all replication - competent retroviruses encode genes for three major proteins : gag , pol , and env .
complex retroviruses , such as human immunodeficiency virus type 1 ( hiv-1 ) , encode additional regulatory and accessory proteins required for efficient replication in host cell or the infected host organism .
gag , an essential retroviral protein , is necessary and sufficient for the assembly , budding , and release of virus - like particles ( vlps ) in all types of retroviruses except the spumaviruses .
gag is synthesized on cytosolic ribosomes and is assembled as a polyprotein precursor . during and/or shortly after budding and release , the polyprotein is cleaved into several domains by the viral protease ( figure 1 ) as reviewed in [ 13 ] .
the major domains of the precursor gag are the matrix ( ma ) , capsid ( ca ) , and nucleocapsid ( nc ) .
the primary role of the n - terminal ma domain is targeting of the gag precursor protein to the site of assembly , typically the plasma membrane ( pm ) .
in general , electrostatic interactions between basic amino acid residues in ma and the acidic inner leaflet of the pm are important for gag - membrane targeting [ 4 , 5 ] . in the case of hiv-1 , the n - terminal myristate group and a cluster of basic residues in the ma domain of the hiv-1 gag that interacts with phosphatidylinositol-4,5-bisphosphate ( pi(4,5)p2 ) together
although the gag - membrane targeting of both murine leukemia virus ( mlv ) and mason - pfizer monkey virus ( mpmv ) is also affected by pi(4,5)p2 modulation [ 8 , 9 ] , it has been reported that the membrane targeting of rous sarcoma virus ( rsv ) and human t - lymphotropic virus type 1 ( htlv-1 ) is largely independent of pi(4,5)p2 [ 10 , 11 ] .
the ma domain also plays a role in the incorporation of the env glycoprotein into virions .
the ca domain is important for gag - gag interactions during virus assembly and constitutes the outer part of the viral core after gag processing by the viral protease [ 1214 ] .
this small , basic domain is responsible for the binding and incorporation of the viral rna genome into virions , which is mediated by gag interactions with genomic rna .
many studies demonstrate that the major site of hiv-1 assembly is the pm [ 1518 ] , although late endosomes could be a platform for virus assembly under specific conditions . in primary macrophages
however , studies have recently suggested that the above vesicles are not late endosomes but rather membrane invaginations connected to the pm [ 2022 ] .
in addition to gag , the other major structural retroviral protein is the env glycoprotein .
env proteins are required for virus entry into target cells and are thus essential for forming infectious retroviral particles . in this paper
, we discuss current knowledge about the biosynthesis , intracellular trafficking , and virion incorporation of retroviral env proteins , as well as the membrane microdomains involved in virus assembly and/or transfer .
retroviral env glycoproteins are synthesized from a spliced form of the viral genomic rna as reviewed in [ 2325 ] ( figure 1 ) .
translation of the env protein occurs on ribosomes bound to the endoplasmic reticulum ( er ) and starts with the leader sequence , which contains a small , n - terminal hydrophobic signal peptide .
the env protein is cotranslationally inserted into the lumen of the rough er . in the er , the leader sequence is removed by cellular signal peptidases .
in addition , env polypeptides are n- and o - glycosylated and subsequently trimmed [ 26 , 27 ] .
the hydrophobic transmembrane ( tm ) domain prevents env proteins from being fully released into the lumen of the er [ 28 , 29 ] .
the amino acid sequence following the tm is referred to as the cytoplasmic tail ( ct ) , which varies from 30 to around 150 residues , depending on the virus .
