Search tips
Search criteria 


Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Cell Host Microbe. Author manuscript; available in PMC 2011 January 1.
Published in final edited form as:
PMCID: PMC2921950

Cytoplasmic Viral Replication Complexes


Many viruses that replicate in the cytoplasm compartmentalize their genome replication and transcription in organelle-like structures that enhance replication efficiency and protection from host defenses. In particular, recent studies with diverse positive-strand RNA viruses have further elucidated the ultrastructure of membrane-bounded RNA replication complexes and their close coordination with virion assembly and budding. The structure, function and assembly of some positive-strand RNA virus replication complexes have parallels and potential evolutionary links with the replicative cores of double-strand RNA virus and retrovirus virions, and more general similarities with the replication factories of cytoplasmic DNA viruses.

Keywords: Virus genome replication, membrane vesicles, compartmentalization, replication complex, virus replication factory, electron microscope tomography


While eukaryotic cells sequester and organize their genome replication and transcription in the nucleus, many RNA and some DNA viruses carry out viral genome replication and transcription in the cytoplasm. To establish efficient genome replication and shield it from host defenses, including crucial intrinsic and innate defenses, many or most of these cytoplasmically replicating viruses organize their genome replication and transcription in organelle-like compartments (Novoa et al., 2005). These replication compartments or factories often are associated with the sites of subsequent stages in the viral replication cycle, including particle formation and virus budding.

Recently, substantial advances have been made in characterizing the cytoplasmic replication compartments of positive-strand RNA viruses. Positive-strand RNA viruses package their genomes as messenger sense, single stranded RNA and replicate those genomes solely through RNA intermediates. For a diverse set of positive-strand RNA viruses, three-dimensional, high resolution imaging by electron microscope (EM) tomography, in combination with other complementary approaches, have revealed critical aspects of the structure and organization of membrane-bounded RNA replication compartments and their close spatial and functional relationships with virus translation and virion assembly and budding sites. This review summarizes selected recent findings in this area and their relation to other RNA and DNA viruses, including implications for potential evolutionary relationships between the genome replication processes of at least some positive-strand RNA viruses and those of double strand (ds)RNA and reverse-transcribing viruses. We regret that space limitations prevent highlighting all of the valuable contributions to these fields (Mackenzie, 2005; Miller and Krijnse-Locker, 2008; Novoa et al., 2005; Salonen et al., 2005).


Positive-strand RNA viruses encompass over one-third of known virus genera (ICTV, 2005) and include many medically and practically important human, animal and plant pathogens. At the outset of infection, after their initial delivery to the cytoplasm, positive-strand RNA virus genomes are used as templates for viral protein synthesis. Among the first viral proteins to accumulate are RNA replication proteins that redirect the viral genome from functioning as an mRNA to serving as a template for synthesizing complementary negative-strand RNA, which then becomes the template for new positive-strand genomic RNAs and subgenomic mRNAs.

Pioneering ultrastructural and other studies with a variety of positive-strand RNA viruses established that viral RNA synthesis was associated with membranes and, moreover, with virus-specific membrane rearrangements such as single and double-membrane vesicles and invaginations (e.g., (Bienz et al., 1987; Bienz et al., 1983; Froshauer et al., 1988; Grimley et al., 1968; Hatta et al., 1973; Russo and Martelli, 1972)). Subsequent work confirmed that RNA replication by all positive-strand RNA viruses studied to date was linked to virus-induced, often extensive rearrangements of specific intracellular membranes (Denison, 2008; Mackenzie, 2005; Salonen et al., 2005). Below we summarize recent findings with a diverse set of positive-strand RNA viruses that reveal new understandings of the structure, function and assembly of these complexes, their roles in coordinating successive steps in RNA replication and beyond, their relation to dsRNA and DNA virus factories, and their evolutionary implications.


Picornaviruses are a large family of human and animal viruses whose best-studied member is poliovirus (PV) (Racaniello, 2007). The PV genome expresses a single polyprotein that is processed by viral proteases into functional intermediate precursors and fully cleaved end products. Over half of the genome encodes RNA replication factors, including the RNA-dependent RNA polymerase 3D, the 3B/VPg protein primer for RNA synthesis, and the 2C NTPase. Additionally, the 2B, 2C and 3A proteins interact with each other and the other replication proteins (Yin et al., 2007) and mediate membrane association of these RNA replication proteins (Fujita et al., 2007; Teterina et al., 1997; Teterina et al., 2006).

Interaction with the PV RNA replication proteins extensively reorganizes endoplasmic reticulum (ER), Golgi and lysosomal membranes into 50 – 400 nm singleand double-membrane-bounded vesicles (Bienz et al., 1990; Cho et al., 1994; Egger et al., 2000; Schlegel et al., 1996). When these membrane-associated replication complexes are extracted from infected cells, the vesicles adopt a rosette-like appearance (Egger and Bienz, 2002). The viral replication proteins localize to the rosette center on the exposed surface of the double vesicle membrane (Bienz et al., 1990). PV-induced membrane vesicles have been linked to COPII-dependent vesicle trafficking (Rust et al., 2001) and to activation of cellular Arf GTPases that modulate membrane trafficking (Belov et al., 2007). Recent studies further showed that viral modulation of Arf GTPase function locally enriches the membranes targeted by picornaviruses and flaviviruses (see also below) in phosphatidylinositol-4-phosphate, promoting RNA replication, potentially by facilitating recruitment of relevant viral and perhaps cellular factors and by modulating membrane curvature (Hsu et al., 2010).

The double-membrane vesicles associated with PV infection resemble double-membrane structures generated during autophagy (Jackson et al., 2005; Schlegel et al., 1996; Suhy et al., 2000; Taylor and Kirkegaard, 2008). Based on this and other work, evidence is accumulating that picornaviruses and certain other viruses induce and subvert the host autophagy pathway to support their replication, progeny virus export, or both (Dreux and Chisari, 2010; Jackson et al., 2005; Kirkegaard, 2009; Taylor and Kirkegaard, 2008).


Brome mosaic virus (BMV), a member of the alphavirus-like superfamily, conducts its RNA replication on perinuclear ER membranes (Restrepo-Hartwig and Ahlquist, 1996) in ~60 nm vesicular invaginations similar to those induced on other target membranes by many viruses in and beyond the alphavirus-like superfamily (Ahlquist, 2006; Schwartz et al., 2002) (Figure 1). BMV encodes two large RNA replication proteins: 2apol, the viral RNA-dependent RNA polymerase, and 1a, a multifunctional protein with 5' RNA capping and RNA NTPase/helicase domains (Ahola and Ahlquist, 1999; Ahola et al., 2000; Kong et al., 1999; Wang et al., 2005).

