Infectivity of Moloney murine leukemia virus defective in late assembly events is restored by late assembly domains of other retroviruses

The p12 region of the Moloney murine leukemia virus (M-MuLV) Gag protein contains a PPPY motif important for efficient virion assembly and release. To probe the function of the PPPY motif, a series of insertions of homologous and heterologous motifs from other retroviruses were introduced at various positions in a mutant gag gene lacking the PPPY motif. The assembly defects of the PPPY deletion mutant could be rescued by insertion of a wild-type PPPY motif and flanking sequences at several ectopic positions in the Gag protein. The late assembly domain (L-domain) of Rous sarcoma virus (RSV) or human immunodeficiency virus type 1 (HIV-1) could also fully or partially restore M-MuLV assembly when introduced into matrix, p12, or nucleocapsid domains of the mutant M-MuLV Gag protein lacking the PPPY motif. Strikingly, mutant viruses carrying the RSV or the HIV-1 L-domain at the original location of the deleted PPPY motif were replication competent in rodent cells. These data suggest that the PPPY motif of M-MuLV acts in a partially position-independent manner and is functionally interchangeable with L-domains of other retroviruses. Electron microscopy studies revealed that deletion of the entire p12 region resulted in the formation of tube-like rather than spherical particles. Remarkably, the PPPY deletion mutant formed chain structures composed of multiple viral particles linked on the cell surface. Many of the mutants with heterologous L-domains released virions with wild-type morphology.     

Analysis of bovine leukemia virus gag membrane targeting and late domain function

Assembly of retrovirus-like particles only requires the expression of the Gag polyprotein precursor. We have exploited this in the development of a model system for studying the virus particle assembly pathway for bovine leukemia virus (BLV). BLV is closely related to the human T-cell leukemia viruses (HTLVs), and all are members of the Deltaretrovirus genus of the Retroviridae family. Overexpression of a BLV Gag polyprotein containing a carboxy-terminal influenza virus hemagglutinin (HA) epitope tag in mammalian cells led to the robust production of virus-like particles (VLPs). Site-directed mutations were introduced into HA-tagged Gag to test the usefulness of this model system for studying certain aspects of the virus assembly pathway. First, mutations that disrupted the amino-terminal glycine residue that is important for Gag myristylation led to a drastic reduction in VLP production. Predictably, the nature of the VLP production defect was correlated to Gag membrane localization. Second, mutation of the PPPY motif (located in the MA domain) greatly reduced VLP production in the absence of the viral protease. This reduction in VLP production was more severe in the presence of an active viral protease. Examination of particles by electron microscopy revealed an abundance of particles that began to pinch off from the plasma membrane but were not completely released from the cell surface, indicating that the PPPY motif functions as a late domain (L domain).     

Mutations in the PPPY motif of vesicular stomatitis virus matrix protein reduce virus budding by inhibiting a late step in virion release

The N terminus of the matrix (M) protein of vesicular stomatitis virus (VSV) and of other rhabdoviruses contains a highly conserved PPPY sequence (or PY motif) similar to the late (L) domains in the Gag proteins of some retroviruses. These L domains in retroviral Gag proteins are required for efficient release of virus particles. In this report, we show that mutations in the PPPY sequence of the VSV M protein reduce virus yield by blocking a late stage in virus budding. We also observed a delay in the ability of mutant viruses to cause inhibition of host gene expression compared to wild-type (WT) VSV. The effect of PY mutations on virus budding appears to be due to a block at a stage just prior to virion release, since electron microscopic examination of PPPA mutant-infected cells showed a large number of assembled virions at the plasma membrane trapped in the process of budding. Deletion of the glycoprotein (G) in addition to these mutations further reduced the virus yield to less than 1% of WT levels, and very few particles were assembled at the cell surface. This observation suggested that G protein aids in the initial stage of budding, presumably during the formation of the bud site. Overall, our results confirm that the PPPY sequence of the VSV M protein possesses L domain activity analogous to that of the retroviral Gag proteins.     

