AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 7, Number 3, 1991 Mary Ann Liebert, Inc., Publishers
pi 7 and pl7-Containing gag Precursors of Input Human Immunodeficiency Virus Are Transported into the Nuclei of Infected Cells
NATHALIE SHAROVA and ALICE BUKRINSKAYA
ABSTRACT Subcellular localization of input human immunodeficiency virus type 1 (HIV-1) gag proteins was determined in infected H9 and Jurkat tat cells. Infected cells were fractionated at intervals, and the proteins in cell fraction were identified by immunoblotting using pooled sera from acquired immunodeficiency syndrome (AIDS) patients and monoclonal antibodies. Cycloheximide was added at 0 time to prove that the proteins detected were not nascent ones. Gag proteins p55, p41. p39 (in the most essential relative concentrations), and p 17 were found in the cell nuclei for at least 4 h after infection. However, p24 was not found in the cell nuclei and was accumulated in the nuclear washing buffer. The data presented confirm the presence of karyotypic signal at the N terminus of p55 gag precursor. The potential role of nuclear localization of gag precursor is discussed.
INTRODUCTION
of virus
particles. If this is true for HIV, the gag proteins are expected to be transported into cell nuclei. In fact, recombinant gag proteins of simian immunodeficient virus (SIV) and HIV-1 expressed in insect cells were detected in cell nuclei.7,8
THE(HIV-l)p24, pl7, p7,
gag proteins OF human immunodeficiency virus type 1
and p6 are the products of proteolytic of the gag precursor processing p55. The polyprotein is myristoylated at the amino terminus and targeted to the plasma membrane of infected cells where assembly and budding of virus particles occurs. '~3 Uncleaved precursor is included in the virus particle and is processed within it by a virally encoded aspartic protease; cleavage is performed through intermediate precursor proteins.1,4 Recently three intermediate products of cleavage have been described, two of them, p41a and p39, from the N terminus of p55 and p41b from the C terminus.5 When the gag precursor expressed by the recombinant vaccinia virus was processed by the recombinant bacterial protease, the intermediate precursor p39 appeared within 30 min after mixing of two cellular lysates while p24 and pl7 were not revealed until 2 h or
later.6
The fate of parental gag proteins in infected cells is unknown. By analogy with the other envelope viruses, these proteins could play an important role in the replicative cycle, being involved into transcriptive complexes and in early steps of morphogenesis
METHODS The aim of this study was to determine the subcellular localization of HIV-1 gag proteins of input HIV-1 virus in two kinds of cells, H9 and Jurkat tat. HIV-1 produced by H9/IIIB cells was pelleted through 30% glycerol from culture fluid at 20 000 rpm for 1 h in an SW27 bucket rotor, resuspended in phosphate buffered saline (PBS), pH 7,2 and used for infection of lymphoid cells H9 and Jurkat tat at a multiplicity of infection about 0.2-0.5 synoytium forming unit per cell. After 1 h adsorption at 37°C, the virus was removed, the cells were washed with PBS, and incubated in RPMI-1640 supplemented with 10% of fetal calf serum and 300 mg/ml glutamine L at 37CC in C02 atmosphere. At intervals, the cells were washed with PBS, homogenized in the presence of phenylmethyl sulfonyl fluorine (PMSF), the nuclei were pelleted at 800 g and washed
D.I. Ivanovsky Institute of Virology, USSR Academy of Medical Sciences, and Central Institute of Medicine Advanced Training, USSR Health Ministry, Moscow, USSR.
303
SHAROVA AND BUKRINSKAYA
304 gpI20 I
2
3
p65
I
4
1*
j>55
gp4I p39 p3I
f>24
plî
gpI20
p65 P55
Sp4I p39
_
p3T p24 Pl7
V/\ 12
3
í
i.
i
L P65 -
p55
L gp^1 i~
p39
j"
p3I
T p24
L pI7 FIG. 1. Input HIV-1 proteins in cellular fractions of infected H9 cells. (A)HIV-l proteins in cytoplasmic fractions. Infected cells were fractionated 2 h after infection, and viral proteins were revealed by immunoblotting using pooled sera of AIDS patients. Lane 1, nuclear washings; lane 2, fraction P20; lane 3, fraction S20; lane 4, input HIV-1. Arrow shows the position of gp 120 expressed by recombinant vaccinia virus. (B) Scanogram of HIV-1 proteins in the nuclei 2, 4, and 24 h after infection. The proteins were revealed by immunoblotting using pooled sera of AIDS patients. The top scan represents viral proteins of input HIV. (C) HIV-1 proteins in the nuclei 2 h after infection revealed by monoclonal antibodies to p24 (lanes 1,2) and p 17 (lanes 4,5). Lanes 3,6, proteins of input virus revealed by monoclonal antibodies to p24 and p 17, respectively. Lane 7, proteins of input virus revealed by pooled sera of AIDS patients.