Figure 1
BMV-induced perinuclear ER RNA replication vesicles and model of the BMV RNA replication complex

In addition to direct roles in RNA synthesis, 1a is the master organizer of RNA replication complex assembly. 1a directs ER membrane association (den Boon et al., 2001; Restrepo-Hartwig and Ahlquist, 1999) and, even in the absence of other viral factors, induces ER membrane invagination to form replication vesicles (Schwartz et al., 2002) (Figure 1). When present, BMV genomic RNA replication templates and 2apol are recruited by 1a to the ER (Chen et al., 2001; Janda and Ahlquist, 1998; Schwartz et al., 2002). 1a recruits 2apol through interaction of 1a's C-terminus with an N-proximal region preceding 2apol’s polymerase domain (Chen and Ahlquist, 2000; Kao and Ahlquist, 1992). Recent results indicate that 2apol recruitment occurs prior to and is inhibited by 1a’s induction of replication vesicles (Liu et al., 2009). By contrast, 1a recruitment of viral genomic RNA templates is closely linked to replication vesicle formation (Liu et al., 2009). Template recognition and recruitment is mediated by conserved RNA sequence elements (Baumstark and Ahlquist, 2001; Chen et al., 2001; Sullivan and Ahlquist, 1999) and requires an active 1a NTPase/helicase domain, apparently to translocate the RNA into pre-formed vesicles (Wang et al., 2005). In these sites, both the initial positive-strand template RNA and all subsequent negative-strand RNAs are strongly protected from nuclease and presumably other cytoplasmic factors (Schwartz et al., 2002; Sullivan and Ahlquist, 1999).

EM and quantitative biochemical analyses show that a single ~60 nm BMV replication vesicle contains one or a few positive and negative strand RNA molecules, ~10–20 2apol proteins, and ~200–400 1a proteins (Schwartz et al., 2002). This level of 1a is sufficient to coat the interior of the replication vesicle. Accordingly, since 1a is strongly membrane associated and self-interacts through multiple regions (Kao and Ahlquist, 1992; O'Reilly et al., 1995), BMV replication compartments have been proposed to contain a 1a protein shell lining the vesicle interior, suggesting a simple explanation for 1a’s ability to form these compartments (Figure 1C).


Additional support for a protein shell-supported replication complex model came from similar observations for the replication complex of Flock House virus (FHV), the best-studied member of the animal nodaviruses (Venter and Schneemann, 2008). FHV encodes a single, highly multifunctional RNA replication protein, protein A. Protein A contains an N-terminal trans-membrane domain targeting outer mitochrondrial membranes (Miller and Ahlquist, 2002), an RNA-dependent RNA polymerase domain, and regions that direct self-interaction (Dye et al., 2005) and specific recognition and recruiting of FHV genomic RNAs to mitochondrial membranes (Van Wynsberghe and Ahlquist, 2009; Van Wynsberghe et al., 2007).

Conventional two-dimensional thin section transmission EM and three-dimensional imaging by EM tomography show that FHV induces ~50 nm vesicular invaginations between the inner and outer mitochondrial membranes (Figure 2). The interiors of these vesicles are the sites where protein A and newly synthesized FHV RNAs accumulate (Kopek et al., 2007). Stoichiometry analyses showed that each FHV replication vesicle contains ~100 copies of protein A and one or two genome RNA replication intermediates. These results are consistent with a model for the FHV replication complex very similar to that for BMV (Figure 1C), with a continuous shell of self-interacting, trans-membrane protein A lining the interior of the FHV-induced mitochondrial membrane vesicle. Furthermore, tomographic imaging revealed that every FHV-induced vesicle remains attached to the outer mitochondrial membrane by a ~10nm neck-like connection to the cytoplasm (Kopek et al., 2007) (Figure 2D).

Figure 2
FHV-induced RNA replication vesicles on outer mitochondrial membranes


While BMV and FHV present examples of RNA replication complexes in simple vesicular membrane invaginations, members of the coronavirus and arterivirus families within the order Nidovirales induce more complicated mixtures of convoluted membrane rearrangements and large double-membrane vesicles (Gosert et al., 2002; Pedersen et al., 1999; Snijder et al., 2006; Snijder et al., 2001). Among the best studied are the RNA replication structures in severe acute respiratory syndrome (SARS) coronavirus-infected cells. EM tomography studies of SARS virus-infected cells have revealed that the different membrane structures represent a single network of interconnected ER-derived membranes (Knoops et al., 2008; Knoops et al., 2010) (Figure 3).

Figure 3
EM tomographic three-dimensional reconstruction of SARS coronavirus-induced, ER-derived double-membrane vesicles

The 5’ two-thirds of the ~30 kb genome coronavirus genome, the largest among positive-strand RNA viruses, encodes polyprotein precursors that are processed into 15 or 16 RNA replicase subunits (Snijder et al., 2003; Thiel et al., 2003; Ziebuhr et al., 2000) that localize to the virus-induced membrane structures (Knoops et al., 2008). When appropriately assayed, membrane extracts from SARS coronavirus-infected cells synthesize the typical nested set of coronavirus genomic and subgenomic RNAs. Such in vitro activity is RNAse- and protease-resistant but detergent-sensitive, indicating that the membranes provide a protective environment for RNA replication (van Hemert et al., 2008b). Similar observations were made with membrane extracts from cells infected with the distantly related arterivirus EAV (van Hemert et al., 2008a) which in electron tomography studies were recently found to contain a similar network of interconnected single- and double-membrane structures (Knoops & Snijder, personal communication).

In keeping with these results, dsRNA, the presumptive RNA replication intermediate, predominantly localizes to the interiors of the large, 200–300 nm diameter double-membrane vesicles in coronavirus-infected cells (Knoops et al., 2008). Nevertheless, it is not yet established that these vesicle interiors represent the actual sites of RNA synthesis. The outer membranes of the double-membrane vesicles are interconnected through ~8 nm tubules, but no connections between the vesicle interiors and the cytosol have yet been visualized (Knoops et al., 2008). It thus remains uncertain how ribonucleotides and product RNAs would be exchanged with the cytosol if RNA synthesis occurs inside these double-membrane vesicles. One possible solution is that the coronavirus replication complex might use a protein channel as the equivalent of the neck-like openings in the BMV and FHV replication spherules (Knoops et al., 2008). Three of the 16 SARS RNA replication proteins have integral membrane-spanning domains (Kanjanahaluethai et al., 2007; Oostra et al., 2008) and, in principle, could support the formation of proteinaceous membrane pores to the cytoplasm. Current EM tomography images do not provide sufficient resolution to visualize or rule out the presence of such channels.

