29304). The DNA was resuspended in 50 lL ultra-pure H2O and stored at –20 ◦C. Viral DNA were amplified by PCRusing the TaKaRa Ex Taq Kit (Takara Bio Inc., Japan, #RR001A) ina reaction mixture as per manufacturer’s instructions. For detectionof BKV DNA 215-bp fragment in VP1 gene of BKV was amplifiedusing primers BLP-15 (5′-ACAGCACAGCAAGAATTCCCCTCCC-3′) and BLP-16 (5′-CAAGGGTTCTCCACCTACAGCAA-3′). Twosets of PCR primers were used for detection of SV40 DNA.The STP-1 (5′-CAGGTTCAGGGGGAGGTGTGGG-3′) and STP-2 (5′-GATGGTGGGGAGAAGAACATGG-3′) amplify a 178-bp region of SV40T antigen. SLP-1 (5′-TTGATGTGGGAAGCTGTTACTG-3′) and SLP-4 (5′-ATGAAAATTTGACCCTTGAATG-3′) amplify a 129-bp region of SV40 VP-1.Each primer was used at a final concentration of 2.5 ng/lL. The PCRprogram consisted of 95 ◦C, 1 min; 60 ◦C, 1 min; and 72 ◦C, 1 min for40 cycles with an initial denaturation step of 95 ◦C for 5 min and a finalextension of 72 ◦C for 10 min. The annealing temp was set to 64 ◦C for theSTP-1, -2 primer set. The PCR products were analyzed on a 2% agarosegel containing 0.5% lg/mL ethidium bromide. There was no evidence ofconcurrent JC or SV40 viral excretion by PCR in any of the patients.

For electron microscopy the samples were fixed in a solution of 4%formaldehyde and 1% glutaraldehyde in mono-phosphate buffer, followedby 1% osmium tetroxide in mono-phosphate buffer. The tissue was thendehydrated in increasing concentrations of alcohol, cleared in propylene ox-ide and embedded in epoxy resin (Epon 812). Ultrathin sections from theseblocks were stained with uranyl acetate and lead citrate. Transmission elec-tron microscopy was performed with a JEOL 1200 EX1.

A total of 64 grids were evaluated ultrastructurally (3 from each biopsy and20 from each nephrectomy). An average of 150 tubular cross-sections werepresent per grid. Serial sections of 7 grids with abundant infected tubuleswere also evaluated.

Results

Virions associated with the surface

cytoplasmic membrane

In infected tubules abundant virions were present over thecell surfaces of tubular cells (Figure 1a). The virions wereroughly spherical with a diameter of 40–42 nm. The distri-bution of viruses over the cell surfaces was random and notrestricted to the apical aspects. The viral load on the surfaceof individual cells varied from a few scattered isolated viri-ons to thick clumps of innumerable virions. Some virions at-tached tightly to the cytoplasmic membrane. In viable cellsa minority of virions was associated with flask-like invagi-nations of the membrane as they attached to the cell sur-face. Other virions were located deeper in the cytoplasmand apparently caused the cytoplasmic membrane to wraparound them. In other cases the virions were within a vesi-cle that appeared to have been separated from the surfacemembrane. The vast majority of the vesicular membranes

1384 American Journal of Transplantation 2003; 3: 1383–1392

BK Allograft Nephropathy Ultrastructure

Figure 1: a: Renal tubular cell with BK

virions attaching to the surface cellu-

lar membrane, including microvilli. band c: Entry of BK virions. The virionsattach to the surface membrane and in-duce formation of flask-like invaginationsthat surround the virus. d: The mem-brane encloses the virion and a vesicle isformed. e: The viral loaded vesicles sepa-rate from the inner aspect of the surfacecellular membrane (arrows).

Figure 2: Deeper in the cytoplasm the viral-loaded vesicles

fuse with irregular tubulo-vesicular structures. Two virions areseen still on the surface of the cell (top right).

were smooth (i.e. lacking a clathrin-like cytoplasmic coat).Most vesicles were spherical and measured 60 nm inaverage diameter. The membranes surrounded the virustightly, although there was a 10–12-nm narrow space be-tween the virion and the membrane (Figures 1e and 2).Progressive stages of viral internalization are demonstratedon Figure 1(a–e). Rare vesicles had irregular shapes or acompound appearance, and rarely enclosed two or threevirions. In some cases the latter findings appeared to re-sult from tangential cutting of surface virions, partially en-closed by the cytoplasmic membrane. Extremely rare vesi-cles (less than 0.5%) appeared to show an external fuzzylayer of electron-dense material, in some cases suggest-ing a clathrin coat. In cells with evidence of advanced pro-ductive infection and accumulation of intranuclear virions,there were no significant numbers of virions in pinocytoticvesicles.

