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Laboratorio de Virología, Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pab. II, 4 Piso, C1428BGA, Buenos Aires, Argentina
Correspondence
Luis A. Scolaro
luisco{at}qb.fcen.uba.ar
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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); S RNA encodes the major structural proteins, the nucleoprotein (N) and a glycoprotein precursor (GPC), which is processed post-translationally into the mature glycoproteins, G1 and G2 (Buchmeier, 2002; Meyer et al., 2002). The L segment encodes the viral RNA-dependent RNA polymerase (L) (Salvato et al., 1989) and a small zinc-binding matrix protein (Z) (Strecker et al., 2003). L, N and the viral RNA form the nucleocapsid structures sufficient for transcription and replication of genomic RNA (Lee et al., 2000; Tortorici et al., 2001), while Z has controversial regulatory functions in the viral multiplication cycle (Cornu & de la Torre, 2001; Garcin et al., 1993; Jacamo et al., 2003; Lopez et al., 2001) and has been recently described as the main driving force for virus budding through its putative interaction with the cellular protein TSG101, a component of the vesicular protein sorting machinery (Perez et al., 2003).
Arenaviruses are able to cause persistent infections in several species of rodents that act as reservoirs and in a wide variety of mammalian cell cultures suitable for virus growth. The persistent stage of JUNV infection is characterized by production of low levels of infectivity recovered from supernatants of the cultures that may alternate with the synthesis of defective interfering (DI) particles (Damonte et al., 1983). In some cases, neither virus nor DI particles are produced by the cultures (Ellenberg et al., 2002). In both cases, persistently JUNV-infected cultures proved to be resistant to the multiplication of homologous virus.
In the present study, we characterized a BHK-21 cell line persistently infected with JUNV and assessed the presence of viral genes, virus production and expression of viral antigens, with emphasis on the superinfection exclusion phenomenon.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Viruses.
Stock of JUNV, strain XJCl3, was prepared by infecting BHK-21 cells (m.o.i.=0.05 p.f.u. per cell) and harvesting supernatants at 6 days p.i. Virus titrations were performed by p.f.u. assay in Vero cells. Lymphocytic choriomeningitis virus (LCMV) WE strain, Tacaribe (TCRV) TRLV11573 strain, Pichinde (PICV) AN3739 strain and vesicular stomatitis virus (VSV) Indiana strain stocks were prepared by infecting Vero cells at 0.05–0.1 p.f.u. per cell and collected according to the virus. 35S-methionine labelling and purification of JUNV was performed as previously described (Ellenberg et al., 2004). Stocks of JUNV and TCRV were purified by ultracentrifugation at 100 000 g for 2 h. The pellet was resuspended in 10 mM Tris/HCl, pH 7.2, 1 mM EDTA and then layered onto a linear 20–60 % sucrose gradient. After centrifugation at 100 000 g for 2 h, fractions were collected. For TSG101 detection in virions, viruses were grown in BHK-21 cells and further purification was performed as previously described (Damonte et al., 1994).
Establishment and superinfection of persistently JUNV-infected cell cultures.
Confluent BHK-21 cell monolayers were infected with JUNV strain XJCl3 at an m.o.i. of 0.1 p.f.u. per cell and thereafter named K3. After infection, cells were maintained in DMEM until the end of the acute phase of infection, around 25–30 days p.i. This study was performed between days 1 and 1005 p.i., more specifically experiments concerning susceptibility and multiplication steps of superinfecting viruses were performed during the third year of persistence. Total virus (cell-associated and extracellular infectivity) was recovered from freeze–thawed cultures (cells plus supernatants), whereas extracellular virus was obtained only from supernatants. The number of cells producing infectious virus was monitored by an infectious centres assay (Castilla et al., 1998).
Interference activity was assayed as described elsewhere (D'Aiutolo & Coto, 1986). Presence of DI particles was estimated by the reduction in virus yield in cultures treated with persistently infected cell supernatants in comparison with MM (mock)-treated cells.
Fusion ability was evaluated by a syncytium formation assay described previously (Castilla & Mersich, 1996).
