Роль белка pif1 оболочки вируса ядерного полиэдроза Spodoptera littoralis в процессе проникновения вируса в клетку

Исследовали роль pif1 – белка оболочки вируса ядерного полиэдроза S. littoralis в процессе проникновения вируса в колончатую клетку кишечника насекомого. Показано, что делетирование pif1 из вирусного генома не блокирует процесс слияния вирусной и клеточной мембран, детектируемого методом восстановле...

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Datum:	2007
Hauptverfasser:	Ревякина, О.Ю., Кихно, И.М.
Format:	Artikel
Sprache:	Russian
Veröffentlicht:	Інститут молекулярної біології і генетики НАН України 2007
Schriftenreihe:	Біополімери і клітина
Schlagworte:	Віруси та клітина
Online Zugang:	https://nasplib.isofts.kiev.ua/handle/123456789/157517
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Zitieren:	Роль белка pif1 оболочки вируса ядерного полиэдроза Spodoptera littoralis в процессе проникновения вируса в клетку / О.Ю. Ревякина, И.М. Кихно // Біополімери і клітина. — 2007. — Т. 23, № 5. — С. 425-432. — Бібліогр.: 25 назв. — рос., англ.

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spelling	nasplib_isofts_kiev_ua-123456789-1575172025-02-23T18:16:53Z Роль белка pif1 оболочки вируса ядерного полиэдроза Spodoptera littoralis в процессе проникновения вируса в клетку A role of Spodoptera littoralis nuclear polyhedrosis virus envelope protein pif 1 in the process of primary infection of insect midgut Роль білкa pif1 оболонки вірусy ядерного поліедрозу Spodoptera littoralis y процесі первинного інфікування клітин кишечника комахи Ревякина, О.Ю. Кихно, И.М. Віруси та клітина Исследовали роль pif1 – белка оболочки вируса ядерного полиэдроза S. littoralis в процессе проникновения вируса в колончатую клетку кишечника насекомого. Показано, что делетирование pif1 из вирусного генома не блокирует процесс слияния вирусной и клеточной мембран, детектируемого методом восстановления флуоресценции октадецил-родамин-В-хлорида. С привлечением техники ОТ-ПЦР показано, что функцией pif1 является обслуживание одного или нескольких этапов жизненного цикла вируса, предшествующих проникновению вирусной ДНК в ядро, предположительно таких, как интернализация нуклеокапсида, его транспортировка, освобождение вирусной ДНК из капсида и ее внедрение в ядро. Досліджували роль pif1 – білка оболонки вірусу ядерного поліедрозу S. littoralis у процесі проникнення вірусу в колончасту клітину кишечника комахи. Показано, що делетування pif1 з вірусного геному не блокує процесу злиття вірусної і клітинної мембран, що детектується за допомогою методу відновлення флуоресценції октадецил-родамін-В-хлориду. Із застосуванням техніки ЗT-ПЛР показано, що функцією pif1 є обслуговування одного або кількох етапів життєвого циклу вірусу, які передують проникненню вірусної ДНК в ядро, прогнозовано таких, як інтерналізація нуклеокапсиду, його транспорт, вивільнення вірусної ДНК з капсиду і її входження у ядро. A role of the envelope protein pif 1 of Spodoptera littoralis nuclear polyhedrosis virus in the process of viral penetration into the midgut columnar cell of the insect has been investigated. It has been shown that pif1 deletion from viral genome does not block viral and cell membrane fusion detected by octadecyl- rhodamin-B-chloride fluorescence dequenching assays. The use of RT PCR techniques allowed to demonstrate that pif1 was predicted to be an executor of some step(s) of viral life cycle upstream of viral DNA entry into the nucleus, presumably such as nucleocapsid internalization, nucleocapsid transport, viral DNA release from capsid and its penetration into the nucleus. 2007 Article Роль белка pif1 оболочки вируса ядерного полиэдроза Spodoptera littoralis в процессе проникновения вируса в клетку / О.Ю. Ревякина, И.М. Кихно // Біополімери і клітина. — 2007. — Т. 23, № 5. — С. 425-432. — Бібліогр.: 25 назв. — рос., англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.00077D https://nasplib.isofts.kiev.ua/handle/123456789/157517 578.841:578.23.3 ru Біополімери і клітина application/pdf application/pdf Інститут молекулярної біології і генетики НАН України
institution	Digital Library of Periodicals of National Academy of Sciences of Ukraine
collection	DSpace DC
language	Russian
topic	Віруси та клітина Віруси та клітина
spellingShingle	Віруси та клітина Віруси та клітина Ревякина, О.Ю. Кихно, И.М. Роль белка pif1 оболочки вируса ядерного полиэдроза Spodoptera littoralis в процессе проникновения вируса в клетку Біополімери і клітина
description	Исследовали роль pif1 – белка оболочки вируса ядерного полиэдроза S. littoralis в процессе проникновения вируса в колончатую клетку кишечника насекомого. Показано, что делетирование pif1 из вирусного генома не блокирует процесс слияния вирусной и клеточной мембран, детектируемого методом восстановления флуоресценции октадецил-родамин-В-хлорида. С привлечением техники ОТ-ПЦР показано, что функцией pif1 является обслуживание одного или нескольких этапов жизненного цикла вируса, предшествующих проникновению вирусной ДНК в ядро, предположительно таких, как интернализация нуклеокапсида, его транспортировка, освобождение вирусной ДНК из капсида и ее внедрение в ядро.