In the vicinity of the cytoplasmic membrane the vesiclescarrying virions were more often found separate from eachother (Figure 1e). In contrast, in deeper locations (400–500 nm from the surface), the vesicles with virionswere more often found in clusters of up to 100 vesicles(Figure 2). The clusters of vesicles, each vesicle loadedwith a single virion, fused with irregularly shaped vesicular-tubular structures (Figure 2). These latter structures weremorphologically consistent with the descriptions of caveo-somes (9,10,12). In rare instances there were recognizablechanges in the cellular cytoskeleton with aggregation of in-termediate and thin filaments in the adjacent cytosol.

Cytoplasmic virions

In infected tubules, only a minority of tubular cells dis-played cytoplasmic viral particles rather than the nuclearviral aggregates characteristic of the mature productive in-fection. In cells with predominant cytoplasmic virions, thelatter were seen in monopinocytotic vesicles, sometimesnear the nucleus, or were localized in complex membrane-bound viral aggregates. The aggregates were of two types:(I) The virions were localized in irregularly shaped vesiclesor were aligned in long tubules with smooth and paral-lel walls (Figure 3). The vesicles and the tubules wereoften continuous. The tubulovesicular aggregates wererandomly distributed in the cytoplasm, and when the sec-tions demonstrated longitudinal images of the tubules,these were long and ran over significant portions ofthe cytoplasm (Figure 3). (II) A tubulo-reticular net-work of delicate undulating tubules that containedlarge numbers of virions mostly arranged in a latticepattern (Figure 4). The second type of viral aggregateswas more often noted in perinuclear locations (Figure 5).They were composed of a few or innumerable virusesthat in some cases formed inclusions as large as the nu-cleus associated to them (Figure 6). The tubulo-vesicular

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Figure 3: The virions accumulate in a complex system of

smooth-walled tubulo-vesicular structures. The virions ap-proximate the nucleus presumably through this tubular system.Accordingly, membrane-bound virions are seen by the nucleus(arrows).

Figure 4: Large numbers of viruses accumulate in complex

tubulo-reticular structures that tend to be close to the nu-

cleus. These viral aggregates closely associate with the Golgi com-plex (G) and are continuous with the rough endoplasmic reticulum(asterisks).

aggregates were seen occasionally in continuity with theRER and in close proximity and/or continuity to the Golgisystem (Figure 4).

The cells with abundant virions attached on the exter-nal aspect of the cell membranes, only rarely containedabundant tubulovesicular or tubuloreticular cytoplasmic ag-gregates. The converse was also true, suggesting thatthese represented two stages (viral entry and trafficking,respectively) in the host cell. The membrane-bound virusesin the cytosol were morphologically identical to the ex-tracellular ones, showing no morphological evidence ofdisassembly.

Figure 5: An aggregate of virions within tubulo-vesicular

structures is seen by the nucleus. Intact perinuclear envelope isseen to the left (arrowheads). Adjacent to the viral aggregate themembranes of the perinuclear envelope become distorted andfuse with the peripheral membranes of the viral aggregate. Thecontents of the latter appear to be in continuity with the perinuclearcisterna (arrow). Another cluster of tubules loaded with virions isseen in the top right area.

Figure 6: Large, ‘inactive’-appearing paranuclear viral aggre-

gates (asterisks). In contrast to the changes seen in Figure 5, inthis case there is no clear evidence of interaction between theviral aggregate and the perinuclear envelope.

In viable cells demonstrating active endocytosis or traffick-ing of virions, all virions were membrane bound with noinstances of virions lying free in the cytosol. This was incontrast to findings in dying cells (see below).