For the superinfection assay, K3 and BHK-21 cells grown during 24 h in 24-well microtitre plates were infected with JUNV, PICV, LCMV, TCRV and VSV at an m.o.i. of 0.1 p.f.u. per cell. Supernatants of arenavirus and VSV cultures were collected at 4 and 2 days p.i., respectively, and infectivity was assayed by p.f.u. technique. Alternatively, supernatants from JUNV- or TCRV-superinfected K3, collected at 4 days p.i, were incubated with MM or MM containing neutralizing anti-JUNV or anti-TCRV rabbit antisera (1 : 50, neutralizing titre: 2400). Vero cells (5x105) were infected with mock and neutralized supernatants and after 3 days p.i. total RNA was extracted. cDNA synthesis was performed employing ARS1 primer and subsequent PCR for detection of JUNV genome was carried out employing ARS1 (5'-CGCACAGTGGATCCTAGGC-3') and 2881 (5'-AGTCCCTTGCTGTTGAAATCCC-3') primers. TCRV7F (5'-TCTTGTCCATATTTGCCTAACTGA-3') and TCRV476R (5'-TTAGGAGAGTGAACAAAGACATCG-3') were employed for the detection of TCRV sequences.
Expression of viral antigens and TSG101.
Viral antigens and TSG101 expression was assessed by indirect immunofluorescence assay (IFA). Cells grown on glass coverslips in 24-well microtitre plates were washed with PBS and fixed with methanol for 10 min at –20 °C for cytoplasmic staining. Next, cells were washed with PBS and incubated with NA05AG12 (anti-N) mAb (1 : 300), GB03BE08 (anti-G1) (1 : 300) or TSG101 (C-2) (sc-7964, Santa Cruz Biotechnology) (1 : 50) for 45 min at 37 °C, followed by incubation with a FITC-conjugated goat anti-mouse affinity purified immunoglobulin G (IgG, Sigma) (1 : 50) for 45 min at 37 °C. The cells were washed thoroughly with PBS after each incubation step. Finally, nuclei were stained with 0.05 % Evans blue and coverslips were mounted on a 90 % glycerol solution in PBS containing 2.5 % 1.4-diazabicyclo (2.2.2) octane (DABCO). Alternatively, cells for double-staining of N and G1 proteins were washed with PBS and incubated with anti-JUNV rabbit hyperimmune serum (1 : 50); monolayers were fixed with methanol and processed as above. FITC-conjugated goat anti-rabbit and TRITC-conjugated goat anti-mouse were employed and cells were mounted as above.
For Western blot (WB) analyses, cell monolayers (105 cells) were washed with PBS and lysed with 25 µl equal parts of PBS and PAGE-SDS loading buffer (0.06 M Tris/HCl pH 6.8, 5 % SDS, 10 % glycerin, 2 % 
-mercaptoethanol, 0.05 % bromophenol blue). Cell lysates were separated by 10 % SDS-PAGE and transferred to a PVDF membrane (Hybond P, Amersham Pharmacia) in a dry system (LKB Instruments, Multiphor II). The viral nucleoprotein, N, was revealed with NA05AG12 (anti-N) mAb (1 : 300), TSG101 was revealed with MAbTSG101 (C-2) (sc-7964, Santa Cruz Biotechnology) (1 : 100) and a peroxidase anti-mouse IgG (ICN ImmunoBiologicals) (1 : 2000) was used as secondary antibody and visualized by a chemiluminescence detection system (ECL, Amersham Pharmacia). Membranes were stripped and revealed with anti-actin antibody JLA20 (1 : 2000) (Calbiochem). Anti-JUNV mAbs NA05AG12 and GB03BE08 were kindly provided by Dr A. Sanchez (Sanchez et al., 1989).
RNA extraction, cDNA synthesis and PCR analysis.
