Nonenveloped Viruses with More Complicated Structural Features - Enveloped Viruses

The hexon protein has two eight-strand β sandwiches to

give the trimer an approximately sixfold symmetry (and

each hexon protein fills the role of two symmetry units).

There are long loops that intertwine to form a triangular

top. These structures can be fitted uniquely into the enve-

lope of density determined by cryoelectron microscopy,

which produces a structure refined to atomic resolution for

most of the capsid. The minor proteins can be fitted into

this structure.

From the 12 vertices of the icosahedron project long

fibers that are anchored in the surface by a unit called the

penton base. Each fiber terminates in a spherical extension

that forms an organ of attachment to a host cell (Fig. 2.12A).

The length of the fiber differs in the different adenoviruses.

NONENVELOPED VIRUSES WITH MORE

COMPLICATED STRUCTURAL FEATURES

In addition to the nonenveloped viruses that possess rela-

tively straightforward icosahedral symmetry or helical sym-

metry, many viruses possess more complicated symmetries

made possible by the utilization of a large number of struc-

tural proteins to form the virion. The tailed bacteriophages

are prominent examples of this (Fig. 2.13). Some of the

tailed bacteriophages possess a head that is a regular icosa-

R Q

hedron (or, in at least one case, an octahedron) connected

to a tail that possesses helical symmetry. Other appendages,

such as baseplates, collars, and tail fibers, may be connected

to the tail. Other tailed bacteriophages have heads that are

assembled using more complicated patterns. For example,

the T-even bacteriophages have a large head, which can be

thought of as being formed of two hemi-icosahedrons pos-

sessing regular icosahedral symmetry, which are elongated

in the form of a prolate ellipsoid by subunits arranged in a

regular net connecting the two icosahedral ends of the head

FIGURE 2.11 The essential features of the orbivirus native core particle.

of the virus.

The asymmetric unit is indicated by the white lines forming a triangle and

the fivefold, threefold, and twofold axes are marked. (A) The inner capsid

layer of the bluetongue virus (BTV) core is composed of 120 molecules

ENVELOPED VIRUSES

of VP3, arranged in what has been called T=2 symmetry. Note the green

subunit A and the red subunit B which fill the asymmetric unit. (B) The

core surface layer is composed of 780 copies of VP7 arranged as 260

Many animal viruses and some plant viruses are envel-

trimers, with T=13 symmetry. The asymmetric unit contains 13 copies of

oped; that is, they have a lipid-containing envelope surround-

VP7, arranged as five trimers, labeled P, Q, R, S, and T, with each trimer a

ing a nucleocapsid. The lipids are derived from the host cell.

different color. Trimer "T" in blue sits on the icosahedral threefold axis and

Although there is some selectivity and reorganization of lipids

thus contributes only a monomer to the asymmetric unit. From Granoff and

during virus formation, the lipid composition in general mir-

Webster (1999), Color Plate 17.

rors the composition of the cellular membrane from which the

envelope was derived. However, the proteins in the nucleo-

groups of peripentonal hexons are found groups of 9 hexons,

capsid, which may possess either helical or icosahedral sym-

which are sixfold coordinated. Each group of 9 hexons forms

metry, and the proteins in the envelope are encoded in the

the surface of one of the triangular faces. Thus, there are 60

virus. The proteinprotein interactions that are responsible for

peripentonal hexons and 180 hexons in groups of nine.

assembly of the mature enveloped virions differ among the dif-

The structure of the hexon trimer has been solved to

ferent families and the structures of the resulting virions differ.

atomic resolution by X-ray crystallography (Fig. 2.12C).

The virions of alphaviruses, and of flaviviruses, are uniform

Fiber

Penton Base

Peripentonal Hexons

Group of 9

Hexons

DNA

IIIa

VII

FIGURE 2.12

Structure of adenovirus particles. (A) Schematic drawing of the outer shell of an adenovirus (left), and

a schematic cross section through an adenovirus particle, showing the locations of minor polypeptide components (right).

The virus is composed of 60 peripentonal hexons at the bases of the fibers at the fivefold vertices, and groups of 9 hexons,

one on each triangular face of the icosahedron. (B) Cryoeletron microscopic reconstruction of an adenovirus virion,

viewed down the threefold axis. (C) Space-filling model of the hexon trimer, with each subunit in a different color. The

atomic structure of the hexon has been solved and fitted into the cryoelectron microscopic reconstruction. The locations

of the minor constitutents, indicated schematically in A, were deduced by subtraction. (A) is from Fields et al. (1996)

p. 80, (B) is from Stewart et al. (1991), and (C) is from Athappilly et al. (1994).