Alternatively or in addition, coronavirus RNA synthesis might occur in the convoluted single membrane structures that adjoin and interconnect with the double-membrane vesicles. These convoluted membranes appear to be the major accumulation sites of the viral replicase subunits and encompass many spaces or compartments with open connections to the cytoplasm (Knoops et al., 2008). Later stages in the maturation of coronavirus-induced membrane rearrangements appear to involve membrane fusion events, suggesting that similar earlier fusions might allow generating the double-membrane vesicles from the interconnected convoluted membranes ((Knoops et al., 2008); E. Snijder and M. Kikkert, personal communication). If so, the double-membrane vesicles may represent repositories that sequester dsRNAs and perhaps other byproducts produced by RNA replication in the convoluted membranes.

Such possible conversion of convoluted membrane replication sites into double-membrane vesicles is reminiscent of some features of BMV RNA replication compartments. By increasing or decreasing the level of BMV 2apol, bromovirus replication compartments can be interconverted between layered membranes with similarities to coronavirus-induced convoluted membranes, and the vesicular invaginations normally associated with bromovirus infection (see above), which in appropriate EM sections appear as complexes of double-membrane vesicles (Schwartz et al., 2004).


Similarly complex replication-associated membrane structures are induced by the Flaviviridae, which include clinically important members such as hepatitis C virus (HCV), yellow fever virus and Dengue virus (DENV). Like poliovirus, the flavivirus genomic RNA encodes a single polyprotein that is cleaved into virion proteins and RNA replication proteins (Bartenschlager and Miller, 2008; Lindenbach and Rice, 2003; Lindenbach et al., 2007). Three of the replication proteins contain membrane-spanning domains (Mackenzie et al., 1998; Miller et al., 2007; Miller et al., 2006), and are responsible for inducing several distinct ER-derived membrane structures: vesicle packets, convoluted membranes and membranes associated with progeny virus assembly (Grief et al., 1997; Mackenzie et al., 1996; Welsch et al., 2009). Recent three-dimensional EM tomography showed that the different flavivirus membrane structures are all part of a single continuous ER-derived membrane network with resembling the coronavirus membrane rearrangements (Welsch et al., 2009) (Figure 4). The flavivirus-induced convoluted membrane rearrangements do not contain detectable dsRNA and have been proposed to be the sites where replication proteins accumulate, are cleaved, and stored for further use in replication complex assembly (Mackenzie et al., 1996; Welsch et al., 2009).

Figure 4
EM tomographic three-dimensional reconstruction of Dengue virus-induced, ER-derived vesicle packets

The flavivirus vesicle packets consist of an outer bounding membrane surrounding a series of inner, ~90 nm vesicles (Figure 4) that contain most of the replication proteins, dsRNA and nascent RNA (Mackenzie et al., 1996). Thus, these inner vesicles are the likely sites of genome replication. Intriguingly, these vesicle packets show features that bridge the coronavirus double-membraned vesicles and the invaginated vesicular RNA replication compartments induced by bromoviruses and nodaviruses. Like the coronavirus RNA replication compartments, in some planes of sectioning the flavivirus vesicle packet interiors appear separated from the cytoplasm by two membranes (Figure 4). However, three dimensional EM tomography revealed that the inner 90 nm vesicles are invaginations of the outer bounding membrane, and like BMV and FHV replication vesicles bear necked connections to the cytoplasm (Welsch et al., 2009). This topological equivalence of flavivirus and nodavirus replication compartments had been proposed earlier (Ahlquist, 2006; Kopek et al., 2007), based on EM tomography analysis showing that certain cross sections through FHV-modified mitochondria bear remarkable resemblance to flavivirus vesicle packets (Figure 2B).


Many viruses coordinate their genome replication with subsequent steps of producing progeny virions. DENV and SARS virus package their virion capsids in membrane envelopes, and for both viruses EM tomography revealed that some or all virion assembly and budding steps occur within or in close proximity to the same continuous membrane networks that support genome replication (Knoops et al., 2008; Welsch et al., 2009) (Figure 4).

For DENV, in some cases, virion particle formation was observed at sites directly apposed to the open necks of the replication vesicles (Welsch et al., 2009). Moreover, for DENV and the related hepatitis C virus, viral capsid proteins accumulate on the surface of characteristic lipid droplets (McLauchlan, 2009; Ogawa et al., 2009; Samsa et al., 2009). Like the membrane structures of the viral replication complex, these lipid droplets originate from the ER and their abundance in the cell is directly linked to virus infection and replication (Samsa et al., 2009).

New coronaviruses virions bud into ER-Golgi intermediate compartments, but early in infection the viral nucleocapsid protein can also be detected at the double-membrane vesicle sites of viral replication (Stertz et al., 2007). Some EM tomography images of late stages of SARS virus infection showed merged replication and budding compartments (Knoops et al., 2008).

PV, BMV and FHV, unlike flavi- and coronaviruses, are non-enveloped “naked” viruses and do not rely on membranes for budding. Nevertheless, strong spatial and functional links between genome replication and virion assembly exist for these viruses also. PV, BMV, and FHV virion formation all require actively replicating genomic RNA (Annamalai and Rao, 2005, 2006; Nugent et al., 1999; Venter et al., 2005). Additional EM tomography results have shown substantial FHV virion accumulation close to mitochondria with FHV genome replication complexes (Lanman et al., 2008).


Beyond close coordination of viral genome replication and virion assembly, for at least some positive-strand RNA viruses the membrane-associated RNA replication complexes themselves show general parallels with virion assembly and structure, and particular parallels with the replicative cores of dsRNA virus and reverse-transcribing virus virions (Ahlquist, 2006) (Figure 5).

Figure 5
Parallels between positive-strand RNA virus, dsRNA virus and retrovirus genome replication

As an example of dsRNA viruses, members of the well-studied Reoviridae family carry out most of their replication steps in cytoplasmic "viroplasms" or virus factories that, while not membrane bounded, nevertheless concentrate virion assembly, RNA replication and other steps in a defined space (Patton et al., 2006). Such dsRNA viruses encapsidate their genomic dsRNAs, together with viral polymerases, in a virion core that is active in RNA synthesis in much the same way as positive-strand RNA virus replication complexes (Figure 5A). For reoviruses, the virion core shell consists of 120 copies of viral protein λ1 and contains 60 copies of the λ2 RNA capping protein and 12 copies of the λ3 polymerase (Reinisch et al., 2000; Zhang et al., 2003). BMV 1a and FHV protein A thus resemble λ1 as high copy number structural components of the RNA synthesis complex. 1a further resembles λ1 and λ2 in having NTPase/helicase and RNA capping domains, respectively, while BMV 2apol resembles λ3 in having a polymerase domain and interacting with its cognate NTPase/helicase domain (Ahlquist, 2006) (Figure 5AB). BMV 1a's roles in replication complex assembly show further similarities with reovirus protein µNS, a multifunctional protein that coordinates the recruitment and assembly of additional reovirus proteins and forms the matrix of the viroplasms within which reovirus replication and virion assembly occur (Arnold et al., 2008; Miller et al., 2010).