A Proline-Rich Motif (PPPY) in the Gag Polyprotein of Mason-Pfizer Monkey Virus Plays a Maturation-Independent Role in Virion Release

Virus assembly represents one of the last steps in the retrovirus life cycle. During this process, Gag polyproteins assemble at specific sites within the cell to form viral capsids and induce membrane extrusion (viral budding) either as assembly progresses (type C virus) or following formation of a complete capsid (type B and type D viruses). Finally, the membrane must undergo a fusion event to pinch off the particle in order to release a complete enveloped virion. Structural elements within the MA region of the Gag polyprotein define the route taken to the plasma membrane and direct the process of virus budding. Results presented here suggest that a distinct region of Gag is necessary for virus release. The pp24 and pp16 proteins of the type D retrovirus Mason-Pfizer monkey virus (M-PMV) are phosphoproteins that are encoded in the gag gene of the virus. The pp16 protein is a C-terminally located cleavage product of pp24 and contains a proline-rich motif (PPPY) that is conserved among the Gag proteins of a wide variety of retroviruses. By performing a functional analysis of this coding region with deletion mutants, we have shown that the pp16 protein is dispensable for capsid assembly but essential for virion release. Moreover, additional experiments indicated that the virus release function of pp16 was abolished by the deletion of only the PPPY motif and could be restored when this motif alone was reinserted into a Gag polyprotein lacking the entire pp16 domain. Single-amino-acid substitutions for any of the residues within this motif confer a similar virion release-defective phenotype. It is unlikely that the function of the proline-rich motif is simply to inhibit premature activation of protease, since the PPPY deletion blocked virion release in the context of a protease-defective provirus. These results demonstrate that in type D retroviruses a PPPY motif plays a key role in a late stage of virus budding that is independent of and occurs prior to virion maturation.     

Phosphorylated serine residues and an arginine-rich domain of the moloney murine leukemia virus p12 protein are required for early events of viral infection

Mutational analyses of the p12 Gag phosphoprotein of Moloney murine leukemia virus have demonstrated its participation in both virus assembly and the early stages of infection. The molecular mechanisms by which p12 functions in these events are still poorly understood. We performed studies to examine the significance of p12 phosphorylation in the viral life cycle. Alanine substitutions were introduced at the potential phosphorylation sites in p12, and the resulting mutants were tested for replication. Mutant viruses with changes at S61 and S78 were severely impaired, whereas the other mutant viruses were viable. S61 was shown to be required for normal levels of phosphorylation of p12 in vivo. These defective mutant viruses showed no apparent alteration to Gag protein processing or reduction in the yield of virions after transient transfection, but the mutants failed to form circular viral DNAs in acutely infected cells. Sequence analysis of revertant clones derived from S(61,65)A mutant virus revealed two classes: one group with a single mutation at a residue adjacent to S61 and another group with mutations introducing new positive charges surrounding S61. In vivo [32P]orthophosphate labeling indicated that the rescue of the S(61,65)A mutant virus did not result in a significant increase in the phosphorylation level of p12. Alanine substitutions of an arginine-rich stretch near S61 (at R-66, -68, -70, and -71) resulted in the same phenotype as the S(61,65)A mutant virus. The restored function of S(61,65)A mutant virus by second or third site mutations may result from a structural change or the addition of positively charged residues in the arginine-rich region.     