RNA was prepared from samples of 5x106 cells grown for 2 days in T25 flasks employing a Nucleospin kit (Macheray-Nagel), following the manufacturer's instructions. The RNA solution was stored at –70 °C. Reverse transcription of viral RNA segments was performed by using 1 µg RNA, and after heating at 95 °C for 5 min in the presence of 2 µM arenavirus-specific primer, 1 µl of a mixture of the four dNTPs (at 10 µM each) in 12 µl was added. This mixture was heated to 65 °C and chilled rapidly on ice. Then 4 µl of 5x RT buffer and 2 µl 0.1 M dithiothreitol were added to the mixture and heated to 42 °C for 2 min. Then 1 µl Superscript II-RNase H–Reverse transcriptase (200 U) (Invitrogen) was added to the reaction mixture and incubated at 42 °C for 1 h. The cDNA generated from this reaction was used as a template in subsequent PCRs. In the case of Fig. 4, primers specific to S genomic complementary RNA sequences were used in cDNA synthesis, primer ARS1 for JUNV and primer TCRV7F for TCRV. For the detection of TCRV sequences, primers TCRV7F/TCRV476R were employed for the amplification of a 469 bp product. Primers used for the detection of JUNV sequences were primers ARS1 and N1 (5'-GGCATCCTTCAGAACAT-3'), generating a 186 bp amplification fragment comprising the 3' end of the S RNA containing N coding sequences (Tortorici et al., 2001).
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Adsorption and internalization assays.
Adsorbed and internalized infectivity were analysed in BHK-21 and K3 cells infected with JUNV, TCRV or LCMV (m.o.i. of 5 p.f.u. per cell) as previously described (Castilla et al., 1998). Briefly, at different times post-adsorption at 4 °C, bound virus was quantified by p.f.u. after freeze–thawing of the cells. Combined associated radioactivity was monitored by a binding assay employing purified 35S-methionine-labelled JUNV (107 p.f.u. ml–1, 60 000 c.p.m. µl–1) as previously described (Ellenberg et al., 2004). Briefly, cells were infected at 4 °C for different times, washed extensively with cold PBS and lysed with 0.1 M NaOH, 1 % SDS, and cell-bound radioactivity was quantified. Associated and internalized infectivity were monitored after infection and incubation of cells for different times at 37 °C. Cells were treated with proteinase K, centrifuged and lysed as above. Radioactivity was quantified in a liquid scintillation counter (Wallac 1409 DSA, Perkin-Elmer).
TSG101 siRNA experiments.
TSG101 depletion was accomplished by using short interfering RNAs (siRNAs). BHK-21 and K3 cells in 24-well plates were transfected twice with TS101-specific or non-specific siRNAs (60 nM) at 24 h intervals using Lipofectamine 2000 (Invitrogen). Cells were infected 24 h post-transfection at 1 p.f.u. per cell. Extracellular and total virus were harvested at 24 h p.i. The target sequences for the TS101-specific siRNAs were 5'-CCUCCAGUCUUCUCUCGUCTT-3' (sense) and 5'-GACGAGAGAAGACUGGAGGTT-3' (antisense ) (Invitrogen) (Garrus et al., 2001).
Electron microscopy.
BHK-21-, K3- and JUNV-infected BHK-21 cells (48 hours p.i. m.o.i.=5 p.f.u. per cell) grown in 6-well microtitre plates were washed with cold PBS and fixed with 1.5 % glutaraldehyde-PBS (0.2 M) for 4 h at 4 °C. Cells were scraped and incubated overnight at 4 °C with 0.32 M sucrose. Cells were pelleted and further incubated with 1.5 % OsO4, 0.32 M sucrose for 2 h at 4 °C. After that, cells were pelleted and washed with distilled water. Cells were incubated overnight with 2 % uranyl acetate and dehydrated with a series of ethanol gradients followed by propylene oxide, embedded in Epon 812 resin mixture (TAAB), and polymerized at 70 °C for 2 days. Ultrathin sections were restained with 2 % uranyl acetate and observed in an electronic microscope (C10, Zeiss).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, a peak of infectivity, reaching 3x107 p.f.u. ml–1, was detected in the supernatant of JUNV-infected cells during the first week of infection. After 2 weeks p.i., infectivity production dropped to values ranging from 103 to 104 p.f.u. ml–1. By this time, the interference activity due to the presence of DI particles, which had been undetectable (<5 %) up to that moment, increased significantly, achieving 87 % at 17 days p.i. (Fig. 1a). During the first year p.i., the highest levels of DI particles were coincident with low levels of infectivity and vice-versa, following a characteristic cyclical pattern typical of JUNV persistence in vitro. However, after this period, infectivity decreased steadily with the course of infection reaching 102–103 p.f.u. ml–1 by the second year p.i., while percentage of interference activity kept fluctuating between 20 and 80 % despite the levels of infectivity detected (Fig. 1). The interference activity observed was specific for JUNV multiplication since VSV multiplication was not diminished by pre-treatment with K3 supernatants, ruling out unspecific action of interferon (data not shown).