format	Article
author	Ревякина, О.Ю. Кихно, И.М.
author_facet	Ревякина, О.Ю. Кихно, И.М.
author_sort	Ревякина, О.Ю.
title	Роль белка pif1 оболочки вируса ядерного полиэдроза Spodoptera littoralis в процессе проникновения вируса в клетку
title_short	Роль белка pif1 оболочки вируса ядерного полиэдроза Spodoptera littoralis в процессе проникновения вируса в клетку
title_full	Роль белка pif1 оболочки вируса ядерного полиэдроза Spodoptera littoralis в процессе проникновения вируса в клетку
title_fullStr	Роль белка pif1 оболочки вируса ядерного полиэдроза Spodoptera littoralis в процессе проникновения вируса в клетку
title_full_unstemmed	Роль белка pif1 оболочки вируса ядерного полиэдроза Spodoptera littoralis в процессе проникновения вируса в клетку
title_sort	роль белка pif1 оболочки вируса ядерного полиэдроза spodoptera littoralis в процессе проникновения вируса в клетку
publisher	Інститут молекулярної біології і генетики НАН України
publishDate	2007
topic_facet	Віруси та клітина
url	https://nasplib.isofts.kiev.ua/handle/123456789/157517
citation_txt	Роль белка pif1 оболочки вируса ядерного полиэдроза Spodoptera littoralis в процессе проникновения вируса в клетку / О.Ю. Ревякина, И.М. Кихно // Біополімери і клітина. — 2007. — Т. 23, № 5. — С. 425-432. — Бібліогр.: 25 назв. — рос., англ.
series	Біополімери і клітина
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first_indexed	2025-11-24T06:36:18Z
last_indexed	2025-11-24T06:36:18Z
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fulltext	VIRUSES AND CELL A role of Spodoptera littoralis nu clear polyhedrosis vi rus en ve lope pro tein pif1 in the pro cess of pri mary in fec tion of in sect midgut O. Yu. Reviakina, I. M. Kikhno In sti tute of mo lec u lar bi ol ogy and ge net ics NAS of Ukraine Academicain Zabolotnog str., 150, Kyiv, 03680 Ukraine i.m.kikhno@imbg.org.ua A role of the en ve lope pro tein pif1 of Spodoptera littoralis nu clear polyhedrosis vi rus in the pro cess of vi ral pen e tra tion into the midgut co lum nar cell of the in sect has been in ves ti gated. It has been shown that pif1 de - le tion from vi ral ge nome does not block vi ral and cell mem brane fu sion de tected by octadecyl-rhodamin-B-chlo ride flu o res cence dequenching as says. The use of RT PCR tech niques al lowed dem on strat ing that pif1 was pre dicted to be an ex ec u tor of some step(s) of vi ral life cy cle up stream of vi ral DNA en try into the nu cleus, pre sum ably such as nucleocapsid in ter nal iza tion, nucleocapsid trans port, vi ral DNA re lease from capsid and its pen e tra tion into the nu cleus. Keywords: vi rus en try into the cell, baculovirus, pif1 In tro duc tion. Rep re sen ta tives of dif fer ent vi ral groups have com mon ways of en try into the cell. In par tic u lar, en vel oped vi ruses pen e trate into the cell by means of vi ral and cell mem branes fu sion [1]. This pro cess is main tained by at tach ment and fu sion pro - teins, which are the com po nents of vi ral en ve lope. Al - though fu sion pro teins of dif fer ent vi ral groups are known to be non-ho mol o gous, their struc tural changes in the pro cess of mem brane fu sion have com - mon fea tures, which al lows sup pos ing the com mon con ser va tive model of the pro cess. The de scrip tion of this model has been the ob ject of nu mer ous re views in the re cent years [2]. It should be noted that the ma jor part of data on the mech a nism of fu sion has been ob tained while study - ing small en vel oped RNA-con tain ing vi ruses, spe cific for the pres ence of one fu sion pro tein, which is suf fi - cient for main tain ing all stages of the pro cess. Large en vel oped DNA vi ruses are dif fer ent from small RNA vi ruses in their or i gin and evo lu tion his tory [3]. The pro cess of their en try into the cell is barely stud ied due to its com pli cated na ture, al though the in volve ment of the com plex of vi ral pro teins into the mem brane fu - sion al lows sup pos ing that there may be dis agree ment 425 ISSN 0233-7657. Biopolymers