Additional parallels exist with retrovirus virion assembly (Bieniasz, 2009; Waheed and Freed, 2009) (Figure 5C). When the functions of the cellular ESCRT / multivesicular body sorting pathway are inhibited, e.g., retrovirus virions fail to complete their budding (Morita and Sundquist, 2004). When so arrested, retroviruses remain attached to the plasma membrane by neck-like membrane stalks that are strikingly similar to the necked membrane connections that the spherular RNA replication vesicles of FHV, BMV, alphaviruses and many other positive-strand RNA viruses maintain to the cytoplasm. Moreover, multiple functions of the major retrovirus capsid protein Gag in virion assembly parallel roles of BMV 1a in replication complex spherule formation (Figure 5BC). These similarities include targeting and defining the membrane site of virion/replication complex assembly, binding the target membrane cytoplasmic face as a peripheral membrane protein, inducing target membrane invagination, self-interacting in large numbers within the resulting vesicle, directing viral RNA templates and viral polymerase into these vesicles, and other points (Ahlquist, 2006). Many though not all of these similarities are shared by nodavirus protein A (Dye et al., 2005; Kopek et al., 2007; Miller et al., 2001; Van Wynsberghe and Ahlquist, 2009; Van Wynsberghe et al., 2007).

Particularly striking are the parallels between the above positive-strand RNA virus RNA replication complexes and assembling virions of the foamy retroviruses (Figure 5C). Foamy virus replication is distinct from that of orthoretroviruses such as HIV in several ways (Delelis et al., 2004; Linial, 1999). For example, while orthoretroviruses release virions carrying an RNA genome and delay reverse transcription until after entering a newly infected cell, newly-assembled foamy retrovirus virions reverse transcribe their encapsidated RNA prior to virion release (Moebes et al., 1997). Thus, foamy retrovirus virions are actively involved in genome replication within the same cell in which they assemble, further paralleling positive-strand RNA virus replication complexes such as the BMV and FHV spherules described above.

Another distinction is that, while orthoretoroviruses translate their reverse transciptase (Pol) as fusion protein with Gag, foamy viruses translate Gag and Pol as separate proteins from independent mRNAs. This allows separate regulation of Pol expression and encapsidation, similar to some positive-strand RNA viruses such as BMV. Recruitment of foamy virus Pol depends on C-proximal determinants in the Gag protein sequence (Lee and Linial, 2008), similar to the recruitment of BMV 2apol to RNA replication complexes by the C-terminus of BMV 1a (Chen and Ahlquist, 2000; Kao and Ahlquist, 1992).

From an evolutionary perspective, the similarities of positive-strand RNA virus replication complexes with the replicative cores of dsRNA and retrovirus virions suggest that all of these viruses may have diverged from a common precursor that also used a viral protein shell to organize and sequester the replication of a mRNA-sense genomic RNA template. In subsequent evolution, these viruses would then have diverged with regard to which replication cycle intermediate to export in infectious virions: for retroviruses and dsRNA viruses, the RNA replication complex before and after negative-strand synthesis, respectively, and for positive-strand RNA viruses, the mRNA-sense genomic RNA before assembly into the replication complex (Ahlquist, 2006; Schwartz et al., 2002).


Building novel intracellular structures to support viral replication is an integral part of the life cycle of many if not all viruses. Unlike the RNA viruses discussed above, most DNA viruses, replicate their genomes inside the nucleus. Nevertheless, many of these DNA viruses also assemble cytoplasmic factory-like structures to complete their replication cycles and assemble progeny virus, often in close vicinity to ER membranes where viral proteins are produced (Novoa et al., 2005).

Moreover, unusually among large double-strand DNA viruses, poxviruses such as vaccinia virus carry out their replication entirely in the cytoplasm in membrane-bound viral complexes (Schramm and Krijnse Locker, 2005). Upon infection, vaccinia virus cores are released and accumulate in close proximity to ER membranes. The incoming genomic DNA leaves the core and preferentially associates with the cytosolic side of the ER membranes (Mallardo et al., 2001). DNA replication is initiated in distinct cytoplasmic sites, often referred to as viral factories, formed through gradual envelopment by rough ER membranes. Occasional small gaps in the surrounding membranes have been observed, presumably allowing exchange of molecules between the interior DNA replication compartments and the cytoplasm (Tolonen et al., 2001). After completion of this ER wrapping, the viral DNA replication complexes expand in size, perhaps indicative of active DNA replication (Tolonen et al., 2001).

In further analogy with positive-strand RNA and dsRNA viruses, poxvirus genome replication, transcription and translation, and virus assembly are all coordinated within or associated with the DNA replication factories. Although early viral mRNAs are transcribed in the original viral cores (Mallardo et al., 2001), intermediate and late viral mRNAs concentrate in the viral DNA factories and closely associate with ribosomes and translation initiation factors to produce the many different viral proteins (Katsafanas and Moss, 2007). Late in infection, the ER around the viral factories disassembles, coinciding with a dramatic decrease in DNA synthesis and the formation of virion precursors (Tolonen et al., 2001).


The studies reviewed above have substantially enhanced understanding of the replication structures and pathways of many important viruses, and revealed some common principles. Simultaneously, many fundamental questions remain or have become evident from this work. Among these unresolved questions are the detailed molecular mechanisms by which specific viruses target their replication factors and their RNAs to particular membranes or other intracellular sites to assemble replication complexes or factories, as well as how different viruses orchestrate the varied and often complex membrane rearrangements associated with their replication processes. Related issues include the specific advantages or adaptations associated with the use by diverse viruses of different intracellular sites for similar replication purposes. Different positive-strand RNA viruses, e.g., variously assemble their RNA replication complexes on distinct secretory, endosomal, or organellar membranes, and they and other viruses show a similar diversity in the sites used for virion assembly and/or budding. However, the implications of such choices for replicative efficiency, virus-host interactions, and pathology remain poorly understood. Recent findings on how picornaviruses, flaviviruses and coronaviruses manipulate components of the secretory pathway and related pathways to create novel membrane environments with specific lipid enrichments and other replication-supportive characteristics are examples of these essential research directions (Belov et al., 2007; Hsu et al., 2010; Reggiori et al., 2010). Such efforts will be critical to identify and understand the roles of cellular factors and molecular pathways in efficient viral replication.