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). Cells positive for N also expressed G1 in their membranes. Expression of the fusogenic viral protein, G2, was analysed by a syncytium formation assay at low pH (Fig. 1d). The number of syncytia in K3 cells exposed to low pH diminished with the course of infection in a fashion similar to infectivity. At 10 days p.i. confluent syncytia were obtained throughout numbers the monolayer, whereas the numbers of syncytia per coverslip dropped to 86±15, 66±18, 57±10, 28±7 and 32±12 at 112, 471, 549, 621 and 921 days p.i., respectively. The size of syncytia remained constant at all times analysed, averaging 32±16 nuclei/syncytium.
Susceptibility of K3 cells to homologous and heterologous virus superinfection
In order to test the susceptibility of K3 cells to multiplication of homologous and heterologous viruses, challenge experiments were performed with JUNV (homologous), TCRV (antigenically related arenavirus, heterologous), LCMV and PICV (antigenically non-related arenaviruses, heterologous) and VSV (rhabdovirus, heterologous). For this purpose, K3 cultures were superinfected at an m.o.i. of 0.1 p.f.u. per cell and virus yield in supernatants was assayed at 2 and 4 days p.i. for VSV and arenaviruses, respectively. As can be seen in Fig. 2(a), titres obtained in JUNV- and TCRV-superinfected K3 cells were more than 2 log lower than those observed for infected BHK-21 cells, whereas comparable levels of infectivity were obtained when superinfection was performed with LCMV, PICV or VSV (Fig. 2a). As can be inferred from the total/extracellular virus ratio (Fig. 2b), most virus produced in JUNV-superinfected K3 cells remained cell-associated in a fashion similar to the infectivity recovered from non-superinfected K3 cells.
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). However, when an infectious centre assay was performed, after superinfection at a high m.o.i., an increment of 1 log in the number of virus-producing cells was obtained, suggesting the contribution of an intracellular blockade, rather than of soluble factors, to superinfection exclusion in K3 cells (Fig. 2c).
Multiplication steps of superinfecting virus
Adsorption and penetration.
In order to elucidate the partially blocked step in the multiplication cycle of superinfecting virus, the early stages of binding and internalization were studied by infectivity and radioactivity assays. As can be seen in Fig. 3(a), a diminished level of bound infectivity was detected in JUNV-superinfected K3 cells compared with BHK-21 cells, whereas no differences were observed for LCMV-bound infectivity in K3 cells compared with BHK-21 cultures (Fig. 3b). Experiments performed with radiolabelled JUNV showed that, although adsorption and internalization of virus followed similar kinetics in both types of culture, the amount of associated and penetrated radioactivity was lower in K3 than in BHK-21 cells (Fig. 3c and d). Similarly, when binding and penetration of TCRV to BHK-21 and K3 cells were investigated, diminished levels of adsorption and internalization in K3 cells in comparison with BHK-21 cells could be observed (data not shown).