and cell. 2007. vol. 23. N 5. Translated from Ukrainian. Ó O. YU. REVIAKINA, I. M. KIKHNO, 2007 be tween the mech a nism of this pro cess and gen eral par a digm. Baculoviruses, in sect patho gens, are not in fec tious for mam mals, which makes them a con ve nient model to study the pro cess of pen e tra tion of large DNA vi ruses into the cell. Baculoviruses are spe cific for lon ger evo - lu tion ary his tory, com pared to ver te brate vi ruses, and thus, the pro cess of their pen e tra tion into the cell is of great in ter est due to its ar chaic char ac ter. Be sides, some spe cific fea tures of baculoviruses al low ex pect ing spe cial ways of en try into the cell. One of the unique fea tures of baculoviruses is the ex is tence of two forms of vi rus par ti cles, formed at dif fer ent stages of life cy cle and dif fer ent in or i gin, mem brane con tents, tro pism to dif fer ent cells, and ways of en try into the cell. Bud ded vi rus (BV) par ti cles are formed at the early stages of vi rus life cy cle. BV pen e trates into the cell via endocytosis [4]. En ve lope pro tein gp64, which pro vides at tach ment and fu sion of BV with endosomal mem brane, is spe cific for fea tures of stan - dard fu sion pro tein. The pro cess, main tained by this pro tein, has com mon fea tures with that of other vi - ruses [5], which sup poses its cor re spon dence to the com mon model. BV par ti cles are re spon si ble for sys - temic in fec tion of all or gans and tis sues of the in sect. Poly he dra de rived vi rus (PDV) par ti cles are formed at the fi nal stages of in fec tion. The name PDV in di cates the in clu sion of the par ti cles into the pro tein cap sule called poly he dra. PDV nucleocapsids are re - spon si ble for pri mary in fec tion of midgut co lum nar cells. They pen e trate into the cy to plasm via di rect fu - sion of vi ral en ve lopes and mem branes of co lum nar cell microvilli [6]. Nei ther the mech a nism of the pro - cess is known, nor PDV fu sion pro tein is iden ti fied. PDV of Autographa californica (Ac) nu clear polyhedrosis vi rus (NPV), the most stud ied rep re sen - ta tive of baculoviruses, in cludes circa 44 pro teins, en - coded by the vi rus [7]. At least 4 of them were shown to be ab so lutely re quired in the pro cess of nucleocapsid en try into the cell – 3 per os infectivity fac tors – pif1 [8], pif2 [9], pif3 [10], and p74 [11]. De - le tion of genes of the men tioned pro teins re sults in the loss of PDV infectivity. Genes of these pro teins are pres ent in genomes of all (over 30) se quenced baculoviruses, which pre sup poses the con ser va tive mech a nism of PDV pen e tra tion into the cell. pif3 was de ter mined not to par tic i pate in the pro cesses of at - tach ment and fu sion, its role re mains un clear, whereas pif1, pif2, p74 pro mote at tach ment [10, 12]. Au thors of the men tioned works showed that at tach ment ef fi - ciency of these genes de le tion mu tants is 3-fold lower than that of wild type vi rus. At the same time the at - tached vi rus tends to fu sion with microvillar mem - branes, which is de tected by the octadecyl-rhodamine-B-chlo ride (R18) flu o res cence dequenching as say, which in au thor’s opin ion al lows ex clud ing the pos si bil ity of di rect in volve ment of these pro teins into the fu sion pro cess. It is yet to be dis cov ered why at tach ment and fu sion of 1/3 of vi ral par ti cles does not re sult in pro duc tive in fec tion. Cur rent work is ded i cated to fur ther func tional in - ves ti ga tion of one of the fac tors of Spodoptera littoralis (Spli) PDV infectivity, namely pif1. Fu sion ca pac ity of pif1 de le tion mu tant has been in ves ti gated in in vi tro ex per i ments. The ap pli ca tion of PCR method at tempted to an swer the ques tion – which of the stages of vi ral cy cle is blocked by the ab sence of this gene prod uct? Ma te ri als and Meth ods. Vi ruses and lar vae. Wild-type Spli NPV strain M2 and ear lier ob tained on its ba sis plaque iso late Spli D4 with de le tion of pif1 were used [8]. Spli lar vae of dif fer ent age, grown and ar ti fi cially fed in insectarium were used for ex per i - ments. Lar vae were in fected by feed ing them with poly he dra sus pen sion or pu ri fied PDV in 10% su crose. Pu ri fi ca tion of poly he dra and PDV par ti cles. Poly he dra and PDV par ti cles were pu ri fied ac cord ing to the pro ce dure, de scribed by Braunagel et al. [13]. 