A second class of challenges and opportunities is associated with using growing knowledge in these areas to improve virus control or beneficial uses of viruses. For virus control, growing recognition of the intimate coordination of many successive virus replication steps with each other and with cellular pathways offers many additional points at which to disrupt infection. In this regard, one important area will be further defining the roles and interactions of viral replication compartments as barriers to host defenses, including host systems for detecting viruses through dsRNA, etc., and for impeding virus replication, such as through RNA silencing or certain interferon-stimulated pathways. While the emerging complexities exceed the expectations of earlier stages of investigation, such questions offer challenging but satisfying directions and a fulfilling future for these important areas in the cell biology of virus replication.


We thank members of our laboratory, Eric Snijder, Marjolein Kikkert, Ellie Ehrenfeld and many others for valuable discussions on the areas of this review. Research related to these topics in the authors’ laboratory was supported by NIH grant GM35072. P.A. is an Investigator of the Howard Hughes Medical Institute.


Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.


  • Ahlquist P. Parallels among positive-strand RNA viruses, reverse-transcribing viruses and double-stranded RNA viruses. Nat Rev Microbiol. 2006;4:371–382. [PubMed]
  • Ahola T, Ahlquist P. Putative RNA capping activities encoded by brome mosaic virus: methylation and covalent binding of guanylate by replicase protein 1a. J Virol. 1999;73:10061–10069. [PMC free article] [PubMed]
  • Ahola T, den Boon JA, Ahlquist P. Helicase and capping enzyme active site mutations in brome mosaic virus protein 1a cause defects in template recruitment, negative-strand RNA synthesis, and viral RNA capping. J Virol. 2000;74:8803–8811. [PMC free article] [PubMed]
  • Annamalai P, Rao AL. Replication-independent expression of genome components and capsid protein of brome mosaic virus in planta: a functional role for viral replicase in RNA packaging. Virology. 2005;338:96–111. [PubMed]
  • Annamalai P, Rao AL. Packaging of brome mosaic virus subgenomic RNA is functionally coupled to replication-dependent transcription and translation of coat protein. J Virol. 2006;80:10096–10108. [PMC free article] [PubMed]
  • Arnold MM, Murray KE, Nibert ML. Formation of the factory matrix is an important, though not a sufficient function of nonstructural protein mu NS during reovirus infection. Virology. 2008;375:412–423. [PMC free article] [PubMed]
  • Bartenschlager R, Miller S. Molecular aspects of Dengue virus replication. Future Microbiol. 2008;3:155–165. [PubMed]
  • Baumstark T, Ahlquist P. The brome mosaic virus RNA3 intergenic replication enhancer folds to mimic a tRNA TpsiC-stem loop and is modified in vivo. RNA. 2001;7:1652–1670. [PubMed]
  • Belov GA, Habbersett C, Franco D, Ehrenfeld E. Activation of cellular Arf GTPases by poliovirus protein 3CD correlates with virus replication. J Virol. 2007;81:9259–9267. [PMC free article] [PubMed]
  • Bieniasz PD. The cell biology of HIV-1 virion genesis. Cell Host Microbe. 2009;5:550–558. [PMC free article] [PubMed]
  • Bienz K, Egger D, Pasamontes L. Association of polioviral proteins of the P2 genomic region with the viral replication complex and virus-induced membrane synthesis as visualized by electron microscopic immunocytochemistry and autoradiography. Virology. 1987;160:220–226. [PubMed]
  • Bienz K, Egger D, Rasser Y, Bossart W. Intracellular distribution of poliovirus proteins and the induction of virus-specific cytoplasmic structures. Virology. 1983;131:39–48. [PubMed]
  • Bienz K, Egger D, Troxler M, Pasamontes L. Structural organization of poliovirus RNA replication is mediated by viral proteins of the P2 genomic region. J Virol. 1990;64:1156–1163. [PMC free article] [PubMed]
  • Chen J, Ahlquist P. Brome mosaic virus polymerase-like protein 2a is directed to the endoplasmic reticulum by helicase-like viral protein 1a. J Virol. 2000;74:4310–4318. [PMC free article] [PubMed]
  • Chen J, Noueiry A, Ahlquist P. Brome mosaic virus Protein 1a recruits viral RNA2 to RNA replication through a 5' proximal RNA2 signal. J Virol. 2001;75:3207–3219. [PMC free article] [PubMed]
  • Cho MW, Teterina N, Egger D, Bienz K, Ehrenfeld E. Membrane rearrangement and vesicle induction by recombinant poliovirus 2C and 2BC in human cells. Virology. 1994;202:129–145. [PubMed]
  • Delelis O, Lehmann-Che J, Saib A. Foamy viruses--a world apart. Curr Opin Microbiol. 2004;7:400–406. [PubMed]
  • den Boon JA, Chen J, Ahlquist P. Identification of sequences in Brome mosaic virus replicase protein 1a that mediate association with endoplasmic reticulum membranes. J Virol. 2001;75:12370–12381. [PMC free article] [PubMed]
  • Denison MR. Seeking membranes: positive-strand RNA virus replication complexes. PLoS Biol. 2008;6:e270. [PMC free article] [PubMed]
  • Dreux M, Chisari FV. Viruses and the autophagy machinery. Cell Cycle. 2010;9 [PubMed]
  • Dye BT, Miller DJ, Ahlquist P. In vivo self-interaction of nodavirus RNA replicase protein a revealed by fluorescence resonance energy transfer. J Virol. 2005;79:8909–8919. [PMC free article] [PubMed]
  • Egger D, Bienz K. Recombination of poliovirus RNA proceeds in mixed replication complexes originating from distinct replication start sites. J Virol. 2002;76:10960–10971. [PMC free article] [PubMed]