the hiv-1 accessory protein vpu binds to the cd4 receptor through its cytoplasmic domain and downregulates the receptor by transporting it to the proteasome for degradation , thereby preventing premature interactions between env and its receptor [ 3436 ] . in the golgi ,
cleavage of the retroviral env precursor occurs at a polybasic ( e.g. , k / r - x - k / r - r ) motif by cellular proteases such as furin or closely related enzymes probably within or near the trans - golgi network ( tgn ) [ 3743 ] . for hiv-1 , the surface glycoprotein gp120 and the tm glycoprotein gp41 , which bind together noncovalently ,
gp160 processing is essential for the activation of env fusogenicity and virus infectivity [ 38 , 42 , 4446 ] . similarly ,
cleavage of env is also essential for membrane fusion and virus infectivity in mlv [ 39 , 4750 ] , in rsv [ 51 , 52 ] , and in mouse mammary tumor virus ( mmtv ) .
a recent report showed that cleavage of mlv env by furin also plays an important role in env intracellular trafficking and incorporation .
although most retroviral env proteins including that of hiv-1 are associated with intracellular membranes [ 5557 ] , at least part of the gp120/gp41 trimer complex traffics through the secretory pathway to the pm .
it has been suggested that ap-1 , one of adaptor proteins for clathrin - coated vesicle formation , is involved in the correct sorting of hiv-1 env from the tgn to the pm , [ 58 , 59 ] .
it has been reported that intracellular ctla-4-containing secretory granules are involved in the trafficking of hiv-1 env to the pm although the subsequent trafficking of env after the golgi is not well understood . after reaching the pm , like those of other lentiviruses , hiv-1 env undergoes rapid endocytosis , which is mediated by the interaction between the 2 subunit of the clathrin adaptor ap-2 and a membrane - proximal , tyr - based motif ( yxxl ) in the gp41 ct
although some of the endocytosed env is recycled back to the pm , most retroviral env is associated with intracellular membranes [ 63 , 64 ] .
maintaining low levels of env at the cell surface allows the infected cells to evade the host immune response and to avoid induction of env - mediated apoptosis .
gammaretroviruses such as mlv and mpmv also have dileucine- and tyr - based motifs in their env ct .
these motifs are important to regulate intracellular trafficking of env of both retroviruses via interactions with clathrin adaptors [ 65 , 66 ] . as for pseudotyping of gammaretroviruses
, it has been reported that the feline endogenous retrovirus rd114 env does not allow pseudotyping with viral cores from lentiviruses such as siv , whereas the rd114 env is incorporated into mlv virions [ 6769 ] .
intracellular trafficking of gag and env was examined using a set of chimeric viruses between mlv and rd114 .
interestingly , it was found that the rd114 env was mainly localized along the secretory pathway , whereas the mlv env was mostly localized in endosomes , and that intracellular localization was dependent on specific motifs in the env ct .
in addition , subsequent work revealed that an acidic cluster in the rd114 env ct regulates assembly of not only the rd114 env but also the mlv env through the interaction with a host factor , phosphofurin acidic - cluster - sorting protein 1 .
several models have been proposed for the incorporation of retroviral env glycoprotein into virions as reviewed in [ 23 , 70 ] ( figure 2 ) .
passive incorporation is the simplest model for the incorporation of env proteins into virus particles ( figure 2(a ) ) .
first , viral pseudotyping with a foreign glycoprotein can occur easily in many cases although there are some exceptions , one of which is the exclusion of hiv-1 or siv env with the long ct from most retrovirus cores . with respect to hiv-1 , the virus can be pseudotyped with env glycoproteins not only from several other retroviruses but also with those from other virus families such as ortho ( para ) myxoviruses and flaviviruses [ 7184 ] .
second , retroviruses allow passive incorporation of host membrane proteins into virus particles [ 8587 ] .
most cellular proteins are incorporated into the retrovirus envelope without significant sorting [ 88 , 89 ] . finally , in the case of hiv-1
, several studies have demonstrated that the gp41 ct can be removed without affecting incorporation of the env into virions , although this has been shown to occur only for some laboratory cell lines such as hela or 293 t [ 9094 ] .
although several lines of evidence support the passive incorporation model for retroviral env , there is much evidence indicating that env incorporation into virions is regulated by direct interactions between gag and env proteins ( figure 2(b ) ) .