40 g of dead lar vae were ho mogen ised in 100 ml of 0.1% SDS so lu tion, ho mog e nate was fil tered sev eral times through the cot ton tis sue. Poly he dra were pre - cip i tated by centrifugation at 6 000 rpm for 10 min. Pel let was re sus pend ed in 0.15 M NaCl so lu tion and cen tri fuged once more at the same con di tions. Pel let was re sus pend ed in wa ter and then lay ered onto 50–60% su crose–TE gra di ent. Af ter 1-hour centrifugation at 25 000 rpm, poly he dra, lo cated on interphase, were col lected. The sus pen sion was di - luted sev eral times with wa ter and poly he dra were pelleted at 10 000 rpm for 20 min. Ob tained pel let was re sus pend ed in wa ter to fi nal con cen tra tion of 5·109 poly he dra per 1 ml. REVIAKINA O. YU., KIKHNO I. M. 426 PDV were re leased from poly he dra by al ka line treat ment. 900 ml of sus pen sion were mixed with 300 ml of 0.2 M Na2CO3 and in cu bated for 15 min at room tem per a ture. Sus pen sion was neu tral ised with 0.1 M of tris-HCl, pH 7.4. In sol u ble poly he dra were elim i - nated by centrifugation at 5 000 rpm for 5 min per - formed in Eppendorf cen tri fuge. Supernatant was lay - ered onto 20–60% su crose–TE gra di ent. Af ter 1 hour centrifugation at 90 000 g a band, con tain ing PDV par - ti cles, at the interphase was re moved. PDV sus pen - sion was di luted 3–4 times with wa ter and vi ral par ti - cles were pelleted at 90 000 g for 30 min. The pel let was care fully re sus pend ed in cold PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1 mM KH2O4, pH 7.4). PDV quan tity was cal cu lated us ing Volkman cri te - ria [14], which states that 1 mg of vi ral pro teins cor re - sponds to 1.8·109 PDV. PDV pro teins con cen tra tion was de ter mined us ing BSA as say kit (Pierse, USA) ac cord ing to the man u fac turer’s in struc tion. Flu o res cent la bel ling of PDV par ti cles. Flu o res - cent la bel R18 was in tro duced into mem brane ac cord - ing to the method de scribed in [15]. 7 ml of R18 eth a - nol so lu tion (1 mg/ml) were added to 500 ml of PDV sus pen sion, con tain ing 1 mg/ml of vi ral pro teins. The sam ple was in cu bated in dark room at room tem per a - ture for 1 hour, di luted 3 times with PBS buffer and pelleted in Eppendorf cen tri fuge at the max i mal speed for 30 min at 4°C. PDV were washed twice in three vol umes of PBS, pel let of la belled PDV was sus - pended in 500 ml of PBS. Mem brane ves i cle iso la tion from in sect midgut cells. Midguts of 7-day-old lar vae were iso lated us ing microsurgical tools and mag ni fy ing glass, and then treated ac cord ing to the method, de scribed in [16]. 500 mg of midgut tis sue prep a ra tion were ho mogen - ised on ice in 200 ml of buffer A (300 mM mannitol, 5 mM EDTA, 17 mM tris-HCl, pH 7.5) with ad di tion of pro te ase in hib i tor cock tail (Promega, USA) in Eppendorf vial us ing pes tle and then nee dles of dif fer - ent di am e ter (23–26 G). Then 4.5 ml of buffer A with pro te ase in hib i tors and 4.5 ml of 24 mM MgCl2 were added with sub se quent in cu ba tion of ho mog e nate on ice for 15 min. Ho mog e nate was cen tri fuged at 5 000 rpm for 15 min at 4°C. Supernatant was re moved and cen tri fuged at 19 000 rpm for 30 min at 4°C. The pel - let was re sus pend ed in the same vol ume of buffer A and MgCl2 and then the pro ce dure of high-speed centrifugation was re peated. Ob tained pel let of mem - brane ves i cles was re sus pend ed in small vol ume of PBS buffer. Mon i tor ing of mem brane fu sion. Fu sion of vi ral and cell mem branes was reg is tered as ear lier de scribed for Ac NPV [17], ac cord ing to the method, based on flu o res cence dequenching as say of R18, in cor po rated into vi ral mem brane in the con cen tra tion lead ing to “self-quench ing” of flu o res cence [18]. 