structures that possess icosahedral symmetry. Poxviruses,

domain. It binds to the viral RNA and encapsidates it to form

rhabdoviruses, and retroviruses also appear to have a regular

the nucleocapsid. For most RNA viruses, nucleocapsids can

structure, but there is flexibility in the composition of the par-

be recognized as distinct structures within the infected cell

ticle and the mature virions do not possess icosahedral sym-

and can be isolated from virions by treatment with detergents

metry. The herpesvirus nucleocapsid is a regular icosahedral

that dissolve the envelope. The nucleocapsids of alphavi-

structure (Fig. 2.5), but the enveloped herpesvirions are not

ruses, and probably flaviviruses and arteriviruses as well, are

regular. Other enveloped viruses are irregular, often pleio-

regular icosahedral structures, and there are no other proteins

morphic, and are heterogeneous in composition to a greater

within the nucleocapsid other than the nucleocapsid protein.

or lesser extent. The structures of different enveloped viruses

In contrast, the nucleocapsids of all minus-strand viruses are

that illustrate these various points are described next.

helical and contain, in addition to the major nucleocapsid

protein, two or more minor proteins that possess enzymatic

activity. As described, the nucleocapsids of minus-strand

The Nucleocapsid

RNA viruses remain intact within the cell during the entire

The nucleocapsids of enveloped RNA viruses are fairly

infection cycle and serve as machines that make viral RNA.

simple structures that contain only one major structural

The coronaviruses also have helical nucleocapsids, but being

protein, often referred to as the nucleocapsid protein or core

plus-strand RNA viruses they do not need to carry enzymes

protein. This protein is usually quite basic or has a basic

in the virion to initiate infection. The helical nucleocapsids

A. Enterobacteria phage T2

Head

DNA

152 capsomeres

Collar

Neck

Contractile sheath

Fibers

Core

Spikes

Base

plate

Surface view, tail extended

Cutaway view, tail contracted

100 nm

100 nm

Electron micrograph

B. Enterobacteria phage T7

Head

72 Capsomeres

DNA

T=7

Fibers

Tail

100 nm

Surface view

Cutaway view

100 nm

Electron micrograph

C. Lambda-like Phage

Head

72 Capsomeres

T=7

DNA

Tail

Fibers

Surface view

Cutaway view

Electron micrograph

FIGURE 2.13 Morphology of some bacteriophages (members of the Caudovirales). (A) Enterobacteria phage T2,

in the family Myoviridae. The head is an elongated pentagonal structure. (B) Enterobacteria phage T7, a member of the

Podoviridae. (C) Enterobacteria phage λ, a member of the Siphoviridae. All electron micrographs are stained with uranyl

acetate, and all bars shown are 100 nm. Phage diagrams are adapted from Murphy et al. (1995) pp. 51, 60, 55. Electron

micrographs of T2 and T7 were kindly provided by Dr. H.-W. Ackermann, Laval University, Quebec. The electron

micrograph of Ur-Lambda (note the long kinked tail fibers) was kindly provided by Dr. Roger Hendrix.

of (-) RNA viruses appear disordered within the envelope of

lumen of the ER so that they do not aggregate prior to folding.

all viruses except the rhabdoviruses, in which they are coiled

During folding, the solubility of the proteins is increased by

in a regular fashion (see later).

hiding hydrophobic domains within the interior of the protein

The nucleocapsids of retroviruses also appear to be

and leaving hydrophilic domains at the surface.

fairly simple structures. They are formed from one major

The glycoproteins possess a number of important functions

precursor protein, the Gag polyprotein, that is cleaved dur-

in addition to their structural functions. They carry the attach-

ing maturation into four or five components. The precursor

ment domains by which the virus binds to a susceptible cell.

nucleocapsid is spherically symmetric but lacks icosahedral

This activity is thought to be related to the ability of many

symmetry. The mature nucleocapsid produced by cleavage

viruses, nonenveloped as well as enveloped, to bind to and

of Gag may or may not be spherical symmetric. The nucleo-

agglutinate red blood cells, a process called hemagglutina-

capsid also contains minor proteins, produced by cleavage of

tion. The protein possessing hemagglutinating activity is often

GagProPol, as described in Chapter 1. These minor pro-

called the hemagglutinin or HA. The viral glycoproteins also

teins include the protease, RT, RNase H, and integrase that

possess a fusion activity that promotes the fusion of the mem-

are required to cleave the polyprotein precursors, to make a

brane of the virus with a membrane of the cell. The protein

cDNA copy of the viral RNA, and to integrate this cDNA

possessing this activity is sometimes called the fusion protein,

copy into the host chromosome.