INPUT HIV-I gag PROTEINS ARE TRANSPORTED INTO CELL NUCLEI
containing 1% Triton X-100. The cytocentrifuged at 20 000 g, and the proteins of the supernatant (fraction S20) were precipitated by acetone. The pellet (fraction P20) was resuspended in PBS. The proteins of cellular fractions were analyzed by immunoblotting. Briefly, the proteins were separated by electrophoresis on a 12% polyacrylamide gel slab in the presence of sodium dodecyl sulfate and were electrophoretically transferred to a nitrocellulose sheet. The pooled sera from AIDS patients and monoclonal antibodies to p24 and pl7, kindly supplied by Dr. H. Gelderblom, were used to identify the proteins. Figure 1 shows the intracellular localization of the HIV-1 proteins input into infected H9 cells. It is seen that the fraction S20 contains mainly p24 with fractions P20, p24, p55, and traces of p39. Nuclear washings are enriched with p24 alone. three times with PBS
plasm
was
Meanwhile, the nuclei of infected cells contain gag precursors p55, p41, and p39 but not p24 (Fig. 1A,B). Relative concentra-
tions of the gag precursors did not correlate with that in input virus, concentration of p39 ranking the highest. In fact, the amount of p55 in the nuclei 2 h after infection was only 13% of the amount in the input virus while the amount of p41 was 33% and the amount of p39 reached 60%. The respective concentrations in the nuclei were gradually increased over 2 h to 24 hours after infection (Fig. IB). Traces of proteins encoded by gene pol p65 (reverse transcriptase) and p31 (apparently integrase) were also seen in the nuclei in 24 h after infection. pl7 was not detected in the nuclei when the pooled sera of AIDS patients were applied, most likely due to the low titer of specific antibodies to this protein. However, this protein was revealed by monoclonal antibodies to pl7 (Fig. 1C).
305
Almost identical gag protein patterns were found in the fractions of Jurkat tat cells (Fig. 2). Fraction P20 contained p55 and p24 (lane I) while the nuclear washings contained only p24 (lane 7). The gag precursors p55, p41, and p39 were observed in the nuclei in increased concentrations from 40 min to 2 h after infection, when these proteins and especially p39 were found in essential quantity. In contrast to the nuclei of H9 cells, pl7 was revealed in large amounts comparable to that of p55 and p39 (lane 6). The amount was significantly increased from 40 min to 2 h after infection despite its very low amount in the input virus. p24 again was not detected in the nuclei. To show that the nuclear proteins are the proteins of the input virus, but not the nascent ones, the experiment has been performed in the presence of 100 pg/ml of cycloheximide added just after cell infection. As seen in Figure 2 (lanes 8-11), cycloheximide does not alter the protein pattern in the cytoplasm and nuclei 4 h after infection.
RESULTS
Thus, our data show that gag proteins p55, p41, p39, and p 17 of input virus are transported into the nuclei of infected cells and preserved there for several hours. All these proteins have the common N terminus, and their nuclear localization confirms the existence of a karyotypic signal at N terminus of gag precursor.7,8 The signal might be functional or accessible in the absence of the N-Gly myristate group,8 which suggests that the parental gag proteins lose their myristate group upon entering the cell cytoplasm or that myristoylation was not complete and
4 !
p55~ p4l_ P39 ;
rt
•
P24~
pI7-
FIG.2. InputHIV-1 proteins in cellular fractions of infected Jurkat-tat cells and effect of cycloheximide. Lanes 1-3, fraction P20 (1) and nuclei (2,3) from the cells fractionated 40 min after infection. Lanes 4,5, input virus. Lanes 6,7, nuclei (6) and nuclear washings (7) from the cells fractionated 2 h after infection. Lanes 8-11, nuclei (8) and fraction P20 (10) from the cells fractionated 4 h after infection and the same fractions (9 and 11 respectively) from the cells treated with 100 p.g/ml cycloheximide immediately
after infection.