The role of ITCH protein in human T-cell leukemia virus type 1 release

Human T-cell leukemia virus type 1 (HTLV-1) has two late domain (LD) motifs, PPPY and PTAP, which are important for viral budding. Mutations in the PPPY motif are more deleterious for viral release than changes in the PTAP motif. Several reports have shown that the interaction of PPPY with the WW domains of a Nedd4 (neuronal precursor cell-expressed developmentally down-regulated-4) family ubiquitin ligase (UL) is a critical event in virus release. We tested nine members of the Nedd4 family ULs and found that ITCH is the main contributor to HTLV-1 budding. ITCH overexpression strongly inhibited release and infectivity of wild-type (wt) HTLV-1, but rescued the release of infectious virions with certain mutations in the PPPY motif. Electron microscopy showed either fewer or misshapen virus particles when wt HTLV-1 was produced in the presence of overexpressed ITCH, whereas mutants with changes in the PPPY motif yielded normal looking particles at wt level. The other ULs had significantly weaker or no effects on HTLV-1 release and infectivity except for SMURF-1, which caused enhanced release of wt and all PPPY(-) mutant particles. These particles were poorly infectious and showed abnormal morphology by electron microscopy. Budding and infectivity defects due to overexpression of ITCH and SMURF-1 were correlated with higher than normal ubiquitination of Gag. Only silencing of ITCH, but not of WWP1, WWP2, and Nedd4, resulted in a reduction of HTLV-1 budding from 293T cells. The binding efficiencies between the HTLV-1 LD and WW domains of different ULs as measured by mammalian two-hybrid interaction did not correlate with the strength of their effect on HTLV-1 budding.     

The C terminus of human immunodeficiency virus type 1 matrix protein is involved in early steps of the virus life cycle.

Deletion mutations at the C terminus of the matrix (MA) protein of human immunodeficiency virus type 1 (HIV-1) were generated by site-directed mutagenesis. The resultant mutant viruses had a severe defect in virus infectivity. This defect did not involve late steps of the virus life cycle, as the synthesis and processing of the Gag polyprotein and the assembly and release of mutant virions were not greatly affected. The incorporation of viral proteins and the viral RNA genome was similar for mutant and wild-type virions. In contrast, the early steps of the virus life cycle were severely affected, as the synthesis of viral DNA postinfection was dramatically reduced in mutant-virus-infected cells. One stretch of amino acids that was deleted in one of the mutants has significant homology with a region in VP1 of the picornavirus family. This region of VP1 is presumably involved in poliovirus penetration into cells. These results suggest that in addition to its functional role in virus assembly, the MA protein of HIV-1, and possibly of other retroviruses, plays an important role in virus entry.     

pH-independent murine leukemia virus ecotropic envelope-mediated cell fusion: implications for the role of the R peptide and p12E TM in viral entry.

Murine leukemia virus ecotropic and amphotropic envelope expression vectors were genetically engineered to generate truncations of the p15E TM cytoplasmic tail. The ecotropic construct CEET has the entire cytoplasmic tail of TM deleted, while the CEETR construct has only the R peptide portion of the tail deleted, thereby producing a TM subunit (p12E) that is identical to the one found in mature virions. The analogous amphotropic constructs were called CAET and CAETR. These envelopes, as opposed to their p15E TM counterparts, mediate cell-to-cell fusion at neutral pH in both transformed and nontransformed cell lines. Though the TM cytoplasmic domain is not required, its presence appears to augment such cell-to-cell fusion. This envelope-dependent fusion requires the presence of the viral receptor on the cell surface. Ecotropic virions bearing the p12E TM contain wild-type levels of the envelope complex and have near-normal titers. In contrast, virions which lack the cytoplasmic domain of TM (e.g., CEET) have 10- to 100-fold-lower titers but contain normal amounts of envelope. Both of the corresponding amphotropic virions contain normal amounts of envelope but have 10- to 100-fold-lower titers. Using immunofluorescent detection of envelope to monitor the fate of receptor-bound virions, we found that ecotropic murine leukemia virus envelope disappears from the cell surface while amphotropic envelope persists on the cell surface after virus binding. This pattern of immunofluorescence is consistent with the proposed routes of cell entry for these viruses, i.e., by endocytosis and direct fusion, respectively. In this assay, ecotropic virions bearing the genetically engineered p12E TM also appear to be internalized despite the ability of their envelope to mediate fusion at neutral pH in the same target cells. Our results show that direct fusion at neutral pH is a natural consequence of the surface expression of the mature ecotropic envelope and its receptor. We propose that the processing of the R peptide from the envelope TM (p15E) to yield p12E, at the time of virus budding or within virions, renders the envelope competent to fuse.     