or F. The glycoproteins, being external on the virus, are also

The two families of enveloped DNA viruses that we

primary targets of the humoral immune system, in which cir-

consider here, the poxviruses and the herpesviruses, contain

culating antibodies are directed against viruses; many of these

large genomes and complicated virus structures. The nucleo-

are neutralizing antibodies that inactivate the virus.

capsids of herpesviruses are regular icosahedrons but those

The glycoproteins of some enveloped viruses also contain

of poxviruses are complicated structures containing a core

enzymatic activities. Many orthomyxoviruses and paramyxo-

and associated lateral bodies.

viruses possess a neuraminidase that will remove sialic acid

from glycoproteins. The primary receptor for these viruses is

sialic acid. The neuraminidase may allow the virus to pene-

Envelope Glycoproteins

trate through mucus to reach a susceptible cell. It also removes

The external proteins of enveloped virions are virus-

sialic acid from the viral glycoproteins so that these glyco-

encoded proteins that are anchored in the lipid bilayer of the

proteins or the mature virions do not aggregate, and from the

virus or whose precursors are anchored in the lipid bilayer.

surface of an infected cell, thereby preventing released virions

In the vast majority of cases these proteins are glycoproteins,

from binding to it. The viral protein possessing neuraminidase

although examples are known that do not contain bound car-

activity may be called NA, or in the case of a protein that is

bohydrate. These proteins are translated from viral mRNAs

both a neuraminidase and hemagglutinin, HN.

and transported by the usual cellular processes to reach the

The structure of most enveloped viruses is not as rigor-

membrane at which budding will occur. When budding is at

ously constrained as that of icosahedral virus particles. The

the cell plasma membrane, the glycoproteins are transported

glycoproteins are not required to form an impenetrable shell,

via the Golgi apparatus to the cell surface. Some enveloped

which is instead a function of the lipid bilayer. They appear

viruses mature at intracellular membranes, and in these cases

to tolerate mutations more readily than do proteins that must

the glycoproteins are directed to the appropriate place in the

form a tight icosahedral shell and appear to evolve rapidly in

cell. Both Type I integral membrane proteins, in which the

response to immune pressure. However, the integrity of the

N terminus of the protein is outside the lipid bilayer and the

lipid bilayer is essential for virus infectivity, and enveloped

C terminus is inside the bilayer, and Type II integral mem-

viruses are very sensitive to detergents.

brane proteins, which have the inverse orientation with the C

terminus outside, are known for different viruses. Many viral

Other Structural Proteins in Enveloped

glycoproteins are produced as precursor molecules that are

Viruses

cleaved by cellular proteases during the maturation process.

Following synthesis of viral glycoproteins, during which

In some enveloped viruses, there is a structural protein

they are transported into the lumen of the endoplasmic reticu-

that underlies the lipid envelope but which does not form part

lum (ER) in an unfolded state, they must fold to assume their

of the nucleocapsid. Several families of minus-strand RNA

proper conformation, and assume their proper oxidation state

viruses possess such a protein, called the matrix protein. This

by formation of the correct disulfide bonds. This process often

protein may serve as an adapter between the nucleocapsid and

occurs very quickly, but for some viral glycoproteins it can

the envelope. It may also have regulatory functions in viral

take hours. Folding is often assisted by chaperonins present

RNA replication. The herpesviruses also have proteins under-

in the endoplasmic reticulum. It is believed that at least one

lying the envelope that form a thick layer called the tegument.

function of the carbohydrate chains attached to the protein is

The thickness of the tegument is not uniform within a virion,

to increase the solubility of the unfolded glycoproteins in the

giving rise to some irregularity in its structure. The tegument

proteins perform important functions early after infection of a

microscopy, which has been used to determine the structures

cell by a herpesvirus (see Chapter 7).

of several alphaviruses to 725 Å (Fig. 2.5).

More detailed reconstructions of Sindbis virus and Ross

River virus (RRV) have been derived from a combination

Structure of Alphaviruses

of cryoelectron microscopy of the intact virion and X-ray

The alphaviruses, a genus in the family Togaviridae, are

crystallography of alphavirus structural proteins. A cuta-

exceptional among enveloped RNA viruses in the regular-

way view of RRV at about 25-Å resolution is shown in Fig.

ity of their virions, which are uniform icosahedral particles.