10 ml of mem - brane ves i cle sus pen sion (1 mg/ml) were mixed with dif fer ent amounts of M2 or D4 PDV (from 1·109 to 1·1010), sam ple vol umes were ad justed to 200 ml by PBS buffer. The sam ples were in cu bated in dark room at 27°C for 4 hours, with pe ri odic shak ing. Then each sam ple vol ume was ad justed to 1 ml by PBS buffer and in ten sity of flu o res cence was mea sured us ing Cary eclipse spectrofluorimeter (Varian, USA), ex ci - ta tion wave length 560 nm and emis sion wave length 590 nm. In ten sity of flu o res cence for each sam ple was mea sured one more time af ter ad di tion of tri ton X-100 to fi nal con cen tra tion of 1%. Flu o res cence dequenching per cent age was cal cu lated us ing stan - dard for mula: dequenching % = 100·(Fs – F0) / (Ft – F0), where Fs – flu o res cence in ten sity of sam ple; F0 – flu o res cence in ten sity of la beled PDV; Ft – flu o res - cence in ten sity of sam ple af ter ad di tion of tri ton X-100. Iso la tion of RNA from midgut cells. Midgut tis sue cells, iso lated from in fected lar vae, were used as the source RNA. RNAs were iso lated us ing RNA easy Pro tect mini kit (Qiagen, USA) ac cord ing to the man - u fac turer’s in struc tion. Am pli fi ca tion of egt gene re gion us ing RT-PCR. RNA prep a ra tions, iso lated from midguts of in fected lar vae, were used to ob tain cDNA gene cop ies us ing re verse tran scrip tase. The fol low ing prim ers, ho mol o - gous to N-ter mi nal re gion of Spli NPV egt gene, were used in PCR: di rect – TCACAGTCTGGTGGT and re - verse –CGCTGAGACCTTTGA. Re ac tions of re - verse tran scrip tion and am pli fi ca tion were car ried out us ing One Step RT-PCR kit (Qiagen) in ac cor dance to the man u fac turer’s in struc tions. A ROLE OF SPODOPTERA LITTORALIS NU CLEAR POLYHEDROSIS VI RUS EN VE LOPE PRO TEIN PIF1 427 Re sults and Dis cus sion. In ves ti ga tion of fu sion ki net ics of Spli M2 and D4 PDV with mem branes of midgut co lum nar cells. Flu o res cence dequenching as - say of R18 is widely used in study ing of mem brane fu - sion. This as say is based on the fact that in cor po ra tion of fluorochrome mol e cules R18 into mem brane lipid bilayer in cer tain con cen tra tion re sults in the ef fect of flu o res cence “self-quench ing”, caused by the in ter ac - tion of neigh bour ing mol e cules. The fu sion of la - belled and non-la belled mem branes leads to re dis tri - bu tion of R18 be tween do nor and re cip i ent mem - branes, which re sults in “di lu tion” of the la bel, de crease in its con cen tra tion and, con se quently, in flu - o res cence in ten sity de crease. Flu o res cence dequenching, reg is tered by spectrofluorimeter, was used to study the fu sion ki net - ics of la belled PDV par ti cles with mem brane ves i cles, iso lated from in sect midgut cells. Dif fer ent amounts of R18-la belled vi ral par ti cles were in cu bated in the mix ture with 10 µg of mem brane ves i cles for 4 hours at 27°C. The in crease in PDV M2 amount from 1•109 to 8•109 was ac com pa nied by pro - por tional in crease in mem brane fu sion per cent age, de - tected as in crease in flu o res cence dequenching from 11 to 88% re spec tively (Fig ure 1). Fur ther in crease in PDV amount to 1•1010 re sults in in sig nif i cant pla teau in crease of flu o res cence dequenching to 95%, which ev i dences to sat u ra tion. Sim i lar ten dency to in crease in flu o res cence dequenching due to the in crease in vi - ral par ti cle amount has been ob served for PDV D4 as well, which con firms the fact of mem brane fu sion of de le tion mu tant en ve lope with mem brane ves i cles. At the same time it took al most 2.5 times more vi ral par ti - cles, com par ing to wild type vi rus, to achieve the same level of flu o res cence dequenching. As it has been men tioned above, in ves ti ga tion of pif1 on mod els of