Evidence for guanylate cyclase activity associated with hemagglutinating virus of Japan (Sendai virus).

Purified virions of HVJ (Sendai virus) were found to contain a guanylate cyclase activity that converts GTP to cyclic GMP. Activities of adenylate cyclase and 5'-nucleotidase which are frequently used as marker enzymes of cell membranes were not detected in the virus. Guanylate cyclase and virion-associated activities, neuraminidase and hemagglutinin, were co-purified during a purification of virions. Guanylate cyclase activity was not detected without disruption of the virions with a detergent, Triton X-100 or Nonident P-40. Treatment of intact HVJ with a proteolytic enzyme, trypsin or chymotrypsin, destroyed both neuraminidase and hemagglutinin; however, most of the guanylate cyclase ws retained. Guanylate cyclase activity was found in fractions containing nucleocapsids after sucrose density gradient centrifugation of disrupted virions. These results indicated that the enzyme was tightly bound to cores of HVJ and, therefore, its presence could not be explained by binding of host cell enzyme to the surface of virions. Properties of the virus-derived enzyme and particulate fractions of host cell homogenates were similar. Antiserum against nucleocapsids of HVJ inhibited guanylate cyclase activity of HVJ and particulate fractions of cells such as chorioallantoic membrane and rat liver, while soluble guanylate cyclase was not inhibited by antiserum. The biological significance and origin of guanylate cyclase found in HVJ are obscure and await further study.

Purified virions of HVJ (Sendai virus) were found to contain a guanylate cyclase activity that converts GTP to cyclic GMP. Activities of adenylate cyclase and 5'nucleotidase which are frequently used as marker enzymes of cell membranes were not detected in the virus. Guanylate cyclase and virion-associated activities, neuraminidase and hemagglutinin, were co-purified during a purification of virions. Guanylate cyclase activity was not detected without disruption of the virions with a detergent, Triton X-100 or Nonidet P-40. Treatment of intact HVJ with a proteolytic enzyme, trypsin or chymotrypsin, destroyed both neuraminidase and hemagglutinin; however, most of the guanylate cyclase was retained. Guanylate cyclase activity was found in fractions containing nucleocapsids after sucrose density gradient centrifugation of disrupted virions. These results indicated that the enzyme was tightly bound to cores of HVJ and, therefore, its presence could not be explained by binding of host cell enzyme to the surface of virions. Properties of the virus-derived enzyme and particulate fractions of host cell homogenates were similar. Antiserum against nucleocapsids of HVJ inhibited guanylate cyclase activity of HVJ and particulate fractions of cells such as chorioallantoic membrane and rat liver, while soluble guanylate cyclase was not inhibited by antiserum. The biological significance and origin of guanylate cyclase found in HVJ are obscure and await further study.
Guanylate cyclase, which catalyzes the formation of cyclic GMP from GTP, has been detected in all organisms so far examined (1). However, the presence of guanylate cyclase in viruses has not been reported. In this paper, we describe a tightly bound guanylate cyclase activity in cores of HVJ.' We were unable to detect activities of adenylate cyclase and 5" nucleotidase, used as marker enzymes of cell membrane, in highly purified HVJ. Although the origin and function of guanylate cyclase found in HVJ are not clear, our findings are of interest in view of the presence of protein kinase in viruses (2-7). Our observations suggest an involvement of cyclic GMP in the interaction between host cell and virus.
* This work was supported in part by a research grant from the Ministry of Education, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. I The abbreviation used is: HV.J, hemagglutinating virus of Japan.