2.14A. The nucleocapsid, shown in red and yellow, has a

Virions of two alphaviruses have been crystallized and the

diameter of 400 Å, and is a regular icosahedron with T=4

crystals are regular enough to diffract to 3040-Å resolu-

symmetry. It is formed from 240 copies of a single species of

tion. Higher resolution has been obtained from cryoelectron

capsid protein of size 30 kDa. Note the fivefold and sixfold

N172

R114

S215

T238

M137

W264

L231

H141

D163

A234

G201

I254

FIGURE 2.14 Structure of Ross River virus reconstructed from cryoelectron microscopy. (A) Cutaway view of the

cryoelectron reconstruction illustrating the multilayered structure of the virion. Envelope glycoproteins are shown in

blue, the lipid bilayer in green, the ordered part of the nucleocapsid in yellow, and the remainder of the nucleocapsid

in orange. (B) Ribbon diagram of the X-ray crystallographic structure of the Sindbis virus capsid protein, with β sheets

represented by large arrows. Only the carboxy-terminal domain, which starts at Arg-114, is ordered in crystals. The

active site residues of the autoprotease, Ser-215, His-141, and Asp-163, are shown in red. The carboxy-terminal Trp-

264, which is the P1 residue of the cleavage site, lies within the active site of the enzyme. The seven residues shown

in yellow-green may interact with the cytoplasmic domain of glycoprotein E2 during budding of the nucleocapsid.

by cryoelectron microscopy. (A) and (B) were adapted from Strauss et al. (1995), Figures 4 and 3, respectively, and

coordinated pedestals in yellow that rise above the red back-

define the structure of the shell of the nucleocapsid to atomic

ground of RNA and unstructured parts of the protein. Each

resolution.

of these pedestals is formed by the ordered domains of one

The envelopes of alphaviruses contain 240 copies of

capsid protein molecule. The lipid bilayer is shown in green

each of two virus-encoded glycoproteins, called E1 and E2.

and is positioned between the capsid and the external shell

E2 is first produced as a precursor called PE2. E1 and PE2

of glycoproteins, shown in blue. The glycoproteins are also

form a heterodimer shortly after synthesis, and both span

icosahedrally arranged with T=4 symmetry. The complete

the lipid bilayer as Type I integral membrane proteins (hav-

structure is therefore quite regular and the virion has been

ing a membrane-spanning anchor at or near the C terminus).

described as composed of two interacting protein shells with

The C-terminal cytoplasmic extension of PE2 interacts in a

a lipid bilayer sandwiched between.

specific fashion with a nucleocapsid protein so that there is

The structure of the ordered part of the capsid protein of

a one-to-one correspondance between a capsid protein and a

Sindbis virus has been solved to atomic resolution by con-

glycoprotein heterodimer. The 240 glycoprotein heterodim-

ventional X-ray crystallography and this structure is shown

ers form a T=4 icosahedral lattice on the surface of the par-

in Fig. 2.14B. The first 113 residues are disordered and

ticle by interacting with one another and with the capsid

the structure is formed by residues 114264. This ordered

proteins. Because of the glycoproteincapsid protein inter-

domain has a structure that is very different from the eight-

actions, the icosahedral lattices of the nucleocapsid and the

fold β sandwich described earlier (compare Fig. 2.14B with

glycoproteins are coordinated.

Figs. 2.3B and 2.4). Instead, its fold resembles that of chy-

At some time during transport of the glycoprotein

motrypsin, and it has an active site that consists of a catalytic

heterodimers to the cell surface, PE2 is cleaved by a cellular

triad whose geometry is identical to that of chymotrypsin.

protease called furin to form E2. E1 and E2 remain associ-

The capsid protein is an active protease that cleaves itself

ated as a heterodimer. If cleavage is prevented, noninfec-

from a polyprotein precursor. After cleavage, the C-terminal

tious particles are produced that contain PE2 and E1.

tryptophan-264 remains in the active site and the enzymatic

In the virion, three glycoprotein heterodimers associate to

activity of the protein is lost.

form a trimeric structure called a spike, easily seen in Figs. 2.5

The interactions between the capsid protein subunits that

and 2.14A. It is not known if the spike assembles during virus

lead to formation of the T=4 icosahedral lattice have been

assembly or if heterodimers trimerize during their transport to

deduced by fitting the electron density of the capsid protein

the cell surface. A reconstruction of a spike of Sindbis virus

at 2.5-Å resolution into the electron density of the nucleo-

at a resolution better than 10 Å is shown in Fig. 2.15A. In this

capsid found by cryoelectron microscopy. Such a recon-

reconstruction, the electron density of E1 has been replaced

struction, based on a cryoEM structure of Sindbis virus at a

by the E1 structure of the related Semliki Forest virus deter-

resolution of better than 10 Å, is shown in Fig. 2.14C. The fit

mined to atomic resolution by X-ray crystallography. The

of the capsid protein is unique and the combined approaches

three copies of E1 project upwards at an angle of about 45°

of X-ray crystallography and cryoelectron microscopy thus

and are shown in three colors because they have slightly dif-

A. Sindbis Virus

B. Dengue Virus

FIGURE 2.15 Comparison of (A) the spike structure of mature Sindbis virus (an alphavirus) with (B) the spike of

immature dengue virus (a flavivirus). The Cα backbones of the three E1 (Sindbis) and E (dengue) glycoprotein ectodomains

are shown in red, green, and blue, as they were fitted into the cryoelectron density envelope. The E1 and E densities have been

zeroed out, leaving the gray envelope that corresponds to E2 for Sindbis and prM for dengue. The density corresponding to

the lipid bilayer is shown in bright green. Adapted from Figure 5 in Y. Zhang et al. (2003) with permission.