SHAROVA AND BUKRINSKAYA
306 that gag molecules accumulating in the nucleus were never acylated. However, we cannot exclude the theory that myristoylation does not prevent the nuclear transport of input protein
molecules. The question arises about the potential role of nuclear localization of gag proteins in viral life cycle. Gag precursors are hardly essential for nuclear targeting of reverse transcripts since they are located in immature viral particles deprived of infectious activity. pl7 is known to serve as an isometric scaffold for HIV virions,9,10 and from this point of view, it may interact with genome RNA or provirus DNA or with RNA binding proteins p7 and p6. So far, karyotypic signal at the N terminus of pi 7 might be crucial for the transport of transcriptive complexes into the nuclei and their preservation there until integration events occur. The long period of input viral gag proteins preservation in the nuclei support this concept. As for p24, this protein appears to have no role in the matrix formed by pl7 and is a single viral protein which is solubilized after the treatment of HIV virions with detergents. '! Thus, it is not surprising that p24 is released into cytosol after fusion of viral envelope with cell plasma membrane. The difference in pl7 transport into the nuclei was observed in two cell lines: pl7 was readily accumulated in Jurkat tat cell nuclei, while only traces of it were revealed by monoclonal antibody to pl7 in H9 cell nuclei. This difference reflects the apparent diversity of viral replicative ability within a given cell
line. Cell tropism, replication and cytopathic effect activity of HIV, are known to vary significantly in different cell cultures.12
REFERENCES 1. Mervis RJ, Ahmad N, Lillehoj EP, Raum MG, Salazar RFH, Chan HW, and Venkatesan S: The gag gene products of HIV-1: alignment within the gag open reading frame, identification of posttranslational modifications, and evidence for alternative gag precursors. J Virol 1988;62:3993-4002. 2. Gelderblom HR, Ozel M, and Pauli G: Morphogenesis and mor-
phology
of HIV. Structure-functional relations. Arch Virol
precursor
processing
1989;106:1-13. 3. Gottlinger HJ, Sodroski JG, and Haseltine WA: Role of capsid
infectivity
and myristoylation in morphogenesis and of HIV-1. Proc Nati Acad Sei (USA) 1989;86:5781-
5785. 4. Erickson-Viitanen S, Manfredi J, Viitanen P, Tribe DE, Tritch R, Hutchison CA, Loeb DD, and Swanstrom R: Cleavage of HIV-1 gag polyprotein synthesized in vitro: sequential cleavage by the viral protease. AIDS Res Human Retroviruses 1989;5:577-591. 5. Gowda SD, Stein BS, and Ehgleman EG: Identification of protein intermediates in the processing of the p55 HIV-1 gag precursor in cells infected with recombinant vaccinia virus. J Biol Chem
1989;264:8459-8462. 6.
7.
8.
Bukrinskaya AG, Garayev MM, Vzorov AN, Shulenin S, and Sharova NK: Processing of HIV-1 gag precursor includes N terminal p39 which penetrates into the nuclei. Abstr Vlllth Int Congr Virology, Berlin, 1990. DelchambreM, GheysenD, Thines D, ThitiartC, JacobsE, Verdin E, Horth M, Burny A, and Bex F: The gag precursor of SIV assembles into virus-like particles. EMBO J 1989;8:2653-2660. Gheysen D, Jacobs E, de ForestaF, ThiriartC, Francotte M, Thines D, and De Wilde M: Assembly and release of HIV-1 precursor Pt55s"s virus-like particles from recombinant baculovirus-infected
insect cells. Cell 1989;59:103-112. 9. Marx PA and Munn RJ: Computer emulation on thin section electron microscopy predicts an envelope-associated icosadeltahedral capsid for human deficiency virus. Lab Invest 1988;58:112120. 10. Ozel M, Pauli G, and Gelderblom HR: The organization of envelope projections on the surface of HIV. Arch Virol 1988;100: 255-266. 11. Bukrinskaya AG and Sharova NK: Unusual features of protein interaction in HIV virions. Arch Virol 1990;110:287-293. 12. Cloyd MW and Moore BE: Spectrum of biological properties of HIV-1 isolates. Virology 1990;174:103-116.