Virions and the nucleus

Viral aggregates or vesicles loaded with virions were com-monly seen in the vicinity of the nucleus. In many cells



Figure 7: (a,b,c) a: Membrane-bound virions are located close

to the nucleus (arrow) and within the perinuclear cisterna (ar-

rowhead). A non-membrane-bound virion is also seen in the nu-cleus (open arrow). In the vast majority of cases the virions foundwithin the nucleus were membrane bound (b and c).

the viral aggregates were closely associated to the per-inuclear cisternae, and in occasional nuclei the viral parti-cles were seen within the perinuclear cisterna (Figure 7).In these cases, it was common to see apparent fusionof the membranes surrounding the virions and the perinu-clear membranes (Figure 5). In association with the perinu-clear viral aggregates, there were excess membranes and,rarely, aggregates of empty membranous tubulo-reticularaggregates (Figure 8). The viral particles in the perinuclearcisterna and inside the nucleus proper did not associatespecifically with the nuclear pores, although in rare casesthe virions were located in the vicinity of nuclear pore ar-eas. In some nuclei, close to the perinuclear viral aggre-gates there were small aggregates of virions loosely sur-rounded by membranes, suggesting that this appearancecould have resulted from tangential cutting of virions lo-cated in the perinuclear cisterna and herniating into thenucleus (Figure 7). Occasional nuclei contained fibrillar ormicrotubular arrays in association with the viral particles(Figure 8). Rarefaction of the chromatin with thick granulesand aggregates was seen in cells with evidence of perin-uclear viral activity. Occasional cells showed formation ofsmall non-membrane-bound viral aggregates, presumablyof daughter viruses. In numerous cases, despite the pres-ence of very abundant paranuclear viral aggregates, there

Figure 8: Several virions are seen in the perinuclear cisterna

(arrows). The perinuclear cytoplasm contains an accumulationof excess membranes (arrowheads) and tubulo-reticular struc-tures devoid of virions (asterisk). There is prominent rarefactionof the chromatin with clumping and formation of thick granules.Insert: Part of another nucleus contains a loose aggregate of par-tially membrane-bound virions associated with microtubules (as-terisks). A nuclear pore is clearly seen (arrowhead) without anyassociation to the virions.

was no evidence of nuclear entry or interaction betweenthe virions and the nuclear membrane (Figure 6). In thosecells it appeared that the process of nuclear entry had beenarrested, resulting in extensive accumulation of perinuclearvirions in cells that appeared viable and otherwise healthy(Figure 6).

Lysis of infected tubular cells

In tubular cells with mature productive infection the nucleiwere markedly enlarged due to the accumulation of daugh-ter viral particles in loose groups or in dense crystalline ar-rays (Figure 9). In many cases the viral aggregates wereseparated from the nuclear membrane by a rim of chro-matin (Figure 9). Numerous cells with abundant nuclearaccumulation of virions showed various stages of cell in-jury and, in the most severe cases, necrosis. The latter wascharacterized by nuclear and cytoplasmic swelling, asso-ciated with generalized disruption of the cell membranes,including rupture of the nuclear envelope (Figure 9). In cellsundergoing necrosis, there were large cytoplasmic aggre-gates of randomly distributed, non-membrane-bound viri-ons. These often attached to fragments of membranes andcould be observed occasionally in lysosomes (Figure 10).In occasional infected cells undergoing dissolution, the nu-clear viral aggregates appeared very dense, with loss ofmorphological detail of the viral particles; these nuclei ap-peared mummified.

Lysis of the infected cells resulted in massive shedding ofvirions into the tubular lumen associated with the forma-tion of casts. Accumulation of viruses was also seen in theintercellular spaces. In heavily infected tubules, most cell


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Figure 9: Infected cell undergoing lysis. A dense aggregate ofprogeny virions is seen in the nucleus (top left). The perinuclearmembranes are disrupted. In the cytoplasm there are marked de-generative changes (extensive membrane vesiculation, mitochon-drial swelling and disruption). Insert: Progeny virions in nucleus.The virions display a crystalline array and are surrounded by rimof chromatin that separates the bulk of virions from the nuclearmembranes.

Figure 10: In necrotic areas the virions were randomly dis-

tributed in the cytoplasmic fragments, usually not membrane

bound (arrows), although often attached to membranes. Acrystallized cluster of virions is seen within a structure consistentwith a lysosome (bottom center).

surfaces of viable and dying cells were covered by virions.Viral particles were also present between the infected cellsand the underlying tubular basement membrane. Evalua-tion of more than a thousand tubular cells containing thetypical nuclear features of a fully developed productive in-

fection showed that virions reach the extracellular spaceonly by cell lysis. Viral particles remain confined within thenuclear membrane until the latter ruptures. In the stage ofcell lysis, there is generalized evidence of loss of integrityof the cellular membranes.