ferent environments. The electron density in gray that remains

ers and trimerization of E1 to form homotrimers. The fusion

after subtracting the density due to E1 is thus the electron den-

peptide is exposed and penetrates the target bilayer of the

sity of E2. E2 projects further upward than does E1 and cov-

host endosomal membrane. Fusion follows by methods dis-

ers the apex of E1, which has the fusion peptide. Thus, E2

cussed in Chapter 1.

covers the fusion peptide with a hydrophobic pocket so that it

does not interact with the hydrophilic environment. The apex

Structure of Flaviviruses

of the E2 spike contains the domains that attach to receptors

on a susceptible cell. Both E1 and E2 have C-terminal mem-

Flaviviruses also possess a regular icosahedral structure

brane-spanning anchors that traverse the lipid bilayer shown

(Fig. 2.5) that has been solved by methods similar to those

in green. The C-terminal domain of E1 is not present in the

used to determine the structure of alphaviruses. The struc-

protein whose structure has been determined because hydro-

tures of alphaviruses and flaviviruses are related and have

phobic domains do not easily crystallize. Thus, the electron

descended from a common ancestral structure. Like alphavi-

density shown traversing the lipid bilayer arises from both E1

ruses, flaviviruses produce two structural glycoproteins,

and E2 and shows that the two membrane spanning anchors

called E and prM (for precursor to M). E is homologous to E1

go through as paired α helical structures (Fig. 2.16).

of alphaviruses. Although no sequence identity is detectable,

Upon entry of an alphavirus into a cell, the acidic pH of

the structures of the two proteins are virtually identical and

endosomal vesicles causes disassembly of E2/E1 heterodim-

are formed with a similar fold (Fig. 2.17). prM and E form a

heterodimer and immature particles can be formed if cleavage

of prM is prevented. The glycoprotein heterodimers in these

immature particles trimerize to form spikes whose structure

is very similar to the spikes of alphaviruses (Fig. 2.15B). The

H363

arrangement of the glycoproteins on the immature virus par-

W409

ticle is illustrated in Fig. 2.18A and a cryoEM reconstruction

that illustrates the surface of the immature dengue virus par-

ticle is shown in Fig. 2.18D. The major differences between

I368

the immature flavivirus particle and the alphavirus particle are

that there are 180 copies of the heterodimer in the flavivirus

particle arranged in a T=3 icosahedral structure rather than

F414

240 heterodimers arranged in a T=4 structure in alphaviruses;

that prM is a smaller molecule than PE2 so that in the flavi-

virus spike there is but a thin trace of density that projects

downward, parallel to E, from the cap that shields the fusion

peptide (Fig. 2.15B) rather than a substantial trace of density

M379

in the alphavirus spike (Fig. 2.15A); and that the C-terminal

regions of prM and E that enter the membrane do so inde-

F428

pendently and do not emerge from the internal side of the

membrane (illustrated in Fig. 2.19B), unlike the alphavirus

membrane spanning regions (Fig. 2.16). There is no evidence

that the membrane glycoproteins interact with the nucleocap-

sid in flaviviruses, and the flavivirus nucleocapsid, assuming

it is a regular icosahedral structure, is not coordinated with the

C390

R439

icosahedral structure formed by the spikes, which is again dif-

ferent from alphaviruses where the C-terminal domain of PE2

interacts with the nucleocapsid.

Following cleavage of prM by furin to form M, there is a dra-

matic rearrangement of the flavivirus glycoproteins such that the

T398

final virion structure is very different from the alphavirus structure.

The heterodimers dissociate and E-E homodimers are formed that

collapse over the lipid bilayer (Fig. 2.19). The E-E homodimers

occur in two totally different environments, either perpendicular

FIGURE 2.16 The E1 and E2 transmembrane helices of Sindbis (an

to the twofold axis where they interact with homodimers in side-

alphavirus) determined from a 9Å resolution cryoelectron microscopy

to-side interactions, forming a herringbone pattern, or parallel to

reconstruction. Shown are E1 residues from 409 to 439 and E2 residues

363 to 398 fitted into the transmembrane density. This is Figure 6 from

a twofold axis where they interact at fivefold and threefold axes

Mukhopadhyay et al. (2006), reprinted with permission.