Characterization of Moloney murine leukemia virus p12 mutants blocked during early events of infection

Mutations affecting either the N- or C-terminal regions of the Gag protein p12 block replication of Moloney murine leukemia virus (M-MuLV). Viruses carrying mutations in this portion of gag can mediate the assembly and release of virions but are unable to successfully carry out the early phase of the M-MuLV life cycle. Wild-type and mutant viruses were found to synthesize similar levels of linear viral DNA in both cytoplasmic and nuclear fractions, and there were no significant differences in either the density or sedimentation of the viral protein-nucleic acid complexes. Analysis of the termini of the linear viral DNAs showed that the 3' ends of the mutant viral DNA were processed normally by the integrase. Further, the preintegration complexes extracted from the cytoplasm of cells infected with the mutant viruses were competent for integration into target DNA in vitro. Nevertheless, no circular viral DNAs were detected in cells infected by the mutants, and functional proviruses were not formed. These results suggest that p12 has an unexpected role in the early phase of the life cycle and is needed after viral DNA synthesis to deliver the incoming DNA to the correct location and in the appropriate state to permit either circularization or integration of the viral DNA in vivo.     

The matrix protein of human immunodeficiency virus type 1 is required for incorporation of viral envelope protein into mature virions.

Accumulating evidence suggests that the matrix (MA) protein of retroviruses plays a key role in virus assembly by directing the intracellular transport and membrane association of the Gag polyprotein. In this report, we show that the MA protein of human immunodeficiency virus type 1 is also critical for the incorporation of viral Env proteins into mature virions. Several deletions introduced in the MA domain (p17) of human immunodeficiency virus type 1 Gag polyprotein did not greatly affect the synthesis and processing of the Gag polyprotein or the formation of virions. Analysis of the viral proteins revealed normal levels of Gag and Pol proteins in these mutant virions, but the Env proteins, gp120 and gp41, were hardly detectable in the mutant virions. Our data suggest that an interaction between the viral Env protein and the MA domain of the Gag polyprotein is required for the selective incorporation of Env proteins during virus assembly. Such an interaction appears to be very sensitive to conformational changes in the MA domain, as five small deletions in two separate regions of p17 equally inhibited viral Env protein incorporation. Mutant viruses were not infectious in T cells. When mutant and wild-type DNAs were cotransfected into T cells, the replication of wild-type virus was also hindered. These results suggest that the incorporation of viral Env protein is a critical step for replication of retroviruses and can be a target for the design of antiviral strategies.     

Alteration of zinc-binding residues of simian immunodeficiency virus p8(NC) results in subtle differences in gag processing and virion maturation associated with degradative loss of mutant NC