The tubular cells appeared to become infected by directcontact with infected cells undergoing lysis. Therefore,in infected tubules the vast majority of cells containedviruses. In contrast, unaffected tubules usually did not con-tain any virions at all. Tubular cell necrosis and destructionof the tubular basement membranes occasionally resultedin large extracellular aggregates of virions. The latter wererarely seen within lysosomes in interstitial macrophages.Intact basement membranes appeared to preclude pene-tration of virions in adjacent (non-infected) tubules.

Vascular and interstitial changes

In two of the ten cases, the endothelial cells and occasionalepithelial cells showed abundant tubulo-reticular ‘viral-like’inclusions of the type described in patients treated withinterferon, HIV-associated nephropathy or lupus nephritis(23,24) (Figure 11). Virions, however, were not seen in en-dothelial cells in any instance.

Prominent accumulation of inflammatory cells was seenin the interstitium. The inflammation mostly consisted oflymphocytes, macrophages and plasma cells (Figure 12).

Figure 11: Endothelial cell with a large tubulo-reticular inclu-

sion. This does not contain virions and is identical to the viral-likeaggregates seen in endothelium of HIV-infected patients. Numer-ous caveolae (a normal organelle of endothelial cells) are seen inall aspects of the endothelial plasma membrane (arrows). Insert:Surface cytoplasmic membrane of an epithelial cell covered by viri-ons. In the cytoplasm a virion is located within a vesicle showing afuzzy lining typical of a clathrin coat, an extremely rare occurrence(0.5%).



Figure 12: Tubular cross-section with obvious cellular dam-

age (consistent with necrosis and apoptosis). There is ex-tensive fragmentation of membranes. Condensed and swollenmitochondria are seen (asterisk). On both sides of the tubulethe interstitium shows abundant inflammatory cells and cellulardebris.

Admixed with the inflammation were cellular debris anddeposition of immature collagen fibers.

Tubular cell injury in non-infected tubules

More than 50% of tubules in each biopsy lacked any viri-ons. However, despite the absence of clear evidence ofinfection, there was evidence of generalized tubular cellinjury. The tubular cells showed loss of nuclear polarity, cy-toplasmic vacuolization or condensation, and loss of brushborder. Numerous tubules were lined by irregularly shaped,overlapping epithelial cells rather than by a single layer of or-derly columnar cells. Tubulitis (lymphocytic tubular inflam-mation) was seen in scattered infected and non-infectedtubules (Figure 12).

Scattered tubular cells showed necrosis, apoptosis or in-termediate features between necrosis and apoptosis. Cellsundergoing necrosis were swollen and showed extensivedisruption of the cell membranes. Apoptotic cells showedcondensation of the chromatin at the nuclear periphery in acrescentic fashion and cytoplasmic condensation. In mostcells the subcellular organelles, in particular the mitochon-dria, showed a spectrum of changes from severe conden-sation to high amplitude swelling and terminal disruption.The endoplasmic reticulum and Golgi showed dissolutioninto small vesicles. In occasional cells the cytoskeleton col-lapsed, particularly at the cell base. Vesiculation and bleb-bing of the cell plasma membrane was more prominent atthe luminal surface, but also occasionally towards the cellbase.

Discussion

BK virus nephritis is an important cause of renal allograftdysfunction and graft loss. Although still a poorly under-

stood infection, several recent studies have shed light onthe clinical aspects of BK allograft nephritis (25,26). Spe-cific functional and molecular studies on the biological be-havior of BK virus at the cellular level are not available,and very little is known about the interactions betweenthe virus and the host cells. In contrast, numerous stud-ies have characterized the cellular interactions of the SV40virus and murine polyoma virus (9–20). For the purpose offurther understanding the pathogenicity of BK virus, it maybe helpful to draw potential analogies with these betterknown polyoma viruses, particularly the SV40.