Spli NPV and Ac NPV re vealed phenotypic man i fes ta - tion of these genes to be equal in both vi ruses, i.e. their de le tion re sults in the loss of in fec tion ca pa bil ity by PDV [8, 10]. Cur rent pa per re veals that de le tion of this gene does not im pede the fu sion of Spli NVP and cell mem - brane, which is in good cor re la tion with the data on Ac NVP [10]. The au thors of the lat ter work showed that flu o res cence dequenching per cent age dur ing fu sion of de le tion mu tant Ac NVP with cell mem branes is re - duced three times, com pared to wild type vi rus. This fact is ex plained by three time de crease in num ber of mu tant vi ral par ti cles, at tached to the microvilli. We have not stud ied ef fi ciency of at tach ment of Spli to mem branes, as it has been per formed for Ac NVP, yet it is pos si ble to sup pose that fu sion ef fi ciency of D4 PDV, which is 2.4 times lower com pared to M2, can be con sid ered as the re sult of de creased ef fi ciency of this vi rus at tach ment. There fore, Spli NVP pif1can be con sid ered as func tional an a logue of pif1 Ac NVP, REVIAKINA O. YU., KIKHNO I. M. 428 Fig.1 De pend ence of flu o res cence dequenching per cent age on the amounts of PDV par ti cles: 1 – M2 PDV; 2 – D4 PDV. In creas ing num ber of la beled R18 PDV M2 or PDV D4 was mixed with 10 mg of mem brane ves i cles and incubated for 4 hours at 27°C. namely at tach ment fac tor. It is also ev i denced in di - rectly by ab so lute con ser va tism of four PDV en try pro teins, which pre sup poses sin gu lar mech a nism of pen e tra tion, as the com mon mech a nism re quires func - tional con ser va tism of its per form ers. In ves ti ga tion of PDV DNA pres ence in the nu clei of in fected midgut cells. As de le tion mu tants, not so ef fi ciently, yet were at tached and fused with the cell mem branes, while in fec tion was blocked, it can be sup posed that, along with the func tion of at tach ment, pif1 par tic i pates in the ini ti a tion of other pro cesses of vi ral life cy cle. It is sup posed the o ret i cally that the func tion of pif1 is the main te nance of some stages of in fec tion, fol low ing the fu sion of mem branes, namely, pen e tra tion of nucleocapsid into cy to plasm of microvillus, trans port to the nu cleus, over com ing of vi ral DNA bar rier of nu clear mem brane, DNA rep li ca - tion, tran scrip tion and trans la tion of vi ral genes, for - ma tion of nucleocapsid, and with drawal of nucleocapsids from the cell. Par tic i pa tion of pif1 in the fol low ing events is un likely, as BV par ti cles with de leted pif1, in jected into the in sect haemolimph, re - sult in nor mal in fec tion pro cess [8]. The facts that pro mot ers of early baculoviral genes are rec og nized by en zy matic sys tems of host cells and that tran scrip tion of these genes is per formed by host DNA-poly mer ase are com monly known. Early pro - mot ers func tion af ter transfection in the in sect cell and do not re quire any prod ucts of vi ral genes for main - tain ing their ac tiv ity [19, 20]. On the ba sis of the abovementioned data on specificities of early baculoviral pro mot ers, we put for ward the sup po si tion that the ab sence of tran scripts of early baculoviral gene in the nu cleus of midgut cells may tes tify to the ab sence of vi ral DNA in the nu cleus. To ver ify the pres ence of vi ral DNA in the nu cleus of in fected cell we car ried out the fol low ing ex per i - ment: 6-day-old Spli NPV lar vae were in fected by feed ing with high con cen trated sus pen sion of Spli NPV M2 or D4 poly he dra (3·109 poly he dra in 1 ml). In 7 hours af ter in fec tion, in sects were de cap i tated, midguts were re moved and re leased from their con - tents. To tal RNA was iso lated from midguts. cDNA pool of ma trix RNA has been ob tained us ing re verse tran scrip tase on the ba sis of these sam ples. Re ac tion of am pli fi ca tion us ing the prim ers, ho mol o gous to N-ter mi nal re gion of early egt gene, was used as the ev i dence of pres