(Fig. 2.18B). A comparison of the immature flavivirus particle,

Semliki Forest E1

C-Terminal Domain

Dimerization Domain

Central Domain

Fusion Peptide

TBE E Protein

Semliki Forest E1 Protein

131

166

289 300

390

413 436

TBE E Protein

138

191

286 301

395

449

496

FIGURE 2.17 Comparison of the folds of the Semliki Forest (alphavirus) E1 protein and the tick-borne encephalitis

(flavivirus) E protein. At the top are shown the X-ray crystal structures of the two proteins. At the bottom is a schematic

of the linear amino acid sequences, color coded to indicate which amino acids contribute to each of the domains. Adapted

from Figure 2 in Lescar et al. (2001) with permission.

the mature flavivirus particle, and the alphavirus particle is

Structure of Other Enveloped Viruses with

shown in Fig. 2.18. The mature flavivirus is smooth, with no

Round Nucleocapsids

surface projections, and is 50 nm in diameter (Fig. 2.18E).

The arteriviruses possess icosahedral nucleocapsids, but

The immature particle is ragged in appearance and is 60 nm

the mature virion does not appear to be regular in structure.

in diameter (Fig. 2.18D). The alphavirus virion shows con-

Detailed structures of these particles are not available.

spicuous spikes and is 70 nm in diameter (Fig. 2.18F). The

The herpesviruses are large DNA viruses that have a T=16

arrangement of E or E1 in the three particles is also shown

icosahedral nucleocapsid (Fig. 2.5). A schematic diagram of

(Figs. 2.18A, B, C), illustrating the differences in their

an intact herpesvirion is shown in Fig. 2.20A. Underneath

arrangement.

the envelope is a protein layer called the tegument. The

Entry of flaviviruses follows pathways similar to those

tegument does not have a uniform thickness, and thus the

used by alphaviruses. The acidic pH of the endosome causes

virion is not uniform. Two electron micrographs of herpes-

the E-E homodimers to reorganize to form E homotrim-

virions are shown in Figs. 2.20B and 2.20C that illustrate the

ers. These trimers must reorient so that the exposed fusion

irregularity of the particle and the differing thickness of the

peptide is projected upwards where it penetrates the host

tegument in different particles.

membrane.

100 nm

FIGURE 2.18 Fitting the X-ray crystal structures of flavivirus E protein and alphavirus E1 protein into the respective

cryoelectron density envelopes of the virions. (A) Dengue E protein fitted into the cryoelectron density of immature prM-

containing particle; (B) dengue E protein fitted into the envelope of the mature virion; (C) the fit of Sindbis alphavirus

E1 into the Sindbis virus envelope; (D) cryoelectron reconstruction of the immature dengue prM-containing particle

at 16-Å resolution; (E) cryoelectron reconstruction of the mature dengue virion at 12-Å resolution; (F) cryoelectron

reconstruction of Ross River alphavirus at 25-Å resolution. Panels A and C were provided by Richard J. Kuhn; panel B

is adapted from Figure 3c in Kuhn et al. (2002) with permission; panel D is adapted from Figure 3b in Y. Zhang et al.

(2003) with permission; panel E is reprinted from Figure 1a in W. Zhang et al. (2003) with permission; panel F is adapted

from Figure 4 in Strauss et al. (1995) with permission.

The retroviruses have a nucleocapsid that forms initially

In gammaretroviruses, the capsid forms during budding,

using spherical symmetry principles. Cleavage of Gag during

and the nucleocapsid is round and centrally located in the

virus maturation results in a nucleocapsid that is not icosa-

mature virion. This process is illustrated in Fig. 2.21C for

hedral and that is often eccentrically located in the virion.

murine leukemia virus. The top micrograph shows a bud-

Fig. 2.21A presents a schematic of a retrovirus particle that

ding particle with a partially assembled capsid. The bottom

illustrates the current model for the location of the various

micrograph shows a mature virion.

proteins after cleavage of Gag and GagPol. Figs. 2.21B, C,

In the lentiviruses, of which HIV is a member, the cap-

and D show electron micrographs of budding virus particles

sid also forms as a distinct structure only during budding.

and of mature extracellular virions for three genera of retrovi-

In the top panel of Fig. 2.21D is shown a budding particle

ruses. Betaretrovirus particles usually mature by the formation

of bovine immunodeficiency virus. After cleavage of Gag

of a nucleocapsid within the cytoplasm that then buds through

to form the mature virion, the capsid usually appears cone

the plasma membrane. This process is shown in Fig. 2.21B for

shaped or bar shaped (bottom panel of Fig. 2.21D).

mouse mammary tumor virus. In the top micrograph in Fig.