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The next step studied was the synthesis of proteins of superinfecting virus. Considering the fact that JUNV and TCRV proteins could not be discriminated by a polyclonal anti-JUNV or anti-TCRV antiserum in an IFA or WB analysis, we looked for the presence of TCRV or pseudotyped (JUNV genome and TCRV envelope) virions in supernatants of TCRV-superinfected K3 cells. To this end, we analysed the presence of JUNV or TCRV RNA in Vero cells infected with supernatants obtained from TCRV-superinfected K3 cells that had been previously treated with anti-JUNV or anti-TCRV polyclonal sera. As can be seen in Fig. 5, the presence of JUNV RNA, but not TCRV RNA, could be detected in the case of Vero cells infected with MEM (mock) or anti-TCRV serum-treated supernatants obtained from JUNV- and TCRV-superinfected K3 cells (lane 4, 6, 7 and 16). The absence of signal and a very faint band in lanes 2 and 3, respectively, suggested that treatment of supernatants with anti-JUNV serum reduced to a great extent the possibility of detection of virions with JUNV RNA spiked with JUNV proteins. Similarly, cells infected with supernatants obtained from TCRV-infected BHK-21 cells treated with anti-TCRV serum did not yield an amplification product (lane 14), whereas MEM-treated supernatant showed the expected band (lane 13). Virions with TCRV genome are readily detected in supernatants from TCRV-superinfected K3 cells (lane 11). When cells were infected with supernatants from TCRV-superinfected K3 cells, previously neutralized with anti-JUNV, the presence of virions with TCRV genome and TCRV envelope could be inferred (lane 15). In contrast, TCRV RNA could not be detected when the supernatant was treated with anti-TCRV serum, indicating the absence of virions with TCRV genome spiked with JUNV proteins (lane 12).
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Budding.
Taking into consideration the fact that virus produced by K3 cells remained mostly cell-associated, budding of virus was studied in order to demonstrate an alteration that would contribute, along with the restriction in protein synthesis, to the low level of infectivity produced and released to the supernatant. For this purpose, we focused on the role of the cellular protein TSG101, which has been demonstrated to participate in the egress of arenaviruses Lassa and LCMV (Perez et al., 2003; Urata et al., 2006). In order to determine the influence of TSG101 in JUNV budding, we evaluated the presence of this protein in BHK-21, K3 and JUNV-superinfected K3 cells. As can be seen in Fig. 6(a), although all cell types displayed a highly punctuate, putative endosomal, TSG101 expression IFA pattern, the amount of this protein was lower in BHK-21 cells in comparison with K3 cells. Similar results were obtained by WB analysis, confirming that acute infection of BHK-21 cells or JUNV superinfection of K3 cells did not modify the TSG101 levels observed for the non-infected or non-superinfected cultures, respectively (Fig. 6b). Association of TSG101 with arenavirus virions was analysed by WB from purified viral particles. As can be seen in Fig. 6(c), TSG101 was found only in JUNV and TCRV purified virions, but not in LCMV and PICV, suggesting specific interaction of this host protein with these two arenaviruses. Involvement of TSG101 in JUNV egression from persistently infected cells was also studied by reduction of the TSG101 level by performing an RNA interference assay (siRNA). As can be seen in Fig. 6(d), the levels of TSG101 were reduced in cells which have been transfected with a dsRNA specific for TSG101 mRNA compared with cells transfected with control siRNA. When we examined infectivity produced in TSG101 siRNA-transfected K3 cells or JUNV-superinfected K3 cells, the ratio of total to extracellular virus fraction produced in these cells was significantly lower than those obtained in control siRNA-transfected cells (Fig. 6e and f). In contrast, a reduction in TSG101 levels in acutely infected BHK-21 cells led to an increase in the fraction of cell-associated infectivity (Fig. 6f).
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). The yield of JUNV-superinfected D10 clone was 2 log lower than that obtained in BHK-21 cells, whereas the virus multiplied equally well in the rest of the clones. Resistance was not associated with the presence of N, as shown for D10 and representative clones B2 and B5, but rather with an increased amount of TSG101 in the former (Fig. 6h).