ence/ab sence of cDNA gene copy in the in ves ti gated sam ples. Pos i tive re sult was reg is - tered dur ing re ac tion of am pli fi ca tion us ing cDNA sam ples of in sects, in fected with M2 (Fig ure 2). Re it - er ated PCR us ing D4-in fected in sects cDNAs re - vealed the ab sence of egt RNA in them. The re sults ob tained re veal that DNA vi rus with de leted pif1 gene does not reach the nu cleus. Thus, it is pos si ble to con - clude that func tion of pif1 is to main tain the stages of in fec tion, pre ced ing the vi ral DNA en try into the nu - cleus, sup pos edly nucleocapsid nto cy to plasm of microvillus, trans port to the nu cleus or vi ral DNA pen e tra tion into the nu cleus. As pif1 is known to be long to en ve lope pro teins, and not a nucleocapsid com po nent, it is hard to sup - pose its di rect par tic i pa tion in trans port or re lease of DNA. In this case it can be sup posed that pif1, at - tached to spe cific re cep tors, is ca pa ble of switch ing cell sig nal ing path ways, pro vid ing the ex e cu tion of given func tions [10]. This hy poth e sis is sup ported by Okhawa et al., who point out the fact that amino acid se quence of Ac NVP pif1 con tains RGD-mo tif, spe - cific to pro tein-lig ands of integrins (as integrins are the transductors of extracellular sig nals). Not dis put - ing with the pos si bil ity of sig nal transduction in the cell via pif1, we would like to pay spe cial at ten tion to the pres ence of mo tif in amino acid se quence as nec es - sary but in suf fi cient fac tor to judge about the func tion - al ity of this mo tif. Ad di tional con fir ma tion to func - tional sig nif i cance of the mo tif is its con ser va tion in pro tein homo logues. At the mo ment, there are pif1 aminoacid se quences of over 30 baculoviruses, avail - able in the Gene Bank da ta base. Their anal y sis re - vealed the ab sence of con ser va tive RGD-mo tif. Amino acid se quence of Spli NVP pif1does not con - tain this mo tif at all, which casts doubts on its func - tional sig nif i cance in Ac NVP as well. Con tin u ing the dis cus sion with Okhawa et al. we would like to un der - line the in abil ity of cross ing out of fu sion func tion from the list of pos si ble pif1 func tions. We found it pos si ble to sup pose that pif1 may be di rect per former of the mem brane fu sion pro cess. Mod ern fu sion par a - digm, ap pli ca ble for both ar ti fi cial lipid bilayers and pro tein-con tain ing bi o log i cal mem branes, sug gests the pres ence of sev eral stages of this pro cess [21]. A ROLE OF SPODOPTERA LITTORALIS NU CLEAR POLYHEDROSIS VI RUS EN VE LOPE PRO TEIN PIF1 429 One of the im por tant stages of the pro cess is the state of semi-fu sion, which is char ac ter ized by mix ing of ex ter nal lay ers of re cip i ent and tar get mem branes, whereas in ter nal lay ers re main in tact. The sub se quent state of in ter nal lay ers mix ing re sults in com plete fu - sion, fu sion pore for ma tion and mix ing of wa ter con - tents of two com part ments, up to that mo ment sep a - rated by mem branes. It is also im por tant that flu o res - cence dequenching of R18 al lows de tect ing dis tri bu tion of fluorochrome be tween lipids of do nor and re cip i ent mem branes, at the same time it is im pos - si ble to de ter mine whether com plete or in com plete mem brane fu sion re flects this pro cess. To dif fer en ti - ate these stages, dis tri bu tion anal y sis of their wa - ter-sol u ble com po nents is used along with study on dis tri bu tion of liposoluble com po nents [22]. In the frames of cur rent model of fu sion the pres ence of one fu sion pro tein is suf fi cient for main tain ing of all stages of pro cess, thus, pif1 can not be con sid ered as a can di date for the role of fu sion pro tein. How ever, the lat est data, ob tained in the in ves ti ga tion of her pes vi - ruses, al low sup pos ing that the me chan ics of fu sion in com plex DNA-con tain ing vi ruses may be dif fer ent from the de scribed one. In her pes vi ruses fu sion