MasonPfizer monkey virus is a betaretrovirus whose

2.21B, preassembled capsids are seen in the cytoplasm. In the

capsid is cone shaped and centrally located in the mature

middle micrograph, budding of the capsid through the plasma

virion. A single amino acid change in the matrix protein

membrane is illustrated. In the bottom micrograph, a mature

MA determines whether the capsid preassembles and then

virion with an eccentrically located capsid is shown.

buds, or whether the capsids assemble during budding.

Immature Virion

Mature Virion

prM(18 kDa)

E dimer

E(~50 kDa)

Membrane

M(8 kDa)

Nucleocapsid

RNA:~11 kb

C protein: 13 kDa

(prM/E)3 heterotrimer

E-E homodimer

III

M-H

E-H

E-H2

FIGURE 2.19 (A) Schematic representation of maturation of dengue virus. In the immature particle, as shown in panels

(A) and (D) of Fig. 2.18, three heterodimers of E and prM come together to form a heterotrimer. Upon cleavage of prM to

M and pr, E collapses onto the surface of the mature particle (panels B and E in Fig. 2.18) as homodimers. (B) Diagram

of the dengue virus E protein in the mature particle as derived from cryoEM. This shows the three domains colored in the

same way as in Figs. 2.17 and 2.18 as well as the locations of the transmembrane and intramembrane helices in blue and

orange for E and M, respectively. Reprinted from Figure 4a in W. Zhang et al. (2003) with permission.

100 nm

FIGURE 2.20 Two views of herpes simplex virus. (A) Cutaway schematic representation showing the outer envelope

with projecting spikes, the irregular inner margin of the envelope due to the tegument, and the icosahedral core containing

162 capsomeres in a T=16 arrangement. One of the triangular faces of the icosahedron is outlined. Adapted from Murphy

et al. (1995) p. 114. (B) Negatively stained electron micrograph of an intracellular particle of bovine herpesvirus.

B. Betaretrovirus

C. Gammaretrovirus

D. Lentivirus

Genome

gag

env

pol

CAP

pro

Pol Proteins

Env Proteins

Gag Proteins

INT

PRO

Lipid Bilayer

RNA

FIGURE 2.21 Structure of retrovirus particles. (A) Schematic cross section through a retrovirus particle. The lipid

bilayer surrounds the particle and has imbedded in it trimeric spikes composed of surface (SU) and transmembrane (TM)

envelope proteins. The internal nonglycosylated proteins are encoded by the gag gene and include NC, the nucleocapsid

protein complexed with the genomic RNA, CA, the major capsid protein, and MA, the matrix protein that lines the inner

surface of the membrane. Other components include RT, the reverse transcriptase, IN, the integrase, and PR, the protease.

Adapted from Coffin et al. (1997), Retroviruses, p. 2. (B) Electron micrographs of mouse mammary tumor virus particles.

Top: intracytoplasmic particles; middle: budding particles; bottom: mature extracytoplasmic particles. (C) Electron

micrographs of murine leukemia virus particles. Top: budding particles; bottom: mature extracytoplasmic particles.

(D) Electron micrographs of bovine immunodeficiency virus. Top: budding particles; bottom: mature extracytoplasmic

particles. Adapted from Coffin et al. (1997), Retroviruses, p. 30.

Thus, the point at which capsids assemble does not reflect

between the nucleocapsid and the glycoproteins. The lack of

a fundamental difference in retroviruses. Preassembly of

such interactions permits these viruses to form pseudotypes,

capsids or assembly during budding appears to depend on

in which glycoproteins from other viruses substitute for

the stability of the capsid in the cell. Stable capsids can pre-

those of the virus in question. Pseudotypes are also formed

assemble. Unstable capsids require interactions with other

by retroviruses.

viral components to form as a recognizable structure.

The structures of paramyxoviruses and orthomyxoviruses

are illustrated schematically in Fig. 2.22. The helical nucleo-

capsids contain a major nucleocapsid protein called N or NP,

Enveloped Viruses with Helical

and the minor proteins P (NS1) and L (PB1, PB2, PA), as shown.