RESULTS
Purification of HVJ a n d Guanylate Cyclase Actiuit-y-Guanylate cyclase and virion-associated activities, neuraminidase and hemagglutinin, were determined during purification of HVJ. As shown in Table I, recoveries of 3 activities were parallel during purification of HVJ from chorioallantoic fluid. Although the specific activity of neuraminidase and guanylate cyclase decreased slightly in this experiment, those of both enzymes in other experiments showed no change or increased slightly and the parallel purification of both enzymes was obtained in all experiments. Guanylate cyclase activity sedimented with virions in the second linear sucrose density gradient centrifugation (Fig. 2 in miniprint), and activities of neuraminidase and hemagglutinin of HVJ were also found in this peak. Electron micrography of pooled samples (fraction 8 to 11) showed virions of HVJ, and fragments of cell structures were not detected (Fig. 3A). Sodium dodecyl sulfatepolyacrylamide gel electrophoresis of purified virions of HVJ showed eight identifiable polypeptides and several minor polypeptides ( Fig. 4), analogous to previous publications (19-23). When the particulate fractions of homogenates of whole chick embryo (specific activity 128 pmol/min/mg) or chorioallantoic membranes (specific activity 1650 pmol/min/mg), prepared in the same manner as virions of HVJ, were subjected to centrifugation as shown in Fig. 2, a peak of guanylate cyclase was found in fractions 2 and 3 (not shown). Furthermore, the activities of adenylate cyclase and 5'-nucleotidase, which are frequently used as marker enzymes of cell membranes, were not detected in the purified preparations of HVJ (Table I). These results indicated that guanylate cyclase activity found in preparations of HVJ was not due to contaminating fragments of cell membranes.
Effect of Proteolytic Enzymes-When virions of HVJ and influenza virus are treated with proteolytic enzymes, the surface glycoproteins are removed without alteration of the internal proteins (20-23). Therefore, to exclude a possible binding of guanylate cyclase of host cell on the surface of the virion, virions of HVJ were treated with trypsin or chymotrypsin (  -~~-" -~-" The limits of detectable activity are 0.02 pmol/min/mg for adenylate cyclase and 0.5 nmol/min/mg for 5'-nucleotidase with 0.5 mg of protein used/assay tube. concentration of 20 pg/ml for 60 min at 37°C successfully removed the surface polypeptides, HN and F. Other polypeptides which are internal appeared to remain intact. Morphologically, the spikes seen on the surface of intact virions were not observed after treatment with trypsin or chymotrypsin (not shown). Under these conditions, 77% of the guanylate cyclase activity was recovered with trypsin treatment and 91$ with chymotrypsin treatment. At a concentration of 100 pg/ ml, trypsin digested almost all polypeptides of virions and no guanylate cyclase activity was found, while 677, of the activity was recovered with chymotrypsin. Following chymotrypsin treatment, fragments of M , = 40,OOO-43,000 were found with Guanylate Cyclase Activity in HVJ TARIX I1

Sumntatv of the fractionation of disrupted virions of HVJ
Purified HVJ was treated with 0.5$ Tween 20 and 1 M KC1 in an pellet at zero time and 60 min at room temperature were neutralized alkaline solution. Aliquots (0.1 ml), immediately (zero time) and after and stored as above. The suspended pellet after 60 min at room ($0 min at room temperature, were added to 0.1 ml of 0.2 M I'ris-HCI temperature was subjected to centrifugation through linear sucrose huffer, pH 7.2, and stored at 4OC until activities were determined. density gradient. Activities of guanylate cyclase anti neuraminidase After 60 min at room temperature. HVJ was centrifuged at 105,oW were determined as described under "Materials and Methods" in the X R for 60 min. The supernatant obtained was neutralized and the miniprint supplement. pellet was suspended with alkali-I'ween20-KCI. Aliquots ofsuspended no other detectable viral polypeptides (Fig. 5). However, it is known whether or not this fragment shows guanylate cyclase activity. The finding suggests the possible location of guanylate cyclase activity within the virion. Fractionation of Polypeptides of the Virion-Virions of HVJ, 1 mg/ml, were treated with 0.5% Tween 20 and 1 M KC1 in alkaline solution to prepare the nucleocapsids as described by Hosaka (11). The results are summarized in Table I1 and Figs. 3 , 6, and 7. When virions were treated with alkali-Tween 20-KCI, guanylate cyclase activity was quite unstable. Forty to fifty per cent of the activity was lost at room temperature after 60 min. After centrifugation of disrupted virions at 100,000 X g for 1 h, most of neuraminidase was recovered in the supernatant, while guanylate cyclase was found in the pellet (Table 11). Polypeptides in the supernatant and pellet analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that all major polypeptides were found in both fractions, however, HN and F were primarily in the supernatant and P, NP, and M in the pellet (Fig. 6, lanes 2-4). Therefore, the pellet was again treated with alkali-Tween 20-KC1 and subjected to centrifugation through a linear sucrose density gradient (Fig. 7 in miniprint). Three visible hands were obtained which correspond to Fractions 11, IV, and VI in Fig. 7.Neuraminidase and hemagglutinin were found in the first band (Fraction 11) and guanylate cyclase in the second and third bands (Fractions IV and VI) with the highest activity in Fraction IV (Table 11). Polypeptide analyses revealed that envelope glycoproteins, HN and F, were present primarily in Fractions I1 and 111 (Fig. 6, lanes 6 and 7) and nucleocapsid proteins, P and NP, in Fractions IV-VI (Fig. 6,   lunes 8-10). High molecular weight polypeptides and polypeptide 5 which are nucleocapsid proteins (24) and two unidentified polypeptides, M , of about 43,000, one of which was assumed to he polypeptide 7, were also present in Fractions IV-VI. Polypeptide M was seen in all fractions obtained. By electron microscopic study, free envelopes were found in Fraction 11, complexes of envelopes and nucleocapsids in Fraction IV, and nucleocapsids without envelopes in Fraction VI (Fig.  3). Results of electrophoretic and electronmicroscopic studies Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of pol.ypeptides isolated hy sequential centrifugation of disrupted HVJ. Experimental procedures were described under "Materials and Methods", in the miniprint supplement, except 12.5' acrylamide and 0.17 bisacrylamide in the separating gel were used. Samples are as follows: I , standard proteins; 2, untreated HVJ: 3 , 105,OOO X g supernatant; 4. 105,OOO X , q pellet; 5-10. Fractions I-VI which correspond to Fig. 7, respectively. suggested the possible association of guanylate cyclase activity with nucleocapsids of HVJ.