although removal of the gp41 ct sequence of hiv-1 has little effect on env incorporation in some cell types , as described above , smaller deletions in ct regions cause severe defects in env incorporation [ 95100 ] .
the ma domain of gag has been shown to be important for env incorporation into virions [ 91 , 92 , 101 , 102 ] .
the defect in env incorporation caused by deletion of the gp41 ct is reversed by several ma mutations , indicating that an interaction between env and the ma domain of gag is required for incorporation of full - length env into virions , at least in the case of hiv-1 [ 93 , 98 ] .
more evidence for direct gag - env interaction comes from the finding that hiv-1 env directs gag budding to the basolateral surface of polarized epithelial madin - darby canine kidney ( mdck ) cells through the ct of hiv-1 env , whereas gag alone buds in a nonpolarized fashion [ 103106 ] . the tyr - based motif in the gp41 ct is also utilized in polarized budding of hiv-1 in lymphocytes .
surprisingly , the polarized budding of hiv-1 in mdck cells could also be promoted by mlv and htlv-1 env through their ct .
it also has been reported that coexpression of pr55 inhibits endocytosis of hiv-1 env through its interaction with the gp41 ct .
another example of the specific gag - env interactions was demonstrated using gag and env proteins of mlv and hiv-1 in rat neurons .
similarly , mlv env is preferentially recruited onto mlv gag through its ct domain in the presence of both mlv and hiv-1 cores although the authors also show an alternative mechanism by which the recruitment to hiv-1 budding sites is independent of the ct domain of mlv env .
furthermore , rsv env is exclusively recruited to rsv budding sites through its ct , suggesting that the interaction between env and gag is direct in the case of this avian retrovirus .
in addition to the circumstantial evidence discussed above , some biochemical data suggest a direct interaction between gag and env . in vitro binding between ma and a gp41 ct - gst fusion protein has been reported for both hiv-1 and siv [ 112 , 113 ] .
peptides corresponding to a large central domain of gp41 ct inhibited the capture of membrane - free pr55 with an anti - p24 antibody .
in addition , a stable , detergent - resistant gp41-pr55 interaction was detected in immature hiv-1 virions .
the retention of gp41 in detergent - treated virions is dependent on the ct region , suggesting a direct or indirect interaction between pr55 and gp41 [ 115 , 116 ] . in the third model
, it is assumed that host cellular factors ( mostly proteins ) play a role in bridging gag and env in virus - infected cells ( figure 2(c ) ) .
several host factors have been reported to bind to gag and/or env of hiv-1 or siv however , only a couple of host factors were shown to be required for env incorporation and/or viral replication . the 47-kda tail - interacting protein ( tip47 )
has been reported to bridge gag and env , allowing efficient env incorporation in hiv-1 [ 117 , 118 ] .
the same group also showed that both the we motif near the n - terminus of the ma domain and the yw motif in the gp41 ct domain are important for interactions between gag or env and tip47 . in a subsequent paper ,
the same group showed that mutations in either the we motif of ma or the yw motif in the gp41 ct caused defects in virus replication in primary monocyte - derived macrophages .
although this finding of an important role for tip47 in env incorporation in hiv-1 has received much attention from retrovirologists , no confirmatory data have been published by other researchers in this field .
human discs large protein ( hdlg1 ) has been reported to interact with the ct of htlv-1 env and to colocalize with both env and gag in virus - infected cells .
subsequent work demonstrated that dlg1 also binds hiv-1 gag and that the expression level of dlg1 is inversely correlated with hiv-1 env expression and incorporation levels of the env proteins , although the mechanism behind this phenomena needs to be investigated .
prenylated rab acceptor 1 ( pra1 ) , which was identified as a rab regulatory protein , was reported to be a binding partner for the siv gp41 ct in a mammalian yeast two - hybrid ( y2h ) assay .
although colocalization of pra1 and siv env was observed , changes in the endogenous levels of pra1 did not affect virus production , env incorporation , or infectivity of siv or hiv-1 .