A large variety of compounds and organisms enter cellsthrough the process of endocytosis (internalization of ex-ternal particles or molecules). The type of vesicle involvedin the process of cell entry and its coat, determines toa large extent the type of cargo that will be transportedand its destination within the cell (27). First described inendothelial cells (see Figure 11), caveolae are small (50–70 nm) flask-shaped vesicles present in the cell surface ofmany cell types (28). Caveolae are rich in lipids and con-tain caveolin-1. They are actively involved in cholesterolcell entry (10). It has recently been recognized that SV40enters the host cell through caveolae (10–13). Ebola virusamong other viruses also uses caveolae (29). In the caseof SV40, the virions first attach to a receptor related toMHC class I molecules and then translocate to specializedmembrane microdomains (lipid rafts), where the virions in-duce the formation of caveolae and therefore their owninternalization (10–13). In this study we have found thatBK virions appear to enter the renal tubular cell in smooth(non-coated) monopinocytotic vesicles that are morpholog-ically consistent with caveolae. The morphological findingsare remarkably similar to SV40 and murine polyoma virushost cell entry. This is in contrast to the manner of entryof JC virus, that has been found to enter cells by followingthe usual endocytic pathway using clathrin-coated vesicles(20). The different pathways of viral cell entry do not ap-pear to directly correlate with the type of receptor, sinceBK, JC and murine polyoma viruses have sialic-acid type re-ceptors, whereas SV40 has receptors related to the MHCsystem (30). It has been previously reported for SV40 thatentry of virions in the host cell is restricted to the apicalaspects of the cell (31). We have not, however, observedthat the pinocytotic vesicles were restricted to the apicalsurface of the tubular cells.

In the case of SV40, caveolae loaded with SV40 virions fusewith a tubulo-vesicular system of pre-existing organellesthat have neutral luminal pH and also contain caveolin-1 (10). These organelles, called caveosomes, start fus-ing with each other and undergo rapid shape changes,including formation of long tubular structures (10). Thesetubules have no attached ribosomes, but they are in con-tinuity with the rough endoplasmic reticulum (32). The ex-tensive network of tubules and vesicles that transport thevirions from one part of the cell to another is also consis-tent with recently described cisternal compartments within


Drachenberg et al.

intracellular transport pathways that include branchingtubular elements that can be several micrometers long(33). Specifically regarding SV40 virions, it has beendemonstrated that, while avoiding lysosomal degradation,they take advantage of a retrograde endocytic pathway thatleads to the RER and from there to the nucleus. This trackretrieves, in usual circumstances, resident proteins thathave escaped from the endoplasmic reticulum to the Golgisystem (12). Based on our morphological findings, it is likelythat BK virus uses similar mechanisms as SV40 to reachthe endoplasmic reticulum, and later the nucleus. We ob-served that the monopinocytotic vesicles loaded with BKvirions aggregated with each other and fused with polymor-phous membranous organelles connected to long smoothtubular and tubuloreticular structures that appeared to carrythe virions towards the paranuclear areas. We observedthat the paranuclear viral aggregates were occasionallycontinuous with the perinuclear cisterna, the rough endo-plasmic reticulum and the Golgi system. Similar findingshave been described also for murine polyoma virus (15).

The mechanism of nuclear entry is not clearly understoodfor any of the polyoma viruses. Some studies have sug-gested that nuclear entry was impaired by inhibitors of thenuclear pore complex system (10,34). Others believe, how-ever, that nuclear pores are too small to allow the entry ofintact virions (16). We did not see evidence of viral entrythrough nuclear pores, nor do we believe pore size couldbe a limiting factor for viral entry (see Figure 8 insert).

Interactions between the perinuclear, membrane-bound,SV40 viral aggregates and the perinuclear cisterna havebeen described in several studies. It has been shown thatfragments of labeled surface cytoplasmic membrane arecarried with the SV40 virions and accumulate in the outermembrane of the nuclear cisterna (17,35). Also, severalstudies have confirmed that during the process of viralDNA nuclear entry, there is fusion of the membranes sur-rounding the virions with the outer nuclear membrane (36–38). Similar to studies of SV40 and murine polyoma virus(17,19,34), we consistently saw BK virions in the perin-uclear cisterna as well as evidence of membrane fusionbetween the perinuclear viral aggregates and the nuclearmembranes. Interestingly, the numbers of virions in theperinuclear cisterna and within the nucleus were extremelysparse, in contrast to the large paranuclear aggregates ofcytoplasmic virions.