In all retroviruses analyzed to date (except for the spumaretroviruses), the Zn(2+)-coordinating residues of nucleocapsid (NC) perform or assist in crucial reactions necessary to complete the retrovirus life cycle. Six replication-defective mutations have been engineered in the two NC Zn(2+) fingers (ZFs) of simian immunodeficiency virus [SIV(Mne)] that change or delete specific Zn(2+)-interacting Cys residues and were studied by using electron microscopy, reversed-phase high-performance liquid chromatography, immunoblotting, and RNA quantification. We focused on phenotypes of produced particles, specifically morphology, Gag polyprotein processing, and genomic RNA packaging. Phenotypes were similar among viruses containing a point or deletion mutation involving the same ZF. Mutations in the proximal ZF (ZF1) resulted in near-normal Gag processing and full-length genomic RNA incorporation and were most similar to wild-type (WT) virions with electron-dense, conical cores. Mutation of the distal ZF, as well as point mutations in both ZFs, resulted in more unprocessed Gag proteins than a deletion or point mutation in ZF1, with an approximate 30% reduction in levels of full-length genomic RNA in virions. These mutant virions contained condensed cores; however, the cores typically appeared less electron dense and more rod shaped than WT virions. Surprisingly, deletion of both ZFs, including the basic linker region between the ZFs, resulted in the most efficient Gag processing. However, genomic RNA packaging was approximately 10% of WT levels, and those particles produced were highly abnormal with respect to size and core morphology. Surprisingly, all NC mutations analyzed demonstrated a significant loss of processed NC in virus particles, suggesting that Zn(2+)-coordinated NC is protected from excessive proteolytic cleavage. Together, these results indicate that Zn(2+) coordination is important for correct Gag precursor processing and NC protein stability. Additionally, SIV particle morphology appears to be the result of proper and complete Gag processing and relies less on full-length genomic RNA incorporation, as dictated by the Zn(2+) coordination in the ZFs of the NC protein.     

Equine infectious anemia virus Gag p9 function in early steps of virus infection and provirus production

We have previously reported that serial truncation of the Gag p9 protein of equine infectious anemia virus (EIAV) revealed a progressive loss in replication phenotypes in transfected cells, such that a proviral mutant (E32) expressing the N-terminal 31 amino acids of p9 produced infectious virus particles similarly to parental provirus, while a proviral mutant (K30) with two fewer amino acids produced replication-defective virus particles, despite containing apparently normal levels of processed Gag and Pol proteins (C. Chen, F. Li, and R. C. Montelaro, J. Virol. 75:9762-9760, 2001). Based on these observations, we sought in the current study to identify the precise defect in K30 virion infection of permissive equine dermal (ED) cells. The results of these experiments clearly demonstrated that K30 virions entered target ED cells and produced early (minus-strand strong-stop) and late (Gag) viral DNA products as efficiently as did the replication-competent E32 mutant and parental EIAV(UK) viruses. However, in contrast to the replication-competent E32 mutant and parental viruses, infection with K30 mutant virus failed to produce detectable two-long-terminal-repeat DNA circles, stable integrated provirus, virus-specific Gag mRNA expression, or intracellular viral protein expression. Taken together, these data demonstrate that the K30 mutant is defective in the ability to produce sufficient nuclear viral DNA to establish a productive infection in ED cells. Thus, these observations indicate for the first time that the EIAV Gag p9 protein performs a critical role in viral DNA production and processing to provirus during EIAV infection, in addition to its previously defined role in viral budding mediated by the p9 L domain.     

Proline residues in human immunodeficiency virus type 1 p6(Gag) exert a cell type-dependent effect on viral replication and virion incorporation of Pol proteins

The C terminus of the HIV-1 Gag protein contains a proline-rich domain termed p6(Gag). This domain has been shown to play a role in efficient virus release and incorporation of Vpr into virions. In a previous study (X. F. Yu, L. Dawson, C. J. Tian, C. Flexner, and M. Dettenhofer, J. Virol. 72:3412-3417, 1998), we observed that the removal of the p6 domain of Gag as well as drastic mutations in the PTAP motif resulted in reduced virion-associated Pol proteins from transfected COS cells. In the present study, amino acid substitutions at residues 5 and 7 of p6(Gag) resulted in a cell type-dependent replication of the mutant virus in CD4(+) T cells; the virus was replication competent in Jurkat cells but restricted in H9 cells and primary blood-derived monocytes. Established Jurkat and H9 cell lines expressing p6(Gag) mutant and parental virus were used to further understand this defect. Mutant virions produced from H9 cells, which displayed no defect in extracellular virion production, showed an approximately 16-fold reduction in Pol protein levels, whereas the levels of Pol proteins were only marginally reduced in mutant virions produced from Jurkat cells. The reduction in the virion-associated Pol proteins could not be accounted for by differences in the levels of intracellular p160(Gag-Pol) or in the interaction between p55(Gag) and p160(Gag-Pol) precursors. Electron microscopic analysis of the p6(Gag) mutant virions showed a predominately immature morphology in the absence of significant defects in Gag proteolytic cleavage. Taken together, these data suggest that the proline-rich motif of p6(Gag) is involved in the late stages of virus maturation, which include the packaging of cleaved Pol proteins in viral particles, a process which may involve cell-type-specific factors.     