a prohibitin 1/prohibitin 2 ( phb1/phb2 ) heterodimer was identified as a binding partner of the gp41 ct of hiv-1 using human t - cell lines and tandem affinity chromatography .
phb1 and phb2 are members of the prohibitin superfamily of proteins , which are localized to several cellular compartments such as the mitochondria , nucleus , and the pm [ 125 , 126 ] .
gp41 ct mutants , in which binding to phb1/phb2 is disrupted , could replicate well in permissive cell types such as mt-4 , but could not replicate efficiently in nonpermissive h9 cells .
further analysis is necessary to elucidate the mechanism by which these proteins regulate virus replication through interactions with env .
luman , a transcription factor that is mainly localized to the er , was found to interact with the gp41 ct of hiv-1 in a y2h screen using a cdna library from human peripheral blood lymphocytes ( pbl ) .
overexpression of a constitutively active form of this protein reduced the intracellular levels of gag and env , leading to a decrease in virus release .
the mechanism for this negative effect on virus assembly involves luman binding to tat , which decreases tat - medicated transcription . by using a y2h screen with human cdna libraries , p115-rhogef , an activator of rho gtpase , was found to interact with the gp41 ct through its c - terminal regulatory domain .
the gp41 mutants that lost the ability to bind p115 showed impaired replication kinetics in t - cell lines such as supt1 , h9 , and jurkat , suggesting that the gp41 ct could modulate the activity of p115-rhogef to support virus replication .
in addition to the host factors described above , calmodulin [ 129132 ] and -catenin [ 133135 ] have been reported to interact with hiv-1 and/or siv .
however , their roles in virus replication , especially with respect to the env functions of both proteins , have not been clearly elucidated .
regardless of whether direct or indirect interactions between retroviral gag and env proteins are required for env incorporation into virions , a great deal of experimental evidence suggests that retroviruses assemble and bud from  membrane microdomains . 
the most well - known microdomains are  lipid raft(s ) ,  which are enriched in cholesterol and sphingolipids [ 136 , 137 ] .
lipid rafts are widely thought to function as a platform for the assembly of protein complexes and to allow various biological processes such as cellular transport and signal transduction to proceed efficiently [ 138 , 139 ] .
lipid rafts are reportedly used as assembly platforms or entry scaffolds in the replication of enveloped viruses such as retroviruses [ 140146 ] .
the association of gag / env with lipid rafts is important for the regulation of env incorporation and pseudotyping [ 143 , 144 , 147 , 148 ] .
evidence that both the hiv-1 pr55 and env proteins are preferentially localized to lipid rafts comes from biochemical studies as well as direct observations by microscopy [ 142 , 149 , 150 ] .
another membrane microdomain for retrovirus assembly is the  tetraspanin - enriched microdomain ( tem )  [ 151154 ] .
tetraspanins are a superfamily of cell surface proteins that are ubiquitously expressed in mammalian cells .
tems have been reported to be involved in the assembly and release of not only hiv-1 , but also htlv-1 and hcv . when both hiv-1 and influenza virus were produced in the same cell
, only hiv-1 colocalized with the tem marker , and its release was inhibited by an anti - cd9 ab , which led to extensive aggregation of tetraspanins .
analysis of dynamics of both lipid rafts and tems by quantitative microscopy has revealed that components of both lipid rafts and tems are recruited during viral assembly to create a new microdomain that is different from preexisting membrane microdomains [ 153 , 157 ] .
there have been three recent reports in which both pseudotyping and microdomain issues were discussed . in the first paper ,
the authors examined hiv-1 assembly under conditions where the env proteins of hiv-1 and ebola virus were coexpressed with hiv-1 gag in the same cell .
interestingly , however , the virions contained either hiv-1 env or ebola virus glycoprotein ( gp ) , but not both env proteins within a single virion .
these results suggest that hiv-1 env and ebola virus gp localized to distinct microdomains on the surface of the same cell . in the second paper ,
the subcellular localization of gag and env proteins was investigated using a combination of three different retroviral env proteins ( rsv env , mlv env , or vesicular stomatitis virus ( vsv ) g ) and two different gag proteins ( rsv or hiv-1 ) .