  • Egger D, Teterina N, Ehrenfeld E, Bienz K. Formation of the poliovirus replication complex requires coupled viral translation, vesicle production, and viral RNA synthesis. J Virol. 2000;74:6570–6580. [PMC free article] [PubMed]
  • Froshauer S, Kartenbeck J, Helenius A. Alphavirus RNA replicase is located on the cytoplasmic surface of endosomes and lysosomes. J Cell Biol. 1988;107:2075–2086. [PMC free article] [PubMed]
  • Fujita K, Krishnakumar SS, Franco D, Paul AV, London E, Wimmer E. Membrane topography of the hydrophobic anchor sequence of poliovirus 3A and 3AB proteins and the functional effect of 3A/3AB membrane association upon RNA replication. Biochemistry. 2007;46:5185–5199. [PMC free article] [PubMed]
  • Gosert R, Kanjanahaluethai A, Egger D, Bienz K, Baker SC. RNA replication of mouse hepatitis virus takes place at double-membrane vesicles. J Virol. 2002;76:3697–3708. [PMC free article] [PubMed]
  • Grief C, Galler R, Cortes LM, Barth OM. Intracellular localisation of dengue-2 RNA in mosquito cell culture using electron microscopic in situ hybridisation. Arch Virol. 1997;142:2347–2357. [PubMed]
  • Grimley PM, Berezesky IK, Friedman RM. Cytoplasmic structures associated with an arbovirus infection: loci of viral ribonucleic acid synthesis. J Virol. 1968;2:1326–1338. [PMC free article] [PubMed]
  • Hatta T, Bullivant S, Matthews RE. Fine structure of vesicles induced in chloroplasts of Chinese cabbage leaves by infection with turnip yellow mosaic virus. J Gen Virol. 1973;20:37–50. [PubMed]
  • Hsu NY, Ilnytska O, Belov G, Santiana M, Chen YH, Takvorian PM, Pau C, van der Schaar H, Kaushik-Basu N, Balla T, et al. Viral reorganization of the secretory pathway generates distinct organelles for RNA replication. Cell. 2010;141:799–811. [PMC free article] [PubMed]
  • ICTV. Virus Taxonomy: VIIIth Report of the International Committee on Taxonomy of Viruses. In: Fauquet CM, Mayo MA, Maniloff U, Desselberger U, Ball LA, editors. 2005.
  • Jackson WT, Giddings TH, Jr, Taylor MP, Mulinyawe S, Rabinovitch M, Kopito RR, Kirkegaard K. Subversion of cellular autophagosomal machinery by RNA viruses. PLoS Biol. 2005;3:e156. [PubMed]
  • Janda M, Ahlquist P. Brome mosaic virus RNA replication protein 1a dramatically increases in vivo stability but not translation of viral genomic RNA3. Proc Natl Acad Sci U S A. 1998;95:2227–2232. [PubMed]
  • Kanjanahaluethai A, Chen Z, Jukneliene D, Baker SC. Membrane topology of murine coronavirus replicase nonstructural protein 3. Virology. 2007;361:391–401. [PMC free article] [PubMed]
  • Kao CC, Ahlquist P. Identification of the domains required for direct interaction of the helicase-like and polymerase-like RNA replication proteins of brome mosaic virus. J Virol. 1992;66:7293–7302. [PMC free article] [PubMed]
  • Katsafanas GC, Moss B. Colocalization of transcription and translation within cytoplasmic poxvirus factories coordinates viral expression and subjugates host functions. Cell Host Microbe. 2007;2:221–228. [PMC free article] [PubMed]
  • Kirkegaard K. Subversion of the cellular autophagy pathway by viruses. Curr Top Microbiol Immunol. 2009;335:323–333. [PubMed]
  • Knoops K, Kikkert M, Worm SH, Zevenhoven-Dobbe JC, van der Meer Y, Koster AJ, Mommaas AM, Snijder EJ. SARS-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum. PLoS Biol. 2008;6:e226. [PubMed]
  • Knoops K, Swett-Tapia C, van den Worm SH, Te Velthuis AJ, Koster AJ, Mommaas AM, Snijder EJ, Kikkert M. Integrity of the early secretory pathway promotes, but is not required for, severe acute respiratory syndrome coronavirus RNA synthesis and virus-induced remodeling of endoplasmic reticulum membranes. J Virol. 2010;84:833–846. [PMC free article] [PubMed]
  • Kong F, Sivakumaran K, Kao C. The N-terminal half of the brome mosaic virus 1a protein has RNA capping-associated activities: specificity for GTP and S-adenosylmethionine. Virology. 1999;259:200–210. [PubMed]
  • Kopek BG, Perkins G, Miller DJ, Ellisman MH, Ahlquist P. Three-dimensional analysis of a viral RNA replication complex reveals a virus-induced mini-organelle. PLoS Biol. 2007;5:e220. [PubMed]
  • Lanman J, Crum J, Deerinck TJ, Gaietta GM, Schneemann A, Sosinsky GE, Ellisman MH, Johnson JE. Visualizing flock house virus infection in Drosophila cells with correlated fluorescence and electron microscopy. J Struct Biol. 2008;161:439–446. [PMC free article] [PubMed]
  • Lee EG, Linial ML. The C terminus of foamy retrovirus Gag contains determinants for encapsidation of Pol protein into virions. J Virol. 2008;82:10803–10810. [PMC free article] [PubMed]
  • Lindenbach BD, Rice CM. Molecular biology of flaviviruses. Adv Virus Res. 2003;59:23–61. [PubMed]
  • Lindenbach BD, Thiel H-J, Rice CM. Flaviviridae: The Viruses and Their Replication. In: Knipe DM, Howley PM, Griffin DE, Lamb RA, Martin MA, Roizman B, Straus SE, editors. Fields Virology. Philadelphia: Lippincott Williams & Wilkins; 2007. pp. 1101–1152.
  • Linial ML. Foamy viruses are unconventional retroviruses. J Virol. 1999;73:1747–1755. [PMC free article] [PubMed]
  • Liu L, Westler WM, den Boon JA, Wang X, Diaz A, Steinberg HA, Ahlquist P. An amphipathic alpha-helix controls multiple roles of brome mosaic virus protein 1a in RNA replication complex assembly and function. PLoS Pathog. 2009;5:e1000351. [PMC free article] [PubMed]
  • Mackenzie J. Wrapping things up about virus RNA replication. Traffic. 2005;6:967–977. [PubMed]
  • Mackenzie JM, Jones MK, Young PR. Immunolocalization of the dengue virus nonstructural glycoprotein NS1 suggests a role in viral RNA replication. Virology. 1996;220:232–240. [PubMed]