Late assembly motifs of human T-cell leukemia virus type 1 and their relative roles in particle release

Three late assembly domain consensus motifs, namely PTAP, PPPY, and LYPXL, have been identified in different retroviruses. They have been shown to interact with the cellular proteins TSG101, Nedd4, and AP2 or AIP, respectively. Human T-cell leukemia virus type 1 (HTLV-1) has a PPPY and a PTAP motif, separated by two amino acids, located at the end of MA, but only the PPPY motif is conserved in the deltaretrovirus group. Like other retroviral peptides carrying the late motif, MA is mono- or di-ubiquitinated. A mutational analysis showed that 90% of PPPY mutant particles were retained in the cell compared to 15% for the wild-type virus. Mutations of the PTAP motif resulted in a 20% decrease in particle release. In single-cycle infectivity assays, the infectious titers of late motif mutants correlated with the amounts of released virus, as determined by an enzyme-linked immunosorbent assay. We observed binding of MA to the WW domains of the Nedd4 family member WWP1 but not to the amino-terminal ubiquitin E2 variant domain of TSG101 in mammalian two-hybrid analyses. The binding to WWP1 was eliminated when the PPPY motif was mutated. However, MA showed binding to TSG101 in the yeast two-hybrid system that was dependent on an intact PTAP motif. A dominant-negative (DN) mutant of WWP1 could inhibit budding of the intact HTLV-1 virus. In contrast, DN TSG101 only affected the release of virus-like particles encoded by Gag expression plasmids. Electron and fluorescent microscopy showed that Gag accumulates in large patches in the membranes of cells expressing viruses with PPPY mutations. Very few tethered immature particles could be detected in these samples, suggesting that budding is impaired at an earlier step than in other retroviruses.     

Human immunodeficiency virus type 1 Gag engages the Bro1 domain of ALIX/AIP1 through the nucleocapsid

Human immunodeficiency virus type 1 (HIV-1) and other retroviruses harbor short peptide motifs in Gag that promote the release of infectious virions. These motifs, known as late assembly (L) domains, recruit a cellular budding machinery that is required for the formation of multivesicular bodies (MVBs). The primary L domain of HIV-1 maps to a PTAP motif in the p6 region of Gag and engages the MVB pathway by binding to Tsg101. Additionally, HIV-1 p6 harbors an auxiliary L domain that binds to the V domain of ALIX, another component of the MVB pathway. We now show that ALIX also binds to the nucleocapsid (NC) domain of HIV-1 Gag and that ALIX and its isolated Bro1 domain can be specifically packaged into viral particles via NC. The interaction with ALIX depended on the zinc fingers of NC, which mediate the specific packaging of genomic viral RNA, but was not disrupted by nuclease treatment. We also observed that HIV-1 zinc finger mutants were defective for particle production and exhibited a similar defect in Gag processing as a PTAP deletion mutant. The effects of the zinc finger and PTAP mutations were not additive, suggesting a functional relationship between NC and p6. However, in contrast to the PTAP deletion mutant, the double mutants could not be rescued by overexpressing ALIX, further supporting the notion that NC plays a role in virus release.     

p6Gag is required for particle production from full-length human immunodeficiency virus type 1 molecular clones expressing protease.