Nucleocapsids

There is a matrix protein M (M1) lining the inside of the lipid

The coronaviruses and the minus-strand RNA viruses

bilayer and also two glycoproteins anchored in the bilayer that

have nucleocapsids with helical symmetry. The structures

form external spikes. The two glycoproteins, called F and HN

of the mature virions are irregular, with the exception of

in paramyxoviruses and HA and NA in orthomyxoviruses,

the rhabdoviruses, and the glycoprotein composition is not

do not form heterodimers but rather form homooligomers so

invariant. Because of the lack of regularity in these viruses,

that there are two different kinds of spikes on the surface of

as well as the lack of symmetry, detailed structural studies of

the virions. HA in the orthomyxoviruses forms homotrim-

virions have not been possible. The lack of regularity arises

ers whereas NA forms homotetramers, and the two types of

in part because in these viruses there is no direct interaction

spikes can be distinguished in the electron microscope if the

Paramyxovirus genome

Names of measles virus

gene products

Symbols

NS1

PB1, PB2, PA

Comparable influenza

virus gene products

A paramyxovirus

particle

Orthomyxovirus

particles

Lipid Bilayer

RNA

FIGURE 2.22 Morphology of orthomyxoviruses and paramyxoviruses. (A) Schematic of the genome organization

of a paramyxovirus, Sendai virus. The names of the gene products and symbols to be used in the diagram are indicated.

Also shown are the comparable gene products of influenza virus, an orthomyxovirus. (B) Schematic cutaway view of

an orthomyxovirus or paramyxovirus particle. The nucleocapsid consists of a helical structure made up of the RNA

complexed with many copies of the nucleocapsid protein. This internal structure also contains a few molecules of the

RNA polymerase L (or PB1, PB2, PA in influenza virus), and P (or NS1). The nucleocapsid is enveloped in a lipid bilayer

derived from the host cell in which are embedded two different glycoproteins, F and HN (or HA and NA in influenza

virus) and which is lined on the inner surface with the matrix protein M. (C) Electron micrograph of a thin section of

a measles particle. This photo was taken by Cynthia S. Goldsmith and obtained from the Public Health Image Library

(PHIL). (D) Electron micrograph of a negatively stained influenza virus particles. This photo was taken by Fred Murphy

and obtained from PHIL.

resolution is high enough. As occurs in many enveloped RNA

The structures of rhabdoviruses and filoviruses are

viruses, F and HA are produced as precursors that are cleaved

illustrated in Fig. 2.23. The rhabdoviruses assemble into

by furin during transport of the proteins. Cleavage is required

bullet-shaped or bacilliform particles in which the helical

to activate the fusion peptide of the virus, which is found at the

nucleocapsid is wound in a regular elongated spiral con-

N terminus of the C-terminal product (see Fig. 1.6). Electron

formation (Figs. 2.23B and C). The virus encodes only five

micrographs of virions are shown in Figs. 2.22C and D. The

proteins (Fig. 2.23A), all of which occur in the virion (Fig.

particles in the preparations shown are round and reasonably

2.23B). The nucleocapsid contains the major nucleocapsid

uniform, but in other preparations the virions are pleomorphic

protein N and the two minor proteins L and NS. The matrix

baglike structures that are not uniform in appearance. In fact,

protein M lines the inner surface of the envelope, and G is an

clinical specimens of some orthomyxoviruses and paramyxo-

external glycoprotein that is anchored in the lipid bilayer of

viruses are often filamentous rather than round, illustrating the

the envelope. Budding is from the plasma membrane (Fig.

flexible nature of the structure of the virion. The micrograph

2.23D).

of the paramyxovirus measles virus shown in Fig. 2.22C is a

The filoviruses are so named because the virion is fila-

thin section and illustrates the lack of higher order structure

mentous. A schematic diagram of a filovirus is shown in Fig.

in the internal helical nucleocapsid. The micrograph of the

2.23E, and electron micrographs of two filoviruses, Marburg

orthomyxovirus influenza A virus shown in Fig. 2.22D is of

virus and Ebola virus, are shown in Figs. 2.23F and G. Notice

a negative-stained preparation and illustrates the spikes that

that in the electron microscope, filovirus virions often take

decorate the virus particle.

the shape of a shephard's crook or the number 6.

Genome organization

Viral proteins

Lipid Bilayer

RNA

Other viral components

500 nm

FIGURE 2.23 Morphology of the Rhabdoviridae and Filoviridae. (A) Genome organization of vesicular stomatitis

virus (VSV), a Vesiculovirus, with the symbols for the various viral components shown below. (B) Cutaway diagram of

a VSV particle. (C) A negatively stained electron micrograph of VSV virions. (D) Surface replica of a chicken embryo

fibroblast infected with VSV. Note that the magnification is approximately 1/10 of that shown in (C). (E) Diagram

of a filovirus, using the same color code for the components. (F) Negatively stained preparation of Marburg virus.

(G) Filamentous forms of Ebola (Reston) virus. (B) is from Simpson and Hauser (1966); (F) and (G) are from Murphy

et al. (1995) p. 289; and (D) is from Birdwell and Strauss (1975).

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