The viral minichromosome has to be uncoated before itcan be replicated in the nucleus. It is, however, not knownif uncoating occurs before or after entrance in the nu-cleus. Some authors believe that rapid virus uncoating oc-curs after nuclear entry (17,39). One study showed thatintact viruses were not seen in the nucleus a few hoursafter infection, and the authors concluded that intranu-clear uncoating is very efficient. However, if uncoating oc-curs within the nucleus, the question of how the progenyviruses resist disassembly is raised. It has been proposed

that once replication starts there may be an inhibition ofthe nuclear uncoating enzymes, or that the progeny virusmay have a protein coat different from the infecting par-ticles (36). On the other hand, more recent studies havesuggested that uncoating of polyoma viruses occurs inthe vicinity of the nucleus or as the virions enter the nu-cleus rather than in the nucleus itself. Regarding murinepolyoma virus, it has been demonstrated that VP1 pro-tein which composes the bulk of the viral capside doesnot enter the nucleus (16). Another study has shown thatVP2 and VP3 proteins, which link the capside to the mini-chromosome, accumulate in the perinuclear tubules carry-ing SV40. This finding supports the idea that disassemblystarts within the tubular system, before the viral geneticmaterial enters the nucleus (12). In the current study, thesparsity of intact intranuclear virions in association with theperinuclear viral aggregates appears to support the con-cept that viral uncoating occurs before nuclear entry of theDNA particle.

In the case of SV40, it has been proposed that in cells withproductive infection and accumulation of large number ofnuclear viral particles, a fibrillary network associated withthe nuclear pores prevents the virions from exiting towardsthe cytoplasm (40). It has been reported that the bulk ofSV40 virions remains associated to the nuclear matrix untilvirus-induced cell lysis occurs (41). This is in concordancewith the fact that non-enveloped virus, such as the poly-oma viruses, are released from the infected cells by celllysis (42). Some studies have shown that small amountsof SV40 virions reach the extracellular medium before celllysis occurs (41,43). Evaluation of a very large number ofinfected cells in the current study indicated that the vastmajority of intranuclear daughter virions remain within thenucleus until cell death by lysis occurs. Cells showing rup-ture of the nuclear membrane (lysis) often contained non-membrane-bound virions in the cytoplasm, or occasionallyin the perinuclear cisterna.

Extensive cell necrosis characterizes infection with SV40(44,45). Similarly, BK virus allograft nephropathy is charac-terized by extensive tubular necrosis (22). Surprisingly, inthis study we observed that, in addition to necrosis of in-fected cells, there was generalized damage of apparentlynon-infected tubular cells. Widespread tubular cell injuryand death appeared in the form of both necrosis and apop-tosis. Several studies have shown that the SV40 virus hascomplex effects on the cell cycle of the host cell. Thelarge T antigen has both apoptotic and anti-apoptotic ef-fects, resulting from binding to the retinoblastoma tumorsuppression proteins and to p53. In addition, the small tantigen inhibits the apoptosis-inducing effects of the largeT antigen (46).

The extensive damage in tubular cells that lack evidence ofviral infection probably represents the background of tubu-lar cell injury on which viral reactivation occurs. In clinicalstudies, tubular injury secondary to immunosuppressant



drug toxicity and immune-mediated damage (rejection) hasbeen associated with BK virus nephropathy (21,26).

It has recently been proposed that quantitative viral loadin plasma correlates with the degree of BK virus nephritis(47,48). This notion is supported by the findings in the cur-rent study. We have observed that in tubules undergoingmassive virally induced tubular cell necrosis, there is disso-lution of basement membranes, with massive spillage ofvirions into the intertubular space and destruction of inter-tubular capillary walls. Through the latter, the virions wouldeasily gain access to the systemic blood flow.

Currently, there is no effective treatment for the BK virusinfection. Understanding of the mechanisms of cell entry,cytoplasmic trafficking and nuclear targeting has importantpotential implications for the prevention and treatment ofBK virus-associated nephropathy (49,50).

The conclusions derived from the current study are purelyobservational. From the morphological standpoint, how-ever, it appears that BK and SV40 polyoma viruses followa similar endocytic pathway for nuclear targeting (11,12).To a significant extent, similar findings have also been de-scribed for murine polyoma virus (15). Additional functionaland molecular studies are necessary to further understandthe interaction between BK and the permissive host cell.

Acknowledgments

We wish to thank Caroline Ryschkewitsch for her help in the performance ofthe molecular studies. We are also grateful to Perry Comegys for excellentphotographic work.

We wish to acknowledge the late Dr Gerald Stoner who was consultedon multiple occasions during the preparation of this work and who was acontinuous source of inspiration for us.

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bk polyoma virus allograft nephropathy: ultrastructural features from viral cell entry to lysis

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