  • Mackenzie JM, Khromykh AA, Jones MK, Westaway EG. Subcellular localization and some biochemical properties of the flavivirus Kunjin nonstructural proteins NS2A and NS4A. Virology. 1998;245:203–215. [PubMed]
  • Mallardo M, Schleich S, Krijnse Locker J. Microtubule-dependent organization of vaccinia virus core-derived early mRNAs into distinct cytoplasmic structures. Mol Biol Cell. 2001;12:3875–3891. [PMC free article] [PubMed]
  • McLauchlan J. Lipid droplets and hepatitis C virus infection. Biochim Biophys Acta. 2009;1791:552–559. [PubMed]
  • Miller CL, Arnold MM, Broering TJ, Hastings CE, Nibert ML. Localization of mammalian orthoreovirus proteins to cytoplasmic factory-like structures via nonoverlapping regions of microNS. J Virol. 2010;84:867–882. [PMC free article] [PubMed]
  • Miller DJ, Ahlquist P. Flock house virus RNA polymerase is a transmembrane protein with amino-terminal sequences sufficient for mitochondrial localization and membrane insertion. J Virol. 2002;76:9856–9867. [PMC free article] [PubMed]
  • Miller DJ, Schwartz MD, Ahlquist P. Flock house virus RNA replicates on outer mitochondrial membranes in Drosophila cells. J Virol. 2001;75:11664–11676. [PMC free article] [PubMed]
  • Miller S, Kastner S, Krijnse-Locker J, Buhler S, Bartenschlager R. The non-structural protein 4A of dengue virus is an integral membrane protein inducing membrane alterations in a 2K-regulated manner. J Biol Chem. 2007;282:8873–8882. [PubMed]
  • Miller S, Krijnse-Locker J. Modification of intracellular membrane structures for virus replication. Nat Rev Microbiol. 2008;6:363–374. [PubMed]
  • Miller S, Sparacio S, Bartenschlager R. Subcellular localization and membrane topology of the Dengue virus type 2 Non-structural protein 4B. J Biol Chem. 2006;281:8854–8863. [PubMed]
  • Moebes A, Enssle J, Bieniasz PD, Heinkelein M, Lindemann D, Bock M, McClure MO, Rethwilm A. Human foamy virus reverse transcription that occurs late in the viral replication cycle. J Virol. 1997;71:7305–7311. [PMC free article] [PubMed]
  • Morita E, Sundquist WI. Retrovirus budding. Annu Rev Cell Dev Biol. 2004;20:395–425. [PubMed]
  • Novoa RR, Calderita G, Arranz R, Fontana J, Granzow H, Risco C. Virus factories: associations of cell organelles for viral replication and morphogenesis. Biol Cell. 2005;97:147–172. [PubMed]
  • Nugent CI, Johnson KL, Sarnow P, Kirkegaard K. Functional coupling between replication and packaging of poliovirus replicon RNA. J Virol. 1999;73:427–435. [PMC free article] [PubMed]
  • O'Reilly EK, Tang N, Ahlquist P, Kao CC. Biochemical and genetic analyses of the interaction between the helicase-like and polymerase-like proteins of the brome mosaic virus. Virology. 1995;214:59–71. [PubMed]
  • Ogawa K, Hishiki T, Shimizu Y, Funami K, Sugiyama K, Miyanari Y, Shimotohno K. Hepatitis C virus utilizes lipid droplet for production of infectious virus. Proc Jpn Acad Ser B Phys Biol Sci. 2009;85:217–228. [PMC free article] [PubMed]
  • Oostra M, Hagemeijer MC, van Gent M, Bekker CP, te Lintelo EG, Rottier PJ, de Haan CA. Topology and membrane anchoring of the coronavirus replication complex: not all hydrophobic domains of nsp3 and nsp6 are membrane spanning. J Virol. 2008;82:12392–12405. [PMC free article] [PubMed]
  • Patton JT, Silvestri LS, Tortorici MA, Vasquez-Del Carpio R, Taraporewala ZF. Rotavirus genome replication and morphogenesis: role of the viroplasm. Curr Top Microbiol Immunol. 2006;309:169–187. [PubMed]
  • Pedersen KW, van der Meer Y, Roos N, Snijder EJ. Open reading frame 1a-encoded subunits of the arterivirus replicase induce endoplasmic reticulum-derived double-membrane vesicles which carry the viral replication complex. J Virol. 1999;73:2016–2026. [PMC free article] [PubMed]
  • Racaniello VR. Picornaviridae: The Viruses and Their Replication. In: Knipe DM, Howley PM, Griffin DE, Lamb RA, Martin MA, Roizman B, Straus SE, editors. Fields Virology. Philadelphia: Lippincott Williams & Wilkins; 2007. pp. 795–838.
  • Reggiori F, Monastyrska I, Verheije MH, Cali T, Ulasli M, Bianchi S, Bernasconi R, de Haan CA, Molinari M. Coronaviruses Hijack the LC3-I-positive EDEMosomes, ER-derived vesicles exporting short-lived ERAD regulators, for replication. Cell Host Microbe. 2010;7:500–508. [PubMed]
  • Reinisch KM, Nibert ML, Harrison SC. Structure of the reovirus core at 3.6 A resolution. Nature. 2000;404:960–967. [PubMed]
  • Restrepo-Hartwig M, Ahlquist P. Brome mosaic virus RNA replication proteins 1a and 2a colocalize and 1a independently localizes on the yeast endoplasmic reticulum. J Virol. 1999;73:10303–10309. [PMC free article] [PubMed]
  • Restrepo-Hartwig MA, Ahlquist P. Brome mosaic virus helicase- and polymerase-like proteins colocalize on the endoplasmic reticulum at sites of viral RNA synthesis. J Virol. 1996;70:8908–8916. [PMC free article] [PubMed]
  • Russo M, Martelli GP. Ultrastructural Observations on Tomato Bushy Stunt Virus in Plant Cells. Virology. 1972;49:122–129. [PubMed]
  • Rust RC, Landmann L, Gosert R, Tang BL, Hong W, Hauri HP, Egger D, Bienz K. Cellular COPII proteins are involved in production of the vesicles that form the poliovirus replication complex. J Virol. 2001;75:9808–9818. [PMC free article] [PubMed]
  • Salonen A, Ahola T, Kaariainen L. Viral RNA replication in association with cellular membranes. Curr Top Microbiol Immunol. 2005;285:139–173. [PubMed]
  • Samsa MM, Mondotte JA, Iglesias NG, Assuncao-Miranda I, Barbosa-Lima G, Da Poian AT, Bozza PT, Gamarnik AV. Dengue virus capsid protein usurps lipid droplets for viral particle formation. PLoS Pathog. 2009;5:e1000632. [PMC free article] [PubMed]