is pro vided by the com - plex of four con ser va tive pro teins, namely gD, gH, gL, and gB, act ing con sec u tively, i.e. gD is at tach ment pro tein, gHL com plex pro vides semi-fu sion, and gB com pletes the pro cess of fu sion, which re sults in the pore for ma tion [23]. Sim i larly to her pes vi ruses it is pos si ble to sup pose that in com plete fu sion may be the ob sta cle for the pen e tra tion of nucleocapsid into the cell, and pif1 is the per former, or one of the per form - ers, of the pro cess of com plete fu sion. It is worth men - tion ing that though fu sion of her pes vi ruses is per - formed by a harder scheme, gen eral fea tures of pro - teins of the com plex are the same as for all fu sion pro teins. One of these fea tures is the pres ence of glycosylation. Un like all other known fu sion pro teins, pif1 is non-glycosylated [8], which casts doubts on its func tion as fu sion pro tein. How ever, there are ev i - dences that glycoproteins are not in volved into the pro cess of PDV fu sion. The treat ment of in fected cells with tunicamycine usu ally re sults in the ab sence or sig nif i cant de crease in infectivity of vi rus, ob tained from these cells, as tunicamycine in ter feres with the pro cess of glycosylation of fu sion pro teins. Whereas it is jus ti - fied for BV [24], treat ment with tunicamycine does not in flu ence infectivity of PDV [25]. More over, some de tails of PDV fu sion pro cess are dif fer ent from those of other vi ruses. For all in ves ti gated vi ruses di - rect fu sion of vi ral and cell mem branes is pH-in de - pend ent. Ef fi ciency of PDV fu sion in creases with in - creas ing pH value, reach ing its max i mum at pH 9–11 [17]. For all known vi ruses the de crease of tem per a - ture to 4°C is com monly ac cepted method of fu sion block ing, yet such a tem per a ture is per mis sive for PDV fu sion with microvillus mem branes [17]. The abovementioned facts ev i dence to the fact that the study on PDV pen e tra tion into the cell will al low dis cov er ing the un known mech a nisms of this pro cess, main tained by the com plex of new type of fu sion pro - tein(s). Fur ther in ves ti ga tion on pro cess of PDV in fec tion by elec tron mi cros copy as well as mon i tor ing of de - gree of mem brane fu sion us ing wa ter-sol u ble dyes will en able to ei ther con firm or re ject the hy poth e sis on pif1 be long ing to new type of fu sion pro teins. REVIAKINA O. YU., KIKHNO I. M. 430 Fig.2 Elec tro pho retic anal y sis of prod ucts of am pli fi ca tion of cDNA cop ies of RNA, iso lated from midguts of in fected lar vae: 1 – marker 1 kb (Fermentas); 2 – pos i tive con trol (am pli fi ca tion of pu - ri fied DNA Spli M2); 3, 4 – in sects, in fected with PDV M2; 5, 6 – in - sects, in fected with PDVD4. Î. Þ. Ðåâÿêèíà, È. Ì. Êèõíî Ðîëü áåëêà pif1 îáîëî÷êè âèðóñà ÿäåðíîãî ïîëèýäðîçà Spodoptera littoralis â ïðîöåññå ïðîíèêíîâåíèÿ âèðóñà â êëåòêó Ðåçþìå Èññëåäîâàëè ðîëü pif1 – áåëêà îáîëî÷êè âèðóñà ÿäåðíîãî ïîëèýäðîçà S. littoralis â ïðîöåññå ïðîíèêíîâåíèÿ âèðóñà â êîëîí÷àòóþ êëåòêó êèøå÷íèêà íàñåêîìîãî. Ïîêàçàíî, ÷òî äåëåòèðîâàíèå pif1 èç âèðóñíîãî ãåíîìà íå áëîêèðóåò ïðîöåññ ñëèÿíèÿ âèðóñíîé è êëåòî÷íîé ìåìáðàí, äåòåêòèðóåìîãî ìåòîäîì âîññòàíîâëåíèÿ ôëóîðåñöåíöèè îêòàäåöèë-ðîäàìèí-Â-õëîðèäà. Ñ ïðèâëå÷åíèåì òåõíèêè ÎÒ-ÏÖÐ ïîêàçàíî, ÷òî ôóíêöèåé pif1 ÿâëÿåòñÿ îáñëóæèâàíèå îäíîãî èëè íåñêîëüêèõ ýòàïîâ æèçíåííîãî öèêëà âèðóñà, ïðåäøåñòâóþùèõ ïðîíèêíîâåíèþ âèðóñíîé ÄÍÊ â ÿäðî, ïðåäïîëîæèòåëüíî òàêèõ, êàê èíòåðíàëèçàöèÿ íóêëåîêàïñèäà, åãî òðàíñïîðòèðîâêà, îñâîáîæäåíèå âèðóñíîé ÄÍÊ èç êàïñèäà è åå âíåäðåíèå â ÿäðî. Êëþ÷åâûå ñëîâà: ïðîíèêíîâåíèå âèðóñà â êëåòêó, áàêóëîâèðóñ, pif1. REFERENCES 1. Hernandez L. D., Hoffman L. R., Wolfsberg T. G., White J. M. Virus-cell and cell-cell fusion // Ann. Rev. Cell Develop. 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Роль белка pif1 оболочки вируса ядерного полиэдроза Spodoptera littoralis в процессе проникновения вируса в клетку

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