DISCUSSION
In the present study, we demonstrated guanylate cyclase activity associated with purified virions of HVJ. The enzyme activity was found after disruption of virions by detergents and was resistant to limited proteolysis of intact HVd with trypsin or chymotrypsin (Fig. 5 ) . After disruption of virions by treatment with alkali-Tween 20 or Triton X-100 in high Guanylate Cyclase Activity in HVJ 2511 salt solution, most of the guanylate cyclase activity was recovered in the pellet upon centrifugation (Table 11). Following further fractionation of the pellet by sucrose density gradient centrifugation, guanylate cyclase activity was found in fractions containing nucleocapsids (Figs. 3 and 7). These results indicate that guanylate cyclase activity is tightly associated with cores of HVJ and its presence can not be accounted for by a simple binding of host enzyme to the surface of virions.
In addition, activities of cell membrane-bound enzymes such as adenylate cyclase and 5'-nucleotidase were not detected in the virus. It is not clear whether guanylate cyclase in HVJ is coded by the virus genome or derived from host cells. It is known that the enveloped virus is formed at the cell surface by budding. In the mechanism of budding, it is assumed that the initial insertion of envelope glycoproteins is followed by their migration in the cell membrane, resulting in the formation of a patch of virus-specific membrane, in which M protein plays an important role in the association of nucleocapsids with envelope glycoproteins at a localized region of plasma membrane which will become the viral envelope (22,23). Although the virus formed by budding can contain host cell proteins, the virus can also exclude these proteins from the region of the bud by an unknown mechanism (26, 27). However, some enzymatic activities including ATPase, nucleoside diphosphate kinase, phosphatase, methyltransferase, and polyadenylating activity were reported in purified Rous sarcoma virus and vesicular stomatitis virus (28, 29), protein kinase in various viruses (2-7), actin-like protein in HVJ (19), and alkaline protease in some viruses (30,31). Some of these proteins appear to be virus-coded (3-5, 32) and others are of an unknown origin. As with most of these enzymes, we were unable to identify a polypeptide which bears the activity of guanylate cyclase in HVJ. However, its distribution of guanylate cyclase activity during isolation of viral polypeptides was so similar to that of M protein of HVJ (Figs. 6 and 7) that guanylate cyclase may be located between the envelope and nucleocapsid, like M protein (22,23).
Mammalian tissues contain two forms of guanylate cyclase, soluble and particulate forms, with different properties (1) and different antigenicity (33). The characteristics of guanylate cyclase activity of HVJ were similar to that of the particulate form of guanylate cyclase and both activities were inhibited more than 90% by antiserum against nucleocapsids of HVJ (Fig. 8). The antiserum showed no effect on the soluble form from avian and mammalian cells. These results indicated that the guanylate cyclase found in HVJ has properties and antigenicity similar to that of the host particulate enzyme and suggested that the enzyme in HVJ could be host cell in origin. However, several proteins from normal host cells and from viruses have been shown to have a common antigenicity (34-38). Guanylate cyclase of HVJ and membrane-bound guanylate cyclase of both chick and rat tissues might be another example of the same antigenicity between viral protein and normal cell protein.