Furthermore, thin sections of JUNV-infected BHK-21 cultures examined by electron microscopy showed numerous released virions, whereas K3 cells displayed budding particles that, in many cases, formed aberrant tubular structures that resembled interconnected virions. Also, particles that remained tethered to the cell membrane and a few released ones could be observed (Fig. 7).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Iapalucci et al., 1994). After the first year and up to 3 years p.i., low levels of infectivity were recovered from K3 cells, whereas the proportion of DI particles fluctuated randomly, suggesting no correlation between both parameters during this period and indicating a progression to a more stable virus–cell system with no presence of DI particles and no production of infectivity, as described for some non-virogenic persistently JUNV-infected Vero cell lines (Ellenberg et al., 2002). In view of these results, K3 may be classified as a ‘carrier’ culture, characterized by a dynamic equilibrium between virus-infected and uninfected cells, virus release and cell replication (Rima & Duprex, 2005). The low levels of infectivity produced by K3 cells were not increased when the cells were superinfected with JUNV or TCRV, whereas superinfection with LCMV, PICV and the non-related VSV led to a virus production similar to that obtained in normal cells, confirming the specificity of the exclusion phenomenon of superinfecting virus observed for persistently JUNV-infected cultures (Damonte et al., 1983). Superinfection exclusion is observed during infections by a broad range of viruses, including human immunodeficiency virus (HIV) (Michel et al., 2005), VSV (Whitaker-Dowling et al., 1983), vaccinia virus (Christen et al., 1990), alphaviruses (Karpf et al., 1997) and measles virus (Ludlow et al., 2005), and has been ascribed to an impairment of different steps of the virus multiplication cycle. In this system, a slight decrease in adsorbed/penetrated JUNV and TCRV to K3 cells was observed. However, this initial disadvantage for the superinfecting virus in K3 cells might be reverted during the transcription/replication step, considering that antigenomic sense TCRV RNA appeared earlier in K3 cells in comparison with BHK-21 cells. Although superinfection did not increase the amount of cells expressing N, the increment in infectious centres and the formation of virions with TCRV genome and non-neutralizable by anti-JUNV serum allowed us to conclude that superinfecting virus was able to carry on the synthesis of its own proteins to produce particles, but to a lesser extent than in the acute infection.
Impairment at the level of JUNV budding in K3 cells was inferred from the fact that the infectivity produced by these cells, whether superinfected or not, remained mostly cell-associated. Imbalance of the cellular protein TSG101, which is responsible for vesicular cargo protein transport and sorting, is known to disturb HIV budding by altering virus ‘pinching off’ from the cell (Demirov et al., 2002; Goila-Gaur et al., 2003). Although the pattern of expression of TSG101 was similar in K3 and BHK-21 cells, the level of synthesis of the protein was markedly higher in persistently JUNV-infected cells. To note, JUNV acute infection of BHK-21 cells did not modify the amount of TSG101 present in the culture. Thus the augmented level of TSG101 in K3 cells would be a consequence of a mechanism developed during persistence, although not permanent, as suggested by the fact that isolated K3 cell clones susceptible to JUNV multiplication expressed normal TSG101. Taking into consideration that TSG101 interacts either with proline-rich motifs P(T/S)AP type or ASAP motifs (Garrus et al., 2001), and that JUNV and TCRV Z proteins have a PTAP and an ASAP motif, respectively, impairment of virus budding by high levels of TSG101 might contribute to the superinfection exclusion phenomenon through the interaction with Z. On the other hand, LCMV WE strain and PICV Z proteins, which share PPPY motifs similar to VSV M protein (Bouamr et al., 2003; Harty et al., 2000; Irie et al., 2004), might overcome this problem by interacting with TSG101 via a third protein capable of binding both proteins. Inefficient budding and abnormal virus egress as observed by electron microscopy in K3 cells might also be a consequence of altered TSG101 stoichiometry. It has been shown that imbalance of TSG101 may lead to downregulation of several cell receptors (Amit et al., 2004; Lu et al., 2003). Taking into consideration that JUNV employs transferrin receptor 1 to adsorb and penetrate cells (Radoshitzky et al., 2007) and that expression of this receptor is altered by TSG101 imbalance (Babst et al., 2000), the slightly reduced adsorption of superinfecting virus to K3 cells may be also a consequence of TSG101 imbalance in these cells. These findings indicate both viral (restricted protein synthesis) and cellular (TSG101 imbalance) factors as participants in JUNV exclusion in K3 cells.
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ACKNOWLEDGEMENTS |
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Received 28 March 2007;
accepted 24 June 2007.
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) and K3 (
) cells were infected with JUNV (a) or LCMV (b) at an m.o.i. of 5 p.f.u. per cell and infectivity adsorbed at different times p.i. at 4 °C was determined. Alternatively, cells were incubated with 35S-labelled JUNV at an m.o.i. of 1 p.f.u. per cell and cell-associated radioactivity was measured after different times at 4 °C (c) or 37 °C (d). Inset figures represent the ratio of values obtained for BHK-21 to K3 cells. K3 cells were employed at 835 days p.i.