  • Schlegel A, Giddings TH, Jr, Ladinsky MS, Kirkegaard K. Cellular origin and ultrastructure of membranes induced during poliovirus infection. J Virol. 1996;70:6576–6588. [PMC free article] [PubMed]
  • Schramm B, Krijnse Locker JK. Cytoplasmic organization of POXvirus DNA replication. Traffic. 2005;6:839–846. [PubMed]
  • Schwartz M, Chen J, Janda M, Sullivan M, den Boon J, Ahlquist P. A positive-strand RNA virus replication complex parallels form and function of retrovirus capsids. Mol Cell. 2002;9:505–514. [PubMed]
  • Schwartz M, Chen J, Lee WM, Janda M, Ahlquist P. Alternate, virus-induced membrane rearrangements support positive-strand RNA virus genome replication. Proc Natl Acad Sci U S A. 2004;101:11263–11268. [PubMed]
  • Snijder EJ, Bredenbeek PJ, Dobbe JC, Thiel V, Ziebuhr J, Poon LL, Guan Y, Rozanov M, Spaan WJ, Gorbalenya AE. Unique and conserved features of genome and proteome of SARS-coronavirus, an early split-off from the coronavirus group 2 lineage. J Mol Biol. 2003;331:991–1004. [PubMed]
  • Snijder EJ, van der Meer Y, Zevenhoven-Dobbe J, Onderwater JJ, van der Meulen J, Koerten HK, Mommaas AM. Ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex. J Virol. 2006;80:5927–5940. [PMC free article] [PubMed]
  • Snijder EJ, van Tol H, Roos N, Pedersen KW. Non-structural proteins 2 and 3 interact to modify host cell membranes during the formation of the arterivirus replication complex. J Gen Virol. 2001;82:985–994. [PubMed]
  • Stertz S, Reichelt M, Spiegel M, Kuri T, Martinez-Sobrido L, Garcia-Sastre A, Weber F, Kochs G. The intracellular sites of early replication and budding of SARS-coronavirus. Virology. 2007;361:304–315. [PubMed]
  • Suhy DA, Giddings TH, Jr, Kirkegaard K. Remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagy-like origin for virus-induced vesicles. J Virol. 2000;74:8953–8965. [PMC free article] [PubMed]
  • Sullivan ML, Ahlquist P. A brome mosaic virus intergenic RNA3 replication signal functions with viral replication protein 1a to dramatically stabilize RNA in vivo. J Virol. 1999;73:2622–2632. [PMC free article] [PubMed]
  • Taylor MP, Kirkegaard K. Potential subversion of autophagosomal pathway by picornaviruses. Autophagy. 2008;4:286–289. [PubMed]
  • Teterina NL, Gorbalenya AE, Egger D, Bienz K, Ehrenfeld E. Poliovirus 2C protein determinants of membrane binding and rearrangements in mammalian cells. J Virol. 1997;71:8962–8972. [PMC free article] [PubMed]
  • Teterina NL, Gorbalenya AE, Egger D, Bienz K, Rinaudo MS, Ehrenfeld E. Testing the modularity of the N-terminal amphipathic helix conserved in picornavirus 2C proteins and hepatitis C NS5A protein. Virology. 2006;344:453–467. [PubMed]
  • Thiel V, Ivanov KA, Putics A, Hertzig T, Schelle B, Bayer S, Weissbrich B, Snijder EJ, Rabenau H, Doerr HW, et al. Mechanisms and enzymes involved in SARS coronavirus genome expression. J Gen Virol. 2003;84:2305–2315. [PubMed]
  • Tolonen N, Doglio L, Schleich S, Krijnse Locker J. Vaccinia virus DNA replication occurs in endoplasmic reticulum-enclosed cytoplasmic mini-nuclei. Mol Biol Cell. 2001;12:2031–2046. [PMC free article] [PubMed]
  • van Hemert MJ, de Wilde AH, Gorbalenya AE, Snijder EJ. The in vitro RNA synthesizing activity of the isolated arterivirus replication/transcription complex is dependent on a host factor. J Biol Chem. 2008a;283:16525–16536. [PubMed]
  • van Hemert MJ, van den Worm SH, Knoops K, Mommaas AM, Gorbalenya AE, Snijder EJ. SARS-coronavirus replication/transcription complexes are membrane-protected and need a host factor for activity in vitro. PLoS Pathog. 2008b;4:e1000054. [PMC free article] [PubMed]
  • Van Wynsberghe PM, Ahlquist P. 5' cis elements direct nodavirus RNA1 recruitment to mitochondrial sites of replication complex formation. J Virol. 2009;83:2976–2988. [PMC free article] [PubMed]
  • Van Wynsberghe PM, Chen HR, Ahlquist P. Nodavirus RNA replication protein a induces membrane association of genomic RNA. J Virol. 2007;81:4633–4644. [PMC free article] [PubMed]
  • Venter PA, Krishna NK, Schneemann A. Capsid protein synthesis from replicating RNA directs specific packaging of the genome of a multipartite, positive-strand RNA virus. J Virol. 2005;79:6239–6248. [PMC free article] [PubMed]
  • Venter PA, Schneemann A. Recent insights into the biology and biomedical applications of Flock House virus. Cell Mol Life Sci. 2008;65:2675–2687. [PMC free article] [PubMed]
  • Waheed AA, Freed EO. Lipids and membrane microdomains in HIV-1 replication. Virus Res. 2009;143:162–176. [PMC free article] [PubMed]
  • Wang X, Lee WM, Watanabe T, Schwartz M, Janda M, Ahlquist P. Brome mosaic virus 1a nucleoside triphosphatase/helicase domain plays crucial roles in recruiting RNA replication templates. J Virol. 2005;79:13747–13758. [PMC free article] [PubMed]
  • Welsch S, Miller S, Romero-Brey I, Merz A, Bleck CK, Walther P, Fuller SD, Antony C, Krijnse-Locker J, Bartenschlager R. Composition and three-dimensional architecture of the dengue virus replication and assembly sites. Cell Host Microbe. 2009;5:365–375. [PubMed]
  • Yin J, Liu Y, Wimmer E, Paul AV. Complete protein linkage map between the P2 and P3 non-structural proteins of poliovirus. J Gen Virol. 2007;88:2259–2267. [PubMed]
  • Zhang X, Walker SB, Chipman PR, Nibert ML, Baker TS. Reovirus polymerase lambda 3 localized by cryo-electron microscopy of virions at a resolution of 7.6 A. Nat Struct Biol. 2003;10:1011–1018. [PMC free article] [PubMed]
  • Ziebuhr J, Snijder EJ, Gorbalenya AE. Virus-encoded proteinases and proteolytic processing in the Nidovirales. J Gen Virol. 2000;81:853–879. [PubMed]