Address represent requests to: Alice Bukrinskaya Institute of Virology Gamaleya Str. 16 123098 Moscow, USSR
pi 7 and pl7-Containing gag Precursors of Input Human Immunodeficiency Virus Are Transported into the Nuclei of Infected Cells
NATHALIE SHAROVA and ALICE BUKRINSKAYA
ABSTRACT Subcellular localization of input human immunodeficiency virus type 1 (HIV-1) gag proteins was determined in infected H9 and Jurkat tat cells. Infected cells were fractionated at intervals, and the proteins in cell fraction were identified by immunoblotting using pooled sera from acquired immunodeficiency syndrome (AIDS) patients and monoclonal antibodies. Cycloheximide was added at 0 time to prove that the proteins detected were not nascent ones. Gag proteins p55, p41. p39 (in the most essential relative concentrations), and p 17 were found in the cell nuclei for at least 4 h after infection. However, p24 was not found in the cell nuclei and was accumulated in the nuclear washing buffer. The data presented confirm the presence of karyotypic signal at the N terminus of p55 gag precursor. The potential role of nuclear localization of gag precursor is discussed.
INTRODUCTION
of virus
particles. If this is true for HIV, the gag proteins are expected to be transported into cell nuclei. In fact, recombinant gag proteins of simian immunodeficient virus (SIV) and HIV-1 expressed in insect cells were detected in cell nuclei.7,8
THE(HIV-l)p24, pl7, p7,
gag proteins OF human immunodeficiency virus type 1
and p6 are the products of proteolytic of the gag precursor processing p55. The polyprotein is myristoylated at the amino terminus and targeted to the plasma membrane of infected cells where assembly and budding of virus particles occurs. '~3 Uncleaved precursor is included in the virus particle and is processed within it by a virally encoded aspartic protease; cleavage is performed through intermediate precursor proteins.1,4 Recently three intermediate products of cleavage have been described, two of them, p41a and p39, from the N terminus of p55 and p41b from the C terminus.5 When the gag precursor expressed by the recombinant vaccinia virus was processed by the recombinant bacterial protease, the intermediate precursor p39 appeared within 30 min after mixing of two cellular lysates while p24 and pl7 were not revealed until 2 h or
later.6
The fate of parental gag proteins in infected cells is unknown. By analogy with the other envelope viruses, these proteins could play an important role in the replicative cycle, being involved into transcriptive complexes and in early steps of morphogenesis
METHODS The aim of this study was to determine the subcellular localization of HIV-1 gag proteins of input HIV-1 virus in two kinds of cells, H9 and Jurkat tat. HIV-1 produced by H9/IIIB cells was pelleted through 30% glycerol from culture fluid at 20 000 rpm for 1 h in an SW27 bucket rotor, resuspended in phosphate buffered saline (PBS), pH 7,2 and used for infection of lymphoid cells H9 and Jurkat tat at a multiplicity of infection about 0.2-0.5 synoytium forming unit per cell. After 1 h adsorption at 37°C, the virus was removed, the cells were washed with PBS, and incubated in RPMI-1640 supplemented with 10% of fetal calf serum and 300 mg/ml glutamine L at 37CC in C02 atmosphere. At intervals, the cells were washed with PBS, homogenized in the presence of phenylmethyl sulfonyl fluorine (PMSF), the nuclei were pelleted at 800 g and washed
D.I. Ivanovsky Institute of Virology, USSR Academy of Medical Sciences, and Central Institute of Medicine Advanced Training, USSR Health Ministry, Moscow, USSR.
303
SHAROVA AND BUKRINSKAYA
304 gpI20 I
2
3
p65
I
4
1*
j>55
gp4I p39 p3I
f>24
plî
gpI20
p65 P55
Sp4I p39
_
p3T p24 Pl7
V/\ 12
3
í
i.
i
L P65 -
p55
L gp^1 i~
p39
j"
p3I
T p24
L pI7 FIG. 1. Input HIV-1 proteins in cellular fractions of infected H9 cells. (A)HIV-l proteins in cytoplasmic fractions. Infected cells were fractionated 2 h after infection, and viral proteins were revealed by immunoblotting using pooled sera of AIDS patients. Lane 1, nuclear washings; lane 2, fraction P20; lane 3, fraction S20; lane 4, input HIV-1. Arrow shows the position of gp 120 expressed by recombinant vaccinia virus. (B) Scanogram of HIV-1 proteins in the nuclei 2, 4, and 24 h after infection. The proteins were revealed by immunoblotting using pooled sera of AIDS patients. The top scan represents viral proteins of input HIV. (C) HIV-1 proteins in the nuclei 2 h after infection revealed by monoclonal antibodies to p24 (lanes 1,2) and p 17 (lanes 4,5). Lanes 3,6, proteins of input virus revealed by monoclonal antibodies to p24 and p 17, respectively. Lane 7, proteins of input virus revealed by pooled sera of AIDS patients.