The human immunodeficiency virus type 1 (HIV-1) Gag protein precursor, Pr55Gag, contains at its C-terminal end a proline-rich, 6-kDa domain designated p6. Two functions have been proposed for p6: incorporation of the HIV-1 accessory protein Vpr into virus particles and virus particle production. To characterize the role of p6 in the HIV-1 life cycle and to map functional domains within p6, we introduced a number of nonsense and single and multiple amino acid substitution mutations into p6. Following the introduction of the mutations into the full-length HIV-1 molecular clone pNL4-3, the effects on Gag protein expression and processing, virus particle production, and virus infectivity were analyzed. The production of mutant virus particles was also examined by transmission electron microscopy. The results indicate that (i) p6 is required for efficient virus particle production from a full-length HIV-1 molecular clone; (ii) a Pro-Thr-Ala-Pro sequence, located between residues 7 and 10 of p6, is critical for virus particle production; (iii) mutations outside the Pro-Thr-Ala-Pro motif have little or no effect on virus assembly and release; (iv) the p6 defect is manifested at a late stage in the budding process; and (v) mutations in p6 that severely reduce virion production in HeLa cells also block or significantly delay the establishment of a productive infection in the CEM (12D-7) T-cell line. We further demonstrate that mutational inactivation of the viral protease reverses the p6 defect, suggesting a functional linkage between p6 and the proteolytic processing of the Gag precursor protein during the budding of progeny virions.     

Genetic evidence for an interaction between human immunodeficiency virus type 1 matrix and alpha-helix 2 of the gp41 cytoplasmic tail

The incorporation of envelope (Env) glycoproteins into virions is an essential step in the retroviral replication cycle. Lentiviruses, including human immunodeficiency virus type 1 (HIV-1), encode Env glycoproteins with unusually long cytoplasmic tails, the functions of which have not been fully elucidated. In this study, we examine the effects on virus replication of a number of mutations in a helical motif (alpha-helix 2) located near the center of the HIV-1 gp41 cytoplasmic tail. We find that, in T-cell lines, small deletions in this domain disrupt the incorporation of Env glycoproteins into virions and markedly impair virus infectivity. Through the analysis of viral revertants, we demonstrate that a single amino acid change (34VI) in the matrix domain of Gag reverses the Env incorporation and infectivity defect imposed by a small deletion near the C terminus of alpha-helix 2. These results provide genetic evidence, in the context of infected T cells, for an interaction between HIV-1 matrix and the gp41 cytoplasmic tail and identify domains of both proteins involved in this putative interaction.     

Effects of a single amino acid substitution within the p2 region of human immunodeficiency virus type 1 on packaging of spliced viral RNA

Human immunodeficiency virus type 1 encapsidates two copies of viral genomic RNA in the form of a dimer. The dimerization process initiates via a 6-nucleotide palindrome that constitutes the loop of a viral RNA stem-loop structure (i.e., stem loop 1 [SL1], also termed the dimerization initiation site [DIS]) located within the 5' untranslated region of the viral genome. We have now shown that deletion of the entire DIS sequence virtually eliminated viral replication but that this impairment was overcome by four second-site mutations located within the matrix (MA), capsid (CA), p2, and nucleocapsid (NC) regions of Gag. Interestingly, defective viral RNA dimerization caused by the DeltaDIS deletion was not significantly corrected by these compensatory mutations, which did, however, allow the mutated viruses to package wild-type levels of this DIS-deleted viral RNA while excluding spliced viral RNA from encapsidation. Further studies demonstrated that the compensatory mutation T12I located within p2, termed MP2, sufficed to prevent spliced viral RNA from being packaged into the DeltaDIS virus. Consistently, the DeltaDIS-MP2 virus displayed significantly higher levels of infectiousness than did the DeltaDIS virus. The importance of position T12 in p2 was further demonstrated by the identification of four point mutations,T12D, T12E, T12G, and T12P, that resulted in encapsidation of spliced viral RNA at significant levels. Taken together, our data demonstrate that selective packaging of viral genomic RNA is influenced by the MP2 mutation and that this represents a major mechanism for rescue of viruses containing the DeltaDIS deletion.