both vsv - g and mlv env were redistributed to the virus budding sites when coexpressed with hiv-1 or rsv gag .
a subsequent paper from the same group showed that the ct of mlv is not required for recruitment of mlv env to hiv-1 budding sites , suggesting that there are no specific interactions between mlv env and hiv-1 gag .
collectively , these results also suggest that retroviral env glycoproteins are not recruited to preexisting membrane platforms but rather that they are actively recruited to newly formed microdomains on the cell surface .  
human retroviruses such as hiv-1 and htlv-1 spread more efficiently between target t cells by cell - cell infection than by cell - free infection [ 159 , 160 ] .
, the  virological synapse ( vs )  as a point of contact between virus - infected cells and uninfected cells [ 161 , 162 ] . the molecular mechanisms of retroviral vs formation are as follows . ( 1 ) with respect to hiv-1 t - cell vs , initial contact between virus - infected cells and uninfected cells occurs through gp120-cd4 binding .
( 2 ) the gp120-cd4 interaction recruits cd4 , coreceptors such as cxcr or ccr5 , adhesion molecules , and filamentous actin into the synaptic area .
( 3 ) the cellular secretory machinery and microtubule organizing centers ( mtoc ) are polarized towards the hiv-1 assembly sites at the pm to form the vs. it has been reported that a so - called microsynapse formed by nanotubes between virus - infected cells and uninfected cells is also involved in cell - cell infection of hiv-1 [ 84 , 163 ] . in cell - cell transfer of htlv-1-infected cells ,
an extracellular matrix structure referred to as the  viral biofilm  was proposed as an alternative to the vs .
in addition to hiv-1 and htlv-1 , the spread of mlv between fibroblasts also occurs via the vs [ 165 , 166 ] .
it is noteworthy that assembly of mlv is directed towards cell - cell contact sites through the interaction of the ct of mlv env with gag [ 167 , 168 ] .
although the concept of cell - cell infection through the vs is now well appreciated , the detailed molecular mechanism of vs assembly and its relevance to viral spread in vivo will require further elucidation through the use of more advanced techniques .
although this paper has introduced several experimental models for retroviral env trafficking and/or incorporation , the correct mechanism for this process is still unclear .
the following questions must be clearly addressed to not only gain a better understanding of this complex biological process , but also to develop new antiretroviral compounds that target env incorporation.what are the structures of the cts of retroviral env proteins ? the answers for this question will give useful information on elucidating a role of the env cts in the env trafficking and/or incorporation in virus - infected cells.what host factor(s )
are necessary for the retroviral env trafficking and/or incorporation into virions?where and how env and gag proteins of retroviruses are recruited to the assembly sites in order to form infectious virus particles ?
the answers for this question will give useful information on elucidating a role of the env cts in the env trafficking and/or incorporation in virus - infected cells .
what host factor(s ) are necessary for the retroviral env trafficking and/or incorporation into virions ?
where and how env and gag proteins of retroviruses are recruited to the assembly sites in order to form infectious virus particles ?
 together with the gag protein , the env glycoprotein is a major retroviral structural protein and is essential for forming infectious virus particles . 
 env is synthesized , processed , and transported to certain microdomains at the plasma membrane and takes advantage of the same host machinery for its trafficking as that used by cellular glycoproteins . 
 incorporation of env into progeny virions is probably mediated by the interaction between env and gag , in some cases with the additional involvement of certain host factors . 
 although several general models have been proposed to explain the incorporation of retroviral env glycoproteins into virions , the actual mechanism for this process is still unclear , partly because structural data on the env protein cytoplasmic tail is lacking . 
 this paper presents the current understanding of the synthesis , trafficking , and virion incorporation of retroviral env proteins . 
1. Introduction
2. Env Biosynthesis and Trafficking to the Plasma Membrane
3. Env Incorporation into Virions
4. Membrane Microdomains
5. Conclusions and Perspectives