INPUT HIV-I gag PROTEINS ARE TRANSPORTED INTO CELL NUCLEI
containing 1% Triton X-100. The cytocentrifuged at 20 000 g, and the proteins of the supernatant (fraction S20) were precipitated by acetone. The pellet (fraction P20) was resuspended in PBS. The proteins of cellular fractions were analyzed by immunoblotting. Briefly, the proteins were separated by electrophoresis on a 12% polyacrylamide gel slab in the presence of sodium dodecyl sulfate and were electrophoretically transferred to a nitrocellulose sheet. The pooled sera from AIDS patients and monoclonal antibodies to p24 and pl7, kindly supplied by Dr. H. Gelderblom, were used to identify the proteins. Figure 1 shows the intracellular localization of the HIV-1 proteins input into infected H9 cells. It is seen that the fraction S20 contains mainly p24 with fractions P20, p24, p55, and traces of p39. Nuclear washings are enriched with p24 alone. three times with PBS
plasm
was
Meanwhile, the nuclei of infected cells contain gag precursors p55, p41, and p39 but not p24 (Fig. 1A,B). Relative concentra-
tions of the gag precursors did not correlate with that in input virus, concentration of p39 ranking the highest. In fact, the amount of p55 in the nuclei 2 h after infection was only 13% of the amount in the input virus while the amount of p41 was 33% and the amount of p39 reached 60%. The respective concentrations in the nuclei were gradually increased over 2 h to 24 hours after infection (Fig. IB). Traces of proteins encoded by gene pol p65 (reverse transcriptase) and p31 (apparently integrase) were also seen in the nuclei in 24 h after infection. pl7 was not detected in the nuclei when the pooled sera of AIDS patients were applied, most likely due to the low titer of specific antibodies to this protein. However, this protein was revealed by monoclonal antibodies to pl7 (Fig. 1C).
305
Almost identical gag protein patterns were found in the fractions of Jurkat tat cells (Fig. 2). Fraction P20 contained p55 and p24 (lane I) while the nuclear washings contained only p24 (lane 7). The gag precursors p55, p41, and p39 were observed in the nuclei in increased concentrations from 40 min to 2 h after infection, when these proteins and especially p39 were found in essential quantity. In contrast to the nuclei of H9 cells, pl7 was revealed in large amounts comparable to that of p55 and p39 (lane 6). The amount was significantly increased from 40 min to 2 h after infection despite its very low amount in the input virus. p24 again was not detected in the nuclei. To show that the nuclear proteins are the proteins of the input virus, but not the nascent ones, the experiment has been performed in the presence of 100 pg/ml of cycloheximide added just after cell infection. As seen in Figure 2 (lanes 8-11), cycloheximide does not alter the protein pattern in the cytoplasm and nuclei 4 h after infection.
RESULTS
Thus, our data show that gag proteins p55, p41, p39, and p 17 of input virus are transported into the nuclei of infected cells and preserved there for several hours. All these proteins have the common N terminus, and their nuclear localization confirms the existence of a karyotypic signal at N terminus of gag precursor.7,8 The signal might be functional or accessible in the absence of the N-Gly myristate group,8 which suggests that the parental gag proteins lose their myristate group upon entering the cell cytoplasm or that myristoylation was not complete and
4 !
p55~ p4l_ P39 ;
rt
•
P24~
pI7-
FIG.2. InputHIV-1 proteins in cellular fractions of infected Jurkat-tat cells and effect of cycloheximide. Lanes 1-3, fraction P20 (1) and nuclei (2,3) from the cells fractionated 40 min after infection. Lanes 4,5, input virus. Lanes 6,7, nuclei (6) and nuclear washings (7) from the cells fractionated 2 h after infection. Lanes 8-11, nuclei (8) and fraction P20 (10) from the cells fractionated 4 h after infection and the same fractions (9 and 11 respectively) from the cells treated with 100 p.g/ml cycloheximide immediately
after infection.
SHAROVA AND BUKRINSKAYA
306 that gag molecules accumulating in the nucleus were never acylated. However, we cannot exclude the theory that myristoylation does not prevent the nuclear transport of input protein
molecules. The question arises about the potential role of nuclear localization of gag proteins in viral life cycle. Gag precursors are hardly essential for nuclear targeting of reverse transcripts since they are located in immature viral particles deprived of infectious activity. pl7 is known to serve as an isometric scaffold for HIV virions,9,10 and from this point of view, it may interact with genome RNA or provirus DNA or with RNA binding proteins p7 and p6. So far, karyotypic signal at the N terminus of pi 7 might be crucial for the transport of transcriptive complexes into the nuclei and their preservation there until integration events occur. The long period of input viral gag proteins preservation in the nuclei support this concept. As for p24, this protein appears to have no role in the matrix formed by pl7 and is a single viral protein which is solubilized after the treatment of HIV virions with detergents. '! Thus, it is not surprising that p24 is released into cytosol after fusion of viral envelope with cell plasma membrane. The difference in pl7 transport into the nuclei was observed in two cell lines: pl7 was readily accumulated in Jurkat tat cell nuclei, while only traces of it were revealed by monoclonal antibody to pl7 in H9 cell nuclei. This difference reflects the apparent diversity of viral replicative ability within a given cell
line. Cell tropism, replication and cytopathic effect activity of HIV, are known to vary significantly in different cell cultures.12
REFERENCES 1. Mervis RJ, Ahmad N, Lillehoj EP, Raum MG, Salazar RFH, Chan HW, and Venkatesan S: The gag gene products of HIV-1: alignment within the gag open reading frame, identification of posttranslational modifications, and evidence for alternative gag precursors. J Virol 1988;62:3993-4002. 2. Gelderblom HR, Ozel M, and Pauli G: Morphogenesis and mor-
phology
of HIV. Structure-functional relations. Arch Virol
precursor
processing
1989;106:1-13. 3. Gottlinger HJ, Sodroski JG, and Haseltine WA: Role of capsid
infectivity
and myristoylation in morphogenesis and of HIV-1. Proc Nati Acad Sei (USA) 1989;86:5781-
5785. 4. Erickson-Viitanen S, Manfredi J, Viitanen P, Tribe DE, Tritch R, Hutchison CA, Loeb DD, and Swanstrom R: Cleavage of HIV-1 gag polyprotein synthesized in vitro: sequential cleavage by the viral protease. AIDS Res Human Retroviruses 1989;5:577-591. 5. Gowda SD, Stein BS, and Ehgleman EG: Identification of protein intermediates in the processing of the p55 HIV-1 gag precursor in cells infected with recombinant vaccinia virus. J Biol Chem
1989;264:8459-8462. 6.
7.
8.
Bukrinskaya AG, Garayev MM, Vzorov AN, Shulenin S, and Sharova NK: Processing of HIV-1 gag precursor includes N terminal p39 which penetrates into the nuclei. Abstr Vlllth Int Congr Virology, Berlin, 1990. DelchambreM, GheysenD, Thines D, ThitiartC, JacobsE, Verdin E, Horth M, Burny A, and Bex F: The gag precursor of SIV assembles into virus-like particles. EMBO J 1989;8:2653-2660. Gheysen D, Jacobs E, de ForestaF, ThiriartC, Francotte M, Thines D, and De Wilde M: Assembly and release of HIV-1 precursor Pt55s"s virus-like particles from recombinant baculovirus-infected
insect cells. Cell 1989;59:103-112. 9. Marx PA and Munn RJ: Computer emulation on thin section electron microscopy predicts an envelope-associated icosadeltahedral capsid for human deficiency virus. Lab Invest 1988;58:112120. 10. Ozel M, Pauli G, and Gelderblom HR: The organization of envelope projections on the surface of HIV. Arch Virol 1988;100: 255-266. 11. Bukrinskaya AG and Sharova NK: Unusual features of protein interaction in HIV virions. Arch Virol 1990;110:287-293. 12. Cloyd MW and Moore BE: Spectrum of biological properties of HIV-1 isolates. Virology 1990;174:103-116.
Address represent requests to: Alice Bukrinskaya Institute of Virology Gamaleya Str. 16 